( F ) Comparison of isogenic β ‐ cateninWT / − and β ‐ cateninHCT116 cells . Upper panel : western blotting showed decreased LC3 ‐ II in HCT116β ‐ catenin cells . Lower panel : western blotting confirmed by quantification using densitometry of the LC3 ‐ II / β ‐ actin ratio ( mean ± s . e . m . of three independent treatments , * P = 0 . 046 ) . Source data for this figure is available on the online supplementary information page .

O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(F) Comparison of isogenic β‐cateninWT/− and β‐cateninHCT116 cells. Upper panel: western blotting showed decreased LC3‐II in HCT116β‐catenin cells. Lower panel: western blotting confirmed by quantification using densitometry of the LC3‐II/β‐actin ratio (mean±s.e.m. of three independent treatments, *P=0.046).Source data for this figure is available on the online supplementary information page.


( A ) Protein expression of p62 increased following β ‐ catenin knockdown under normal and starved conditions in HT29 cells .

O O O O O O B-MEASURED_VAR O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O

(A) Protein expression of p62 increased following β‐catenin knockdown under normal and starved conditions in HT29 cells.


( B ) Comparison of HCT116 expression in isogenic HCT116β ‐ cateninWT / − and HCT116β ‐ catenin − / ΔS45 cells by western blotting showed decreased p62 expression in β ‐ catenin − / ΔS45 cells .

O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O B-MEASURED_VAR O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O

(B) Comparison of HCT116 expression in isogenic HCT116β‐cateninWT/− and HCT116β‐catenin−/ΔS45 cells by western blotting showed decreased p62 expression in β‐catenin−/ΔS45 cells.


( C ) LC3 puncta number per cell during starvation and nutrient addback post starvation . Left panel : LC3 puncta number reduces in non ‐ targeting ( NT ) siRNA control with nutrient addback . Right panel : LC3 puncta number reduces in β ‐ catenin knockdown cells with nutrient addback ( mean ± s . e . m . of three independent treatments ; n > 200 cells in > 20 fields of view per experiment ) . Western blotting for β ‐ catenin confirmed knockdown .

O O O B-MEASURED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O

(C) LC3 puncta number per cell during starvation and nutrient addback post starvation. Left panel: LC3 puncta number reduces in non‐targeting (NT) siRNA control with nutrient addback. Right panel: LC3 puncta number reduces in β‐catenin knockdown cells with nutrient addback (mean±s.e.m. of three independent treatments; n>200 cells in >20 fields of view per experiment). Western blotting for β‐catenin confirmed knockdown.


( D ) Western blotting of LC3 and p62 in HT29 cells following β ‐ catenin knockdown and starvation with lysosomal inhibitors . LC3 ‐ II and p62 protein expression showed an increase with β ‐ catenin siRNA in the presence of autophagy flux inhibitors chloroquine ( 10 μM ) or bafilomycin A1 ( 100 nM ; applied for the final 30 min of the 8 h ) compared to β ‐ catenin knockdown alone . Quantification by densitometry of the LC3 ‐ II / β ‐ actin ratio ( mean ± s . e . m . of four independent experiments ) . See also Supplementary Figure S1A . Source data for this figure is available on the online supplementary information page .

O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(D) Western blotting of LC3 and p62 in HT29 cells following β‐catenin knockdown and starvation with lysosomal inhibitors. LC3‐II and p62 protein expression showed an increase with β‐catenin siRNA in the presence of autophagy flux inhibitors chloroquine (10 μM) or bafilomycin A1 (100 nM; applied for the final 30 min of the 8 h) compared to β‐catenin knockdown alone. Quantification by densitometry of the LC3‐II/β‐actin ratio (mean±s.e.m. of four independent experiments). See also Supplementary Figure S1A.Source data for this figure is available on the online supplementary information page.


( A - D ) LC3 staining ( red ) in mouse intestinal crypt and villus epithelium increased ( white arrowheads ) 2 days post β ‐ catenin deletion in β ‐ catenin − / lox ‐ villin ‐ creERT2 mice ( C and D ) compared to control β ‐ catenin + / lox ‐ villin ‐ creERT2 mice ( A and B ) . Further magnification of the areas marked by white squares is shown ( lower panels ) .

O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A-D) LC3 staining (red) in mouse intestinal crypt and villus epithelium increased (white arrowheads) 2 days post β‐catenin deletion in β‐catenin−/lox‐villin‐creERT2 mice (C and D) compared to control β‐catenin+/lox‐villin‐creERT2 mice (A and B). Further magnification of the areas marked by white squares is shown (lower panels).


( E - H ) p62 staining ( green ) increased in the crypt and villus epithelium2 days after β ‐ catenin deletion in β ‐ catenin − / lox ‐ villin ‐ creERT2 mice ( G and H ) compared to control in β ‐ catenin + / lox ‐ villin ‐ creERT2 mice ( E and F ) . DAPI staining ( blue ) identifies nuclei . β ‐ Catenin deletion was confirmed by immunofluorescence ( Supplementary Figure S1B ) . Red and green channel levels were adjusted post acquisition for clarity ( equal level changes applied across the entire figure ) . Further magnification of the areas marked by white squares is shown ( lower panels ) .

O O O O O B-MEASURED_VAR O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(E-H)p62 staining (green) increased in the crypt and villus epithelium2 days after β‐catenin deletion in β‐catenin−/lox‐villin‐creERT2 mice (G and H) compared to control in β‐catenin+/lox‐villin‐creERT2 mice (E and F). DAPI staining (blue) identifies nuclei. β‐Catenin deletion was confirmed by immunofluorescence ( Supplementary Figure S1B). Red and green channel levels were adjusted post acquisition for clarity (equal level changes applied across the entire figure). Further magnification of the areas marked by white squares is shown (lower panels).


( I and J ) Western blotting on extracts from mouse intestinal epithelial tissue demonstrated increased p62 and LC3 ‐ II protein expression 2 ( I ) and 4 ( J ) days post β ‐ catenin deletion in β ‐ catenin − / lox ‐ villin ‐ creERT2 mice compared to control β ‐ catenin + / lox ‐ villin ‐ creERT2 mice . Source data for this figure is available on the online supplementary information page .

O O O O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O

(I and J) Western blotting on extracts from mouse intestinal epithelial tissue demonstrated increased p62 and LC3‐II protein expression 2 (I) and 4 (J) days post β‐catenin deletion in β‐catenin−/lox‐villin‐creERT2 mice compared to control β‐catenin+/lox‐villin‐creERT2 mice.Source data for this figure is available on the online supplementary information page.


( A ) Relative p62 / SQSTM1 mRNA levels by quantitative real ‐ time ‐ PCR ( qRT - PCR ) increased following β ‐ catenin knockdown in HT29 cells ( mean ± s . e . m . , three independent experiments performed in triplicate , * * * P0 . 001 ) . β ‐ Catenin knockdown was confirmed by western blotting .

O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O

(A) Relative p62/SQSTM1 mRNA levels by quantitative real‐time‐PCR (qRT-PCR) increased following β‐catenin knockdown in HT29 cells (mean±s.e.m., three independent experiments performed in triplicate, ***P0.001). β‐Catenin knockdown was confirmed by western blotting.


( B ) Relative p62 mRNA levels by qRT - PCR decreased with β ‐ cateninS33Yoverexpression after 24 h of starvation in HCT116 β ‐ cateninWT / − cells ( mean ± s . e . m . , three independent experiments performed in triplicate , * * * P0 . 001 ) . β ‐ CateninS33Y overexpression was confirmed by western blotting .

O O O O B-MEASURED_VAR O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O

(B) Relative p62 mRNA levels by qRT-PCR decreased with β‐cateninS33Yoverexpression after 24 h of starvation in HCT116 β‐cateninWT/− cells (mean±s.e.m., three independent experiments performed in triplicate, ***P0.001). β‐CateninS33Y overexpression was confirmed by western blotting.


( C ) The increase in p62 protein expression induced by β ‐ catenin siRNA was attenuated in HT29 cells treated with 10 μg / μl cycloheximide and starvation for 8 and 24 h compared to vehicle control . A complementary experiment using the transcription inhibitor actinomycin D is shown in Supplementary Figure S1C .

O O O O O O B-MEASURED_VAR O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) The increase in p62 protein expression induced by β‐catenin siRNA was attenuated in HT29 cells treated with 10 μg/μl cycloheximide and starvation for 8 and 24 h compared to vehicle control. A complementary experiment using the transcription inhibitor actinomycin D is shown in Supplementary Figure S1C.


( D ) Overexpression of Fzd6 ( green , left panel ) increased p62 ( red , right panel ) protein expression in HEK293T cells . As an internal control , cells not overexpressing Fzd6 are shown in the field of view .

O O O O O B-CONTROLLED_VAR O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O

(D) Overexpression of Fzd6 (green, left panel) increased p62 (red, right panel) protein expression in HEK293T cells. As an internal control, cells not overexpressing Fzd6 are shown in the field of view.


( E ) Doxycycline induction ( 1 μg / ml ) of DNTCF4 increased p62 and LC3 ‐ II protein expression in doxycycline ‐ inducible DNTCF4 LS174T ‐ L8 cells . LGR5 downregulation by DNTCF4 confirmed inhibition of β ‐ catenin / TCF4 signalling .

O O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O B-MEASURED_VAR O O

(E) Doxycycline induction (1 μg/ml) of DNTCF4 increased p62 and LC3‐II protein expression in doxycycline‐inducible DNTCF4 LS174T‐L8 cells. LGR5 downregulation by DNTCF4 confirmed inhibition of β‐catenin/TCF4 signalling.


( F ) Relative p62 mRNA levels ( 48 h ) by qRT - PCR increased following doxycycline induction of DNTCF4 in LS174T ‐ L8 cells ( mean ± s . e . m . , three independent experiments performed in triplicate , * * * P0 . 001 ) .

O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(F) Relative p62 mRNA levels (48 h) by qRT-PCR increased following doxycycline induction of DNTCF4 in LS174T‐L8 cells (mean±s.e.m., three independent experiments performed in triplicate, ***P0.001).


( G ) Upper panel : Wnt3a ( 200 ng / ml ) treatment of HCT116 β ‐ cateninWT / − cells ( 24 h ) decreased LC3 ‐ II and p62 protein expression by western blotting . Lower panel : quantification by densitometry of the LC3 ‐ II / β ‐ actin ratio ( mean ± s . e . m . of three independent experiments ) . Source data for this figure is available on the online supplementary information page .

O O O O O O B-CONTROLLED_VAR O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(G) Upper panel: Wnt3a (200 ng/ml) treatment of HCT116 β‐cateninWT/− cells (24 h) decreased LC3‐II and p62 protein expression by western blotting. Lower panel: quantification by densitometry of the LC3‐II/β‐actin ratio (mean±s.e.m. of three independent experiments).Source data for this figure is available on the online supplementary information page.


( A ) Chromatin immunoprecipitation ( ChIP ) demonstrates β ‐ catenin and TCF4 binding to the p62 promoter . HT29 cells were grown in normal or starvation conditions for 2 h and subjected to ChIP analysis with the indicated antibodies ( IgG was used as a negative control ) . Binding of RNA Pol II , β ‐ catenin and TCF4 to the p62 promoter region was measured by quantitative PCR and expressed as percent enrichment relative to the input chromatin . During nutrient deprivation , binding of RNA Pol II to the p62 promoter increased ; binding of β ‐ catenin to the p62 promoter decreased ; TCF4 binding did not change ( data are from one representative experiment of at least three independent experiments performed in triplicate ) . PCR products subjected to agarose gel electrophoresis are shown in Supplementary Figure S2A .

O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O B-MEASURED_VAR O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O B-MEASURED_VAR O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) Chromatin immunoprecipitation (ChIP) demonstrates β‐catenin and TCF4 binding to the p62 promoter. HT29 cells were grown in normal or starvation conditions for 2 h and subjected to ChIP analysis with the indicated antibodies (IgG was used as a negative control). Binding of RNA Pol II, β‐catenin and TCF4 to the p62 promoter region was measured by quantitative PCR and expressed as percent enrichment relative to the input chromatin. During nutrient deprivation, binding of RNA Pol II to the p62 promoter increased; binding of β‐catenin to the p62 promoter decreased; TCF4 binding did not change (data are from one representative experiment of at least three independent experiments performed in triplicate). PCR products subjected to agarose gel electrophoresis are shown in Supplementary Figure S2A.


( B ) Relative p62 mRNA expression by qRT - PCR increases ∼ 14 ‐ fold after 24 h starvation in HT29 cells ( mean ± s . e . m . , three independent experiments performed in triplicate , * * * P0 . 001 ) .

O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(B) Relative p62 mRNA expression by qRT-PCR increases ∼14‐fold after 24 h starvation in HT29 cells (mean±s.e.m., three independent experiments performed in triplicate, ***P0.001).


( C ) Binding of acetyl ‐ Histone H3 to the p62 promoter increased under starvation conditions , suggesting p62 gene derepression ( data are from one representative experiment of at least three independent experiments performed in triplicate ) .

O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O B-MEASURED_VAR O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O

(C) Binding of acetyl‐Histone H3 to the p62 promoter increased under starvation conditions, suggesting p62 gene derepression (data are from one representative experiment of at least three independent experiments performed in triplicate).


( D ) Binding of β ‐ catenin to the p62 promoter ( relative to β ‐ catenin binding to a non ‐ target gene promoter , GAPDH ) significantly decreased under starvation conditions ( mean ± s . e . m . , three independent experiments performed in triplicate , * * P0 . 01 ) .

O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O B-MEASURED_VAR O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(D) Binding of β‐catenin to the p62 promoter (relative to β‐catenin binding to a non‐target gene promoter, GAPDH) significantly decreased under starvation conditions (mean±s.e.m., three independent experiments performed in triplicate, **P0.01).


( A ) Western blotting showing that autophagy was induced after 2 h starvation , as evidenced by decreased p62 protein and increased LC3 ‐ II protein expression . β ‐ Catenin protein expression decreased over a 24 ‐ h period of starvation in HT29 cells .

O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O

(A) Western blotting showing that autophagy was induced after 2 h starvation, as evidenced by decreased p62 protein and increased LC3‐II protein expression. β‐Catenin protein expression decreased over a 24‐h period of starvation in HT29 cells.


( B ) Autophagy decreased following Atg7 knockdown as shown by increased p62 and decreased LC3 ‐ II protein expression . β ‐ Catenin protein expression increased following Atg7 siRNA . Reduction of LC3 puncta with Atg7 siRNA was confirmed by immunofluorescence and is shown in Supplementary Figure S2B .

O O O O O O B-CONTROLLED_VAR O O O O O B-MEASURED_VAR O O B-MEASURED_VAR O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O B-CONTROLLED_VAR O O O O O O O O O O O O O

(B) Autophagy decreased following Atg7 knockdown as shown by increased p62 and decreased LC3‐II protein expression. β‐Catenin protein expression increased following Atg7 siRNA. Reduction of LC3 puncta with Atg7 siRNA was confirmed by immunofluorescence and is shown in Supplementary Figure S2B.


( C , D ) Reduction of TopFlash activity in HT29 cells after 24 h of autophagy induction using ( C ) starvation or ( D ) 100 nM mTOR inhibitor PP242 ( C and D , mean ± s . e . m . , three independent experiments performed in triplicate , * * * P0 . 001 ) . Autophagy induction was confirmed by western blotting and is shown in Supplementary Figures S2C and S2D ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C, D) Reduction of TopFlash activity in HT29 cells after 24 h of autophagy induction using (C) starvation or (D) 100 nM mTOR inhibitor PP242 (C and D, mean±s.e.m., three independent experiments performed in triplicate, ***P0.001). Autophagy induction was confirmed by western blotting and is shown in Supplementary Figures S2C and S2D).


( E , F ) qRT - PCR shows reduction of Wnt ‐ target gene expression ( E ) Axin2 ( * P = 0 . 017 ) and ( F ) Cyclin D1 ( * * P = 0 . 0036 ) after 8 h starvation ( mean ± s . e . m . , three ( Axin2 ) or six ( Cyclin D1 ) independent experiments performed in triplicate ) .

O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O

(E, F) qRT-PCR shows reduction of Wnt‐target gene expression (E) Axin2 (*P=0.017) and (F) Cyclin D1 (**P=0.0036) after 8 h starvation (mean±s.e.m., three (Axin2) or six (Cyclin D1) independent experiments performed in triplicate).


( G - I ) Inhibition of Wnt ‐ induced TopFlash activity by autophagy induction in ( G ) HCT116 β ‐ cateninWT / − cells ( mean ± s . e . m . , three independent experiments performed in triplicate , * * P0 . 01 ) and ( H ) RKO cells ( mean ± s . e . m . , three independent experiments performed in triplicate , * P0 . 05 ) . ( I ) Reduction of Wnt3a ‐ induced Cyclin D1 gene expression RKO cells after 12 h treatment with autophagy induction using starvation or PP242 ( mean ± s . e . m . , two independent experiments ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O

(G-I) Inhibition of Wnt‐induced TopFlash activity by autophagy induction in (G) HCT116 β‐cateninWT/− cells (mean±s.e.m., three independent experiments performed in triplicate, **P0.01) and (H) RKO cells (mean±s.e.m., three independent experiments performed in triplicate, *P0.05). (I) Reduction of Wnt3a‐induced Cyclin D1 gene expression RKO cells after 12 h treatment with autophagy induction using starvation or PP242 (mean±s.e.m., two independent experiments).


( J , K ) TopFlash activity following Atg7 knockdown in ( J ) HT29 cells ( mean ± s . e . m . , four independent experiments performed in triplicate , * P = 0 . 0488 ) and ( K ) RKO cells with 24 hWnt3a treatment ( mean ± s . e . m . , three independent experiments performed in triplicate , * P = 0 . 0282 ) .

O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(J, K) TopFlash activity following Atg7 knockdown in (J) HT29 cells (mean±s.e.m., four independent experiments performed in triplicate, *P=0.0488) and (K) RKO cells with 24 hWnt3a treatment (mean±s.e.m., three independent experiments performed in triplicate, *P=0.0282).


( L ) A representative western blot of RKO cells following Wnt3a treatment and Atg7 siRNA .

O O O O O O O O O O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O

(L) A representative western blot of RKO cells following Wnt3a treatment and Atg7 siRNA.


( M ) Relative β ‐ catenin mRNA levels did not change after 8 h starvation by qRT - PCR ( mean ± s . e . m . , three independent experiments performed in triplicate ) .

O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(M) Relative β‐catenin mRNA levels did not change after 8 h starvation by qRT-PCR (mean±s.e.m., three independent experiments performed in triplicate).


( N ) Western blot showing prevented β ‐ catenin protein degradation during starvation in the presence of lysosomal autophagy inhibitors chloroquine ( 10 μM ) and bafilomycin A1 ( 100 nM ) compared to starvation alone .

O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O

(N) Western blot showing prevented β‐catenin protein degradation during starvation in the presence of lysosomal autophagy inhibitors chloroquine (10 μM) and bafilomycin A1 (100 nM) compared to starvation alone.


( O ) Inhibition of autophagy using wortmannin ( 50 nM ) prevented β ‐ catenin protein degradation during starvation .

O O O O O O O B-CONTROLLED_VAR O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O

(O) Inhibition of autophagy using wortmannin (50 nM) prevented β‐catenin protein degradation during starvation.


( P ) Western blot analysis of RKO cells expressing a myc - tagged β ‐ catenin mutant ( S33A , S37A , T41A , S45A ) resistant to proteasomal degradation ( myc - β ‐ cateninAAAA ) . RKO cells were subject to nutrient starvation for 2 , 8 and 24 h , and autophagy induction was confirmed by increased LC3 ‐ II and decreased p62 protein expression . Both endogenous β ‐ catenin and myc - β ‐ cateninAAAA protein expression decreased during starvation .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O

(P) Western blot analysis of RKO cells expressing a myc-tagged β‐catenin mutant (S33A, S37A, T41A, S45A) resistant to proteasomal degradation (myc-β‐cateninAAAA). RKO cells were subject to nutrient starvation for 2, 8 and 24 h, and autophagy induction was confirmed by increased LC3‐II and decreased p62 protein expression. Both endogenous β‐catenin and myc-β‐cateninAAAA protein expression decreased during starvation.


( Q ) Proteasome inhibition with MG132 ( 10 μM ) did not prevent starvation ‐ induced decrease in β ‐ catenin protein expression . Source data for this figure is available on the online supplementary information page .

O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O

(Q) Proteasome inhibition with MG132 (10 μM) did not prevent starvation‐induced decrease in β‐catenin protein expression.Source data for this figure is available on the online supplementary information page.


LC3 and β ‐ catenin co ‐ localise in the mouseintestinal epithelium . ( A and B ) Immunofluorescence of LC3 ( red ) and β ‐ catenin ( green ) expression in the intestinal epithelium following 2 daystamoxifen treatment in control ( A : β ‐ catenin + / lox ‐ villin ‐ creERT2 ) and β ‐ catenin deleted ( B : β ‐ catenin − / lox ‐ villin ‐ creERT2 ) mice . ( C and D ) Magnified areas from B revealing co ‐ localisation of β ‐ catenin ( red ) and β ‐ catenin ( green ) . Arrowheads indicate co ‐ localisation of LC3 and β ‐ catenin . ( E ) Linescan analyses from C and D showing staining intensity of indicated co ‐ localised puncta . Red and green channel levels were adjusted post acquisition ( equal changes applied across the entire figure ) and the blue channel was removed for clarity .

B-MEASURED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O B-MEASURED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

LC3 and β‐catenin co‐localise in the mouseintestinal epithelium. (A and B) Immunofluorescence of LC3 (red) and β‐catenin (green) expression in the intestinal epithelium following 2 daystamoxifen treatment in control (A: β‐catenin+/lox‐villin‐creERT2) and β‐catenin deleted (B: β‐catenin−/lox‐villin‐creERT2) mice. (C and D) Magnified areas from B revealing co‐localisation of β‐catenin (red) and β‐catenin (green). Arrowheads indicate co‐localisation of LC3 and β‐catenin. (E) Linescan analyses from C and D showing staining intensity of indicated co‐localised puncta. Red and green channel levels were adjusted post acquisition (equal changes applied across the entire figure) and the blue channel was removed for clarity.


( A ) Co ‐ immunoprecipitation of YFP - LC3 or negative control YFP in HT29 cells . Binding of endogenous β ‐ catenin to YFP - LC3 was detected after 8 h of autophagy induction by starvation and starvation in the presence of lysosomal autophagy flux inhibitor chloroquine ( 10 μM ) . Input and immunodepleted lysates are shown .

O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O

(A) Co‐immunoprecipitation of YFP-LC3 or negative control YFP in HT29 cells. Binding of endogenous β‐catenin to YFP-LC3 was detected after 8 h of autophagy induction by starvation and starvation in the presence of lysosomal autophagy flux inhibitor chloroquine (10 μM). Input and immunodepleted lysates are shown.


( B ) Immunoprecipitation of endogenous LC3 in HT29 cells . IgG was used as a negative control .

O O O O O O B-MEASURED_VAR O O O O O O O O O O O O

(B) Immunoprecipitation of endogenous LC3 in HT29 cells. IgG was used as a negative control.


( D ) Pulldown assays using recombinant GST or GST - LC3B and lysates from HEK293 cells expressing HA - tagged wild ‐ type β ‐ catenin ( HA - β ‐ cateninWT ) or W504A / I507A β ‐ catenin ( HA - β ‐ cateninW504A / I507A ) . HA - β ‐ cateninW504A / I507A exhibited reduced GST - LC3B binding compared to HA - β ‐ cateninWT .

O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O B-MEASURED_VAR O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O

(D) Pulldown assays using recombinant GST or GST-LC3B and lysates from HEK293 cells expressing HA-tagged wild‐type β‐catenin (HA-β‐cateninWT) or W504A/I507A β‐catenin (HA-β‐cateninW504A/I507A). HA-β‐cateninW504A/I507A exhibited reduced GST-LC3B binding compared to HA-β‐cateninWT.


( E ) Recombinant myc - tagged His - LC3 ( His - LC3 - myc ) interacted with recombinant GST - β ‐ cateninWTin vitro , but binding to GST - β ‐ cateninW504A / I507A was reduced ( upper panel ) . Input recombinant proteins visualised with Coomassie blue are shown ( lower panels ) .

O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O

(E) Recombinant myc-tagged His-LC3 (His-LC3-myc) interacted with recombinant GST-β‐cateninWTin vitro, but binding to GST-β‐cateninW504A/I507A was reduced (upper panel). Input recombinant proteins visualised with Coomassie blue are shown (lower panels).


( F ) Co ‐ immunoprecipitation experiments using lysates from HEK293 cells transiently expressing YFP - LC3 with β ‐ cateninWT or β ‐ cateninW504A / I507A starved for 8 h with chloroquine ( 10 μM ) . YFP - LC3 immunoprecipitated β ‐ cateninWT but not β ‐ cateninW504A / I507A . Input lysates are shown and immunoprecipitated p62 and YFP - LC3 served as positive controls .

O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O O O O

(F) Co‐immunoprecipitation experiments using lysates from HEK293 cells transiently expressing YFP-LC3 with β‐cateninWT or β‐cateninW504A/I507A starved for 8 h with chloroquine (10 μM). YFP-LC3 immunoprecipitated β‐cateninWT but not β‐cateninW504A/I507A. Input lysates are shown and immunoprecipitated p62 and YFP-LC3 served as positive controls.


( G ) HA - β ‐ cateninS33A / S37A / T41A / S45A / W504A / I507A ( HA - β ‐ catenin6A ) was more resistant to the starvation ‐ induced reduction in β ‐ catenin protein expression than HA - β ‐ cateninS33Y .

O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O

(G) HA-β‐cateninS33A/S37A/T41A/S45A/W504A/I507A (HA-β‐catenin6A) was more resistant to the starvation‐induced reduction in β‐catenin protein expression than HA-β‐cateninS33Y.


( H ) Relative cell death in HT29 cells subject to nutrient starvation for 24 h . Atg7 knockdown increased cell survival . The increase in cell survival following Atg7 knockdown was significantly reversed by simultaneously depleting β ‐ catenin ( double knockdown of Atg7 and β ‐ catenin ) . Data are the mean ± s . e . m . of three independent experiments performed in triplicate , * * * P0 . 001 ; * * P0 . 01 . Source data for this figure is available on the online supplementary information page .

O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(H) Relative cell death in HT29 cells subject to nutrient starvation for 24 h. Atg7 knockdown increased cell survival. The increase in cell survival following Atg7 knockdown was significantly reversed by simultaneously depleting β‐catenin (double knockdown of Atg7 and β‐catenin). Data are the mean±s.e.m. of three independent experiments performed in triplicate, ***P0.001; **P0.01.Source data for this figure is available on the online supplementary information page.


( b - c ) Western blot for p53 and select p53 targets post - radiation in ( b ) oscillatory and ( c ) rising conditions . Actin is shown as a loading control .

O O O O O O O O B-MEASURED_VAR O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O

(b-c) Western blot for p53 and select p53 targets post-radiation in (b) oscillatory and (c) rising conditions. Actin is shown as a loading control.


( d - e ) Average levels of ( d ) mRNA quantified by RNAseq ( red ) or qRT - PCR ( grey ) or ( e ) protein quantified by mass spectrometry ( light green ) or western blot ( grey ) for select p53 target genes under oscillatory p53 . Data information : , n = 2 biological replicates , error bars represent std dev .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O

(d-e) Average levels of (d) mRNA quantified by RNAseq (red) or qRT-PCR (grey) or (e) protein quantified by mass spectrometry (light green) or western blot (grey) for select p53 target genes under oscillatory p53. Data information: , n=2 biological replicates, error bars represent std dev.


( f - g ) Average levels of ( f ) mRNA quantified by RNAseq ( blue ) or qRT - PCR ( grey ) or ( g ) protein quantified by mass spectrometry ( dark green ) or western blot ( grey ) for select p53 target genes under rising p53 . Data information : n = 2 biological replicates , error bars represent std dev .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O

(f-g) Average levels of (f) mRNA quantified by RNAseq (blue) or qRT-PCR (grey) or (g) protein quantified by mass spectrometry (dark green) or western blot (grey) for select p53 target genes under rising p53. Data information: n=2 biological replicates, error bars represent std dev.


[ Ca2 + ] i changes in individual BMDMs were measured ( A ) using fura - 2 during frustrated phagocytosis ( n = 46 - 113 ) and ( B ) by dropping beads onto adherent cells loaded with Calbryte 520 ( n = 64 - 159 ) , in 1 . 8 mM extracellular Ca2 + ( + Ca2 + o ) or Ca2 + - free medium containing 100 μM EGTA ( - Ca2 + o ) with or without prior loading with 25 μM EGTA / AM or 25 μM BAPTA / AM . The maximum fura - 2 ratio changes ( ∆ 350 / 380 ) or fluorescence changes normalized to initial fluorescence ( ∆ F / F0 ) are plotted .

O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O B-MEASURED_VAR I-MEASURED_VAR O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O B-CONTROLLED_VAR O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

[Ca2+]i changes in individual BMDMs were measured (A) using fura-2 during frustrated phagocytosis (n = 46-113) and (B) by dropping beads onto adherent cells loaded with Calbryte 520 (n = 64-159), in 1.8 mM extracellular Ca2+ (+Ca2+o) or Ca2+-free medium containing 100 μM EGTA (-Ca2+o) with or without prior loading with 25 μM EGTA/AM or 25 μM BAPTA/AM. The maximum fura-2 ratio changes (∆ 350/380) or fluorescence changes normalized to initial fluorescence (∆ F/F0) are plotted.


FcγR - mediated phagocytosis was monitored using beads conjugated to Alexa Fluor 488 ( in green ) . External beads were differentially labelled with anti - IgG ( in red ) and distinguished by their dual labelling ( in yellow ) . Cytochalasin D ( 10 μM , Cyt D ) prevented bead internalization .

O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O

FcγR-mediated phagocytosis was monitored using beads conjugated to Alexa Fluor 488 (in green). External beads were differentially labelled with anti-IgG (in red) and distinguished by their dual labelling (in yellow). Cytochalasin D (10 μM, Cyt D) prevented bead internalization.


Bead uptake in 15 min , under conditions as in ( A , B ) , n = 80 - 91 cells with representative images of individual cells delineated by a dotted line .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Bead uptake in 15 min, under conditions as in (A, B), n = 80-91 cells with representative images of individual cells delineated by a dotted line.


Experiments conducted in - Ca2 + o : ( F ) 1 μM thapsigarin ( Tg ) or 10 μM CPA pre - treatment , all but eliminated the 1 μM ionomycin ( iono ) response ; single - cell Ca2 + traces and mean ∆ F / F0 , n = 450 - 603 cells . ( G ) FcγR activation ( as in B ) evoked Ca2 + oscillations that were blocked with Tg or CPA treatment , whilst bead phagocytosis was not affected ( H ) , n = 312 - 428 cells . ( I ) 1 μM bafilomycin A1 ( Baf ) for 3h , or Ca2 + - binding dextran ( Cal520 - dx ) and non - Ca2 + - binding dextrans ( FITC - dx and Texas red - dx ) trafficked to lysosomes , minimally affected global Ca2 + signals evoked by FcγR activation ( J ) ; in parallel , bead uptake was only reduced by bafilomycin A1 or by chelating Ca2 + within the lysosome with Cal520 - dx ( K ) , n = 100 - 162 cells . All cells are WT BMDM and all beads are 3 - μm IgG - opsonized .

O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O B-CONTROLLED_VAR O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O B-MEASURED_VAR I-MEASURED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Experiments conducted in -Ca2+o: (F) 1 μM thapsigarin (Tg) or 10 μM CPA pre-treatment, all but eliminated the 1 μM ionomycin (iono) response; single-cell Ca2+ traces and mean ∆ F/F0, n = 450-603 cells. (G) FcγR activation (as in B) evoked Ca2+ oscillations that were blocked with Tg or CPA treatment, whilst bead phagocytosis was not affected (H), n = 312-428 cells. (I) 1 μM bafilomycin A1 (Baf) for 3h, or Ca2+- binding dextran (Cal520-dx) and non-Ca2+-binding dextrans (FITC-dx and Texas red-dx) trafficked to lysosomes, minimally affected global Ca2+ signals evoked by FcγR activation (J); in parallel, bead uptake was only reduced by bafilomycin A1 or by chelating Ca2+ within the lysosome with Cal520-dx (K), n = 100-162 cells. All cells are WT BMDM and all beads are 3-μm IgG-opsonized.


The Ca2 + signal elicited by frustrated phagocytosis was significantly reduced in Ned - 19 ( 10 μM ) - treated WT and in all TPC - KO BMDMs ; maximum Ca2 + amplitude , expressed as a percentage of WT control ( n = 314 - 641 cells ) . Phagocytosis of opsonized 3 - µm ( B and C ) and 6 - μm ( D ) beads was inhibited by Ned - 19 ( B ) and in BMDMs from Tpcn - / - mice compared to WT ( n = 134 - 280 cells ) .

O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O

The Ca2+ signal elicited by frustrated phagocytosis was significantly reduced in Ned-19 (10 μM)-treated WT and in all TPC-KO BMDMs; maximum Ca2+ amplitude, expressed as a percentage of WT control (n = 314-641 cells). Phagocytosis of opsonized 3-µm (B and C) and 6-μm (D) beads was inhibited by Ned-19 (B) and in BMDMs from Tpcn-/- mice compared to WT (n = 134-280 cells).


( E ) Phagocytosis of 3 - µm beads was unchanged in MCOLN1 - / - BMDMs , but was impaired using 6 - µm beads ( F ) , n = 89 - 157 .

O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O

(E) Phagocytosis of 3-µm beads was unchanged in MCOLN1-/- BMDMs, but was impaired using 6-µm beads (F), n = 89-157.


Heterologous expression of mouse TPCs tagged with TagRFP - T ( in red ) rescues the phagocytic defect of Tpcn1 - / - and Tpcn2 - / - BMDM , but a pore - dead TPC2 - mutant ( N257A ) does not , n = 39 - 94 . Images taken 40 min post - 3 - μm bead addition .

O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O

Heterologous expression of mouse TPCs tagged with TagRFP-T (in red) rescues the phagocytic defect of Tpcn1-/- and Tpcn2-/- BMDM, but a pore-dead TPC2-mutant (N257A) does not, n = 39-94. Images taken 40 min post- 3-μm bead addition.


Raising cytosolic Ca2 + with ionomycin ( 1 μM ) rescues the Tpcn1 - / - and Tpcn2 - / - phagocytic defect , whilst UTP ( 100 μM ) and ML - SA1 ( 50 μM ) only weakly rescue ( n = 84 - 192 cells ) .

O O B-MEASURED_VAR I-MEASURED_VAR O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O

Raising cytosolic Ca2+ with ionomycin (1 μM) rescues the Tpcn1-/- and Tpcn2-/- phagocytic defect, whilst UTP (100 μM) and ML-SA1 (50 μM) only weakly rescue (n = 84-192 cells).


Internalization of bacteria is reduced in Tpcn - / - BMDM , as assessed by single - cell imaging of BMDM allowed to internalise M . smegmatis tagged with mCherry ( in red ) for 1 h . Internalization of bacteria assessed discontinuously in BMDM populations : ( B ) The resultant growth of bacteria colonies on agar plates following lysis of BMDM infected with M . smegmatis and ( C ) quantification of colony forming units ( CFU ) expressed as a percentage of WT control ( n = 12 ) ;

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Internalization of bacteria is reduced in Tpcn-/- BMDM, as assessed by single-cell imaging of BMDM allowed to internalise M. smegmatis tagged with mCherry (in red) for 1 h. Internalization of bacteria assessed discontinuously in BMDM populations: (B) The resultant growth of bacteria colonies on agar plates following lysis of BMDM infected with M. smegmatis and (C) quantification of colony forming units (CFU) expressed as a percentage of WT control (n = 12);


Real - time internalization of fluorescent bacteria into BMDM populations : ( D ) Relative Fluorescence Units ( RFU ) of internalised mCherry - tagged M . smegmatis ( E ) and mCherry - tagged BCG over time , read using a plate reader ( n = 8 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Real-time internalization of fluorescent bacteria into BMDM populations: (D) Relative Fluorescence Units (RFU) of internalised mCherry-tagged M. smegmatis (E) and mCherry-tagged BCG over time, read using a plate reader (n = 8).


BMDMs were co - loaded with 2 µM Calbryte590 / AM ( 50 min ) and 200 nM Lysotracker Green ( LTG , 5 min ) and , at room temperature , presented with 3 - µm opsonized beads labelled with Alexa Fluor 647 ( blue ) . A three - channel image was recorded every 10 s . The LTG brightness has been enhanced post hoc to visualize the dimmer , peripheral lysosomes . Time Zero is defined as the frame when the first Ca2 + signal was observed . ( A ) Images represent Ca2 + responses or an overlay of LTG and bead fluorescence . Times of each image are relative to the first Ca2 + spike . ( B ) Enlargement of the first contact point between lysosomes and bead ( red arrow ) as depicted by the dashed box , 80 s post - Ca2 + . ( C ) Whole - cell Ca2 + responses from the cell in A . ( D ) Collation of the post - Ca2 + contact time , displayed as a scatter plot of individual cells and the mean ± S . E . M . ( n = 27 cells ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O

BMDMs were co-loaded with 2 µM Calbryte590/AM (50 min) and 200 nM Lysotracker Green (LTG, 5 min) and, at room temperature, presented with 3-µm opsonized beads labelled with Alexa Fluor 647 (blue). A three-channel image was recorded every 10 s. The LTG brightness has been enhanced post hoc to visualize the dimmer, peripheral lysosomes. Time Zero is defined as the frame when the first Ca2+ signal was observed. (A) Images represent Ca2+ responses or an overlay of LTG and bead fluorescence. Times of each image are relative to the first Ca2+ spike. (B) Enlargement of the first contact point between lysosomes and bead (red arrow) as depicted by the dashed box, 80 s post-Ca2+. (C) Whole-cell Ca2+ responses from the cell in A. (D) Collation of the post-Ca2+ contact time, displayed as a scatter plot of individual cells and the mean ± S.E.M. (n = 27 cells).


RAW246 . 7 cells were transfected with either TPC1 - TagRFP - T or TPC2 - TagRFP - T ( red ) and presented with 3 - µm opsonized beads labelled with Alexa Fluor 647 ( blue ) . Acute time - series of bead engagement were collected . Contact between TPCs and beads are depicted in the time - stamped micrographs , where time is arbitrary after the start of the image stack ( E , F ) i . e . 110 s is still prior to engagement . The spatial relationship between TPCs and beads was quantified using a target graticule of 0 . 5 - µm bands centred on the engulfed bead ; the mean fluorescence within each band was normalized to the fluorescence of the maximum band and plotted against this radial distance ( G , H ) ; n = 35 - 36 cells .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

RAW246.7 cells were transfected with either TPC1-TagRFP-T or TPC2-TagRFP-T (red) and presented with 3-µm opsonized beads labelled with Alexa Fluor 647 (blue). Acute time-series of bead engagement were collected. Contact between TPCs and beads are depicted in the time-stamped micrographs, where time is arbitrary after the start of the image stack (E,F) i.e. 110 s is still prior to engagement. The spatial relationship between TPCs and beads was quantified using a target graticule of 0.5-µm bands centred on the engulfed bead; the mean fluorescence within each band was normalized to the fluorescence of the maximum band and plotted against this radial distance (G,H); n = 35-36 cells.


RAW 247 . 6 macrophages loaded with a cytosolic chemical dye ( Cyto dye ) to detect global cytosolic Ca2 + , and heterologously expressing ( B ) TPC1 - G - GECO1 . 2 ( TPC1 - GG ) or ( C - E ) TPC2 - G - GECO1 . 2 ( TPC2 - GG ) to detect peri - endo - lysosomal Ca2 + domains , which were not detected by ( F ) a TPC2 mutant ( D276K ) - G - GECO1 . 2 that blocks ion permeation or ( A ) unfused cytosolic G - GECO1 . 2 ( Cyto GG ) . ( A - F ) Single - cell Ca2 + traces upon FcγR activation by opsonized 3 - μm beads in - Ca2 + o expressed as a percentage of the maximum fluorescence of the indicator , determined by adding 2 μM ionomycin and 10 mM CaCl2 at the end of each experiment ( % Fmax ) ( D ) TPC2 - G - GECO1 . 2 activity was inhibited by 10 µM Ned - 19 , but was unaffected by ( E ) chelating global Ca2 + with EGTA / AM ( 100 μM for 2 min prior to bead addition ) . Summary data of the first spike from cells A - F ( subsequent spikes show the same pattern ; Appendix Fig . S4B ) , n = 87 - 274 cells ; * * * P < 0 . 001 vs Cyto GG , # # # P < 0 . 001 vs TPC2 - GG ctrl , † † † P < 0 . 001 vs Cyto GG . Fusion of G - GECO1 . 2 to TPC2 did not alter the affinity of G - GECO1 . 2 for Ca2 + determined in permeabilized cells ( n = 34 - 71 cells ) .

O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O

RAW 247.6 macrophages loaded with a cytosolic chemical dye (Cyto dye) to detect global cytosolic Ca2+, and heterologously expressing (B) TPC1-G-GECO1.2 (TPC1-GG) or (C-E) TPC2-G-GECO1.2 (TPC2-GG) to detect peri-endo-lysosomal Ca2+ domains, which were not detected by (F) a TPC2 mutant (D276K)-G-GECO1.2 that blocks ion permeation or (A) unfused cytosolic G-GECO1.2 (Cyto GG). (A-F) Single-cell Ca2+ traces upon FcγR activation by opsonized 3-μm beads in -Ca2+o expressed as a percentage of the maximum fluorescence of the indicator, determined by adding 2 μM ionomycin and 10 mM CaCl2 at the end of each experiment (% Fmax) (D) TPC2-G-GECO1.2 activity was inhibited by 10 µM Ned-19, but was unaffected by (E) chelating global Ca2+ with EGTA/AM (100 μM for 2 min prior to bead addition). Summary data of the first spike from cells A-F (subsequent spikes show the same pattern; Appendix Fig. S4B), n = 87-274 cells; ***P<0.001 vs Cyto GG, ### P<0.001 vs TPC2-GG ctrl, ††† P<0.001 vs Cyto GG. Fusion of G-GECO1.2 to TPC2 did not alter the affinity of G-GECO1.2 for Ca2+ determined in permeabilized cells (n = 34-71 cells).


( I ) Single - cell Ca2 + traces upon FcγR activation by opsonized 3 - μm beads in - Ca2 + o expressed as a percentage of Fmax . ( I , J ) Pre - emptying the ER stores with CPA or thapsigargin ( Tg ) abolished the global cytosolic Ca2 + response evoked by beads , but a TPC2 - G ‑ GECO1 . 2 component remained , n = 17 - 121 cells .

O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O

(I) Single-cell Ca2+ traces upon FcγR activation by opsonized 3-μm beads in -Ca2+o expressed as a percentage of Fmax. (I, J) Pre-emptying the ER stores with CPA or thapsigargin (Tg) abolished the global cytosolic Ca2+ response evoked by beads, but a TPC2-G‑GECO1.2 component remained, n = 17 - 121 cells.


No local Ca2 + signal was detected with TPC2 - G - GECO1 . 2 when Ca2 + spiking was evoked by ER - Ca2 + release with 100 μM UTP ( K , L ) or 10 μM CPA ( M ) ; only a bystander response equivalent to unfused cytosolic G - GECO1 . 2 was detected ( n = 121 - 182 cells ; * P < 0 . 05 ) .

O O B-MEASURED_VAR I-MEASURED_VAR O O O O B-CONTROLLED_VAR O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

No local Ca2+ signal was detected with TPC2-G-GECO1.2 when Ca2+ spiking was evoked by ER-Ca2+ release with 100 μM UTP (K, L) or 10 μM CPA (M); only a bystander response equivalent to unfused cytosolic G-GECO1.2 was detected (n = 121-182 cells; *P<0.05).


Translocation of CB , a non - Ca2 + binding CB mutant ( CB mut ) or a triple mTagBFP2 ( BFP ) to the lysosome ( LAMP1 ) , as indicated by the increase in the Pearson ' s correlation coefficient was induced by 250 nM rapalog ( 30 mins ) compared with ethanol ( EtOH ) control - treated RAW 247 . 6 cells . n = 11 - 25 cells ; * * * P < 0 . 001 .

O O B-MEASURED_VAR O O O O O O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Translocation of CB, a non-Ca2+ binding CB mutant (CB mut) or a triple mTagBFP2 (BFP) to the lysosome (LAMP1), as indicated by the increase in the Pearson's correlation coefficient was induced by 250 nM rapalog (30 mins) compared with ethanol (EtOH) control-treated RAW 247.6 cells. n = 11-25 cells; ***P<0.001.


Images of a single RAW 247 . 6 cell displaying yellow puncta indicative of successful re - positioning of CB ( in green ) to the lysosome ( in red ) in the presence of rapalog but not EtOH .

O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O

Images of a single RAW 247.6 cell displaying yellow puncta indicative of successful re-positioning of CB (in green) to the lysosome (in red) in the presence of rapalog but not EtOH.


When CB was brought to the lysosome ( + rapalog ) uptake of beads was inhibited , whilst the non - Ca2 + binding CB ( CB mut ; n = 46 - 187 cells ) or triple mTagBFP2 ( BFP ; n = 38 - 52 cells ) did not block phagocytosis ; * * * P < 0 . 001 .

O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

When CB was brought to the lysosome (+rapalog) uptake of beads was inhibited, whilst the non-Ca2+ binding CB (CB mut; n = 46-187 cells) or triple mTagBFP2 (BFP; n = 38-52 cells) did not block phagocytosis;***P<0.001.


Bead uptake in BMDMs with and without loading of 25 µM EGTA / AM and 10 μM Ned - 19 . EGTA - insensitive phagocytosis of beads was inhibited by Ned - 19 ( n = 75 - 102 cells ) in WT BMDM ; * * * P < 0 . 001 .

O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O

Bead uptake in BMDMs with and without loading of 25 µM EGTA/AM and 10 μM Ned-19. EGTA-insensitive phagocytosis of beads was inhibited by Ned-19 (n= 75-102 cells) in WT BMDM; ***P<0.001.


RAW 247 . 6 macrophages expressing cytosolic G - GECO1 . 2 ( Cyto GG ) , TPC2 - G - GECO1 . 2 ( TPC2 - GG ) or TRPML1 - G - GECO1 . 2 ( TRPML1 - GG ) . ( F ) Single - cell Ca2 + traces upon FcγR activation by opsonized 3 - μm or 6 - μm beads in - Ca2 + o expressed as a percentage of the maximum indicator fluorescence ( % Fmax = 2 μM ionomycin and 10 mM CaCl2 ) . ( G ) Summary of first Ca2 + spike ; n = 69 - 121 ( 3 - μm bead ) , 22 - 46 ( 6 - μm beads ) cells ; * * * P < 0 . 001 , * * P < 0 . 01 vs Cyto GG , # # # P < 0 . 001 TPC2 - GG vs TRPML1 - GG .

O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O

RAW 247.6 macrophages expressing cytosolic G-GECO1.2 (Cyto GG), TPC2-G-GECO1.2 (TPC2-GG) or TRPML1-G-GECO1.2 (TRPML1-GG). (F) Single-cell Ca2+ traces upon FcγR activation by opsonized 3-μm or 6-μm beads in -Ca2+o expressed as a percentage of the maximum indicator fluorescence (% Fmax = 2 μM ionomycin and 10 mM CaCl2). (G) Summary of first Ca2+ spike; n = 69-121 (3-μm bead), 22-46 (6-μm beads) cells; ***P<0.001, **P<0.01 vs Cyto GG, ### P<0.001 TPC2-GG vs TRPML1-GG.


Dynamin inhibitors ( 80 μM Dynasore or 20 μM Dyngo - 4a ) inhibit phagocytosis of opsonized 3 - μm beads in WT BMDM ; n = 101 - 149 cells , * * * P < 0 . 001 .

B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Dynamin inhibitors (80 μM Dynasore or 20 μM Dyngo-4a) inhibit phagocytosis of opsonized 3-μm beads in WT BMDM; n = 101-149 cells, ***P<0.001.


Rescue of the TPC - KO dynamin defect by the dynamin activator , Ryngo - 1 - 23 ( 80 μM ) : mean number of internalised beads per cell as a percentage of the WT control ; n = 128 - 241 cells , * * P < 0 . 01 , * * * P < 0 . 001 .

O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Rescue of the TPC-KO dynamin defect by the dynamin activator, Ryngo-1-23 (80 μM): mean number of internalised beads per cell as a percentage of the WT control; n = 128-241 cells, **P<0.01, ***P<0.001.


In WT BMDM , expression of a dominant - negative dynamin - 2 mutant ( Dyn2 K44A ) reduced phagocytosis ( n = 68 - 184 ) . Expression of wildtype dynamin - 2 ( Dyn2 ) or constitutively active dynamin - 2 ( S764A mutant , mimicking the dephosphorylated state ) in Tpcn1 - / - macrophages restored phagocytosis of 3 - μm beads after 10 min , n = 10 - 76 cells ; * * * P < 0 . 001 vs WT control , # # # P < 0 . 001 vs Tpcn1 - / - control .

O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O

In WT BMDM, expression of a dominant-negative dynamin-2 mutant (Dyn2 K44A) reduced phagocytosis (n = 68-184). Expression of wildtype dynamin-2 (Dyn2) or constitutively active dynamin-2 (S764A mutant, mimicking the dephosphorylated state) in Tpcn1-/- macrophages restored phagocytosis of 3-μm beads after 10 min, n = 10-76 cells; ***P<0.001 vs WT control, ### P<0.001 vs Tpcn1-/- control.


Single WT BMDM expressing HA - tagged dynamin - 2 ( immunolabelled with anti - HA , in red ) undergoing phagocytosis of IgG - 3 - μm beads ( blue ) . Images were taken 10 or 30 min post - addition of beads ; actin ( green ) labelled with Phalloidin Alexa 488 .

O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Single WT BMDM expressing HA-tagged dynamin-2 (immunolabelled with anti-HA, in red) undergoing phagocytosis of IgG-3-μm beads (blue). Images were taken 10 or 30 min post-addition of beads; actin (green) labelled with Phalloidin Alexa 488.


Calcineurin inhibitors , FK506 ( 10 μM ) or cyclosporin A ( CsA , 10 μM ) inhibited phagocytosis in WT cells , n = 65 - 159 cells . Ryngo ( 80 μM ) rescued phagocytosis defects in Tpcn1 - / - BMDM even in the presence of FK506 or CsA , n = 103 - 208 cells .

O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O

Calcineurin inhibitors, FK506 (10 μM) or cyclosporin A (CsA, 10 μM) inhibited phagocytosis in WT cells, n = 65-159 cells. Ryngo (80 μM) rescued phagocytosis defects in Tpcn1-/- BMDM even in the presence of FK506 or CsA, n = 103-208 cells.


Calcineurin activity and cytosolic Ca2 + responses simultaneously monitored in single RAW 247 . 6 using CaNAR2 and jRGECO1a , respectively , upon addition of 3 - μm beads . CaNAR2 signals ( FRET Y / C ) were inhibited by FK506 ( 10 μM ) and Ned - 19 ( 10 μM ) ; ( H ) single - cell traces and ( I ) mean maximum responses . n = 65 - 169 ; * * * P < 0 . 001 vs control .

B-MEASURED_VAR O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Calcineurin activity and cytosolic Ca2+ responses simultaneously monitored in single RAW 247.6 using CaNAR2 and jRGECO1a, respectively, upon addition of 3-μm beads. CaNAR2 signals (FRET Y/C) were inhibited by FK506 (10 μM) and Ned-19 (10 μM); (H) single-cell traces and (I) mean maximum responses. n = 65-169; ***P<0.001 vs control.


Phagocytosis induced dephosphorylation of dynamin - 2 ( ratio of phospho - dynamin to total dynamin - 2 expressed as a percentage of WT at time zero ) in WT BMDM , but not in Tpcn1 - / - BMDM ; * * P < 0 . 01 , * * * P < 0 . 001 ( time - point matched unpaired t - test ) n = 10 - 15 . Measured using in - cell western assay , representative wells of plate image : total dyn2 ( red ) and phospho - dyn2 ( green ) . See also Appendix Figures S6 and S7 .

O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O B-MEASURED_VAR O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O B-MEASURED_VAR O O O O O O O O O O O O

Phagocytosis induced dephosphorylation of dynamin-2 (ratio of phospho-dynamin to total dynamin-2 expressed as a percentage of WT at time zero) in WT BMDM, but not in Tpcn1-/- BMDM; **P<0.01, ***P<0.001 (time-point matched unpaired t-test) n = 10-15. Measured using in-cell western assay, representative wells of plate image: total dyn2 (red) and phospho-dyn2 (green). See also Appendix Figures S6 and S7.


RAW 246 . 7 macrophages expressing the dynein - binding protein ( mTagBFP2 - BicD2 - FKBP12 ) for rapalog - induced lysosome repositioning to the MTOC , also co - expressed HsLAMP1 - mCherry with or without a C - terminal FRB * ( LAMP - FRB * or LAMP , in red ) . Cells were treated with 0 . 1 % ethanol or 250 nM rapalog for 2 h . Only the tripartite complex of FKBP12 - rapalog - FRB induced clustering of lysosomes at the MTOC ( A , B ) . This clustering was selective for lysosomes because ER and mitochondria ( KDEL - GFP and MitoTracker Green ) were not affected ( C ) . ( A , B ) Phagocytosis of IgG - 3 - μm beads was reduced by immobilizing lysosomes at the MTOC , n = 80 - 564 cells ; * * * P < 0 . 001 .

O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

RAW 246.7 macrophages expressing the dynein-binding protein (mTagBFP2-BicD2-FKBP12) for rapalog-induced lysosome repositioning to the MTOC, also co-expressed HsLAMP1-mCherry with or without a C-terminal FRB* (LAMP-FRB* or LAMP, in red). Cells were treated with 0.1% ethanol or 250 nM rapalog for 2 h. Only the tripartite complex of FKBP12-rapalog-FRB induced clustering of lysosomes at the MTOC (A, B). This clustering was selective for lysosomes because ER and mitochondria (KDEL-GFP and MitoTracker Green) were not affected (C). (A, B) Phagocytosis of IgG-3-μm beads was reduced by immobilizing lysosomes at the MTOC, n = 80-564 cells; ***P<0.001.


Activation of dynamin - 2 with Ryngo 1 - 23 or expression of constitutively active dynamin - 2 ( S764A mutant , expression confirmed by the grayscale image ) restored phagocytosis of IgG - 3 - μm beads ( n = 34 - 51 cells ) , but had no effect on the phagocytosis of IgG - 6 - μm beads , n = 36 - 72 cells ( E ) ; * * * P < 0 . 001 EtOH Ctrl vs rapalog Ctrl , # # # P < 0 . 001 rapalog Ctrl vs rapalog Ryngo / Dyn2S764A .

O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR O

Activation of dynamin-2 with Ryngo 1-23 or expression of constitutively active dynamin-2 (S764A mutant, expression confirmed by the grayscale image) restored phagocytosis of IgG-3-μm beads (n = 34-51 cells), but had no effect on the phagocytosis of IgG-6-μm beads, n = 36-72 cells (E); ***P<0.001 EtOH Ctrl vs rapalog Ctrl, ###P<0.001 rapalog Ctrl vs rapalog Ryngo/Dyn2S764A.


( A ) mRNA expressions of PINK1 in CSCs and non - CSCs . Mean ± SEM from 6 patients . Data information : Paired t - test with Bonferroni method , * * p < 0 . 01 , * * * p < 0 . 001 .

O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) mRNA expressions of PINK1 in CSCs and non-CSCs. Mean±SEM from 6 patients. Data information: Paired t-test with Bonferroni method, **p < 0.01, ***p < 0.001.


( B - C ) Immunoblot analysis of PINK1 protein in non - CSCs and CSCs . Representative and mean ± SEM from 9 patients . Data information : Paired t - test with Bonferroni method , * * p < 0 . 01 , * * * p < 0 . 001 .

O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(B-C) Immunoblot analysis of PINK1 protein in non-CSCs and CSCs. Representative and mean±SEM from 9 patients. Data information: Paired t-test with Bonferroni method, **p < 0.01, ***p < 0.001.


( D - E ) Immunoblot analysis of p - PINK1 protein in non - CSCs and CSCs . Representative and mean ± SEM from 7 patients . Data information : Paired t - test with Bonferroni method , * * p < 0 . 01 , * * * p < 0 . 001 .

O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(D-E) Immunoblot analysis of p-PINK1 protein in non-CSCs and CSCs. Representative and mean±SEM from 7 patients. Data information: Paired t-test with Bonferroni method, **p < 0.01, ***p < 0.001.


Immunoblot analysis of Parkin protein in non - CSCs and CSCs .

Immunoblot analysis of Parkin protein in non-CSCs and CSCs.


analysis of Parkin protein in non - CSCs and CSCs . Representative and mean ± SEM from 8 patients . Data information : Paired t - test with Bonferroni method , * * p < 0 . 01 , * * * p < 0 . 001 .

O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

analysis of Parkin protein in non-CSCs and CSCs. Representative and mean±SEM from 8 patients. Data information: Paired t-test with Bonferroni method, **p < 0.01, ***p < 0.001.


( H - I ) PINK1 and Parkin protein levels in mitochondrial fractions and whole cell lyses of CSCs and non - CSCs . Representative and mean ± SEM from 8 patients . Data information : Paired t - test with Bonferroni method , * * p < 0 . 01 , * * * p < 0 . 001 .

O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(H-I) PINK1 and Parkin protein levels in mitochondrial fractions and whole cell lyses of CSCs and non-CSCs. Representative and mean±SEM from 8 patients. Data information: Paired t-test with Bonferroni method, **p < 0.01, ***p < 0.001.


( J - K ) Intracellular levels of p - Parkin protein in non - CSCs and CSCs were analyzed using flow cytometry . Representative histograms and mean ± SEM from 6 patients . Data information : Paired t - test with Bonferroni method , * * p < 0 . 01 , * * * p < 0 . 001 .

O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(J-K) Intracellular levels of p-Parkin protein in non-CSCs and CSCs were analyzed using flow cytometry. Representative histograms and mean±SEM from 6 patients. Data information: Paired t-test with Bonferroni method, **p < 0.01, ***p < 0.001.


Immunoblot analysis of p - Parkin protein in non - CSCs and CSCs .

O O O O O B-MEASURED_VAR O O O O O O O O

Immunoblot analysis of p-Parkin protein in non-CSCs and CSCs.


analysis of p - Parkin protein in non - CSCs and CSCs . Representative and mean ± SEM from 11 patients . Data information : Paired t - test with Bonferroni method , * * p < 0 . 01 , * * * p < 0 . 001 .

O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

analysis of p-Parkin protein in non-CSCs and CSCs. Representative and mean±SEM from 11 patients. Data information: Paired t-test with Bonferroni method, **p < 0.01, ***p < 0.001.


( N - P ) CSCs were treated with Mdivi - 1 ( 50 μM ) and analyzed for sphere formation plus tumorgenicity . Mean ± SEM from 6 experiments . Scale bar 20 mm . Data information : Paired t - test with Bonferroni method , * * p < 0 . 01 , * * * p < 0 . 001 .

O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(N-P) CSCs were treated with Mdivi-1 (50 μM) and analyzed for sphere formation plus tumorgenicity. Mean±SEM from 6 experiments. Scale bar 20 mm. Data information: Paired t-test with Bonferroni method, **p < 0.01, ***p < 0.001.


( Q ) CSCs transfected with PINK1 shRNA or the control were analysed for sphere formation and tumorgenicity . Mean ± SEM from 6 experiments . Data information : Paired t - test with Bonferroni method , * * p < 0 . 01 , * * * p < 0 . 001 .

O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(Q) CSCs transfected with PINK1 shRNA or the control were analysed for sphere formation and tumorgenicity. Mean±SEM from 6 experiments. Data information: Paired t-test with Bonferroni method, **p < 0.01, ***p < 0.001.


( R ) CSCs were treated with DAPT ( 10 μM ) and analysed for sphere formation and tumorgenicity . Mean ± SEM from 6 experiments . Data information : Paired t - test with Bonferroni method , * * p < 0 . 01 , * * * p < 0 . 001 .

O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(R) CSCs were treated with DAPT (10 μM) and analysed for sphere formation and tumorgenicity. Mean±SEM from 6 experiments. Data information: Paired t-test with Bonferroni method, **p < 0.01, ***p < 0.001.


( S ) CSCs were transfected with PINK1 shRNA or control , and analysed for activated Notch1 . Mean ± SEM from 6 patients . Data information : Paired t - test with Bonferroni method , * * p < 0 . 01 , * * * p < 0 . 001 .

O O O O O O O B-CONTROLLED_VAR O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(S) CSCs were transfected with PINK1 shRNA or control, and analysed for activated Notch1. Mean±SEM from 6 patients. Data information: Paired t-test with Bonferroni method, **p < 0.01, ***p < 0.001.


( T ) CSCs with or without transfection of Notch1 ICD were treated with Mdivi - 1 ( 50 μM ) and analyzed for sphere formation plus tumorgenicity . Mean ± SEM from 6 experiments . Data information : Paired t - test with Bonferroni method , * * p < 0 . 01 , * * * p < 0 . 001 .

O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(T) CSCs with or without transfection of Notch1 ICD were treated with Mdivi-1 (50 μM) and analyzed for sphere formation plus tumorgenicity. Mean±SEM from 6 experiments. Data information: Paired t-test with Bonferroni method, **p < 0.01, ***p < 0.001.


( A - B ) Lysosomal DNA was identified with anti - LAMP1 and anti - DNA antibodies in non - CSCs and CSCs . Scale bar 5μm . Representative and mean ± SEM with 50 cells from 5 patients . Data information : Paired t - test with Bonferroni method , * * p < 0 . 01 , * * * p < 0 . 001 .

O O O O O O B-MEASURED_VAR O O O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A-B) Lysosomal DNA was identified with anti-LAMP1 and anti-DNA antibodies in non-CSCs and CSCs. Scale bar 5μm. Representative and mean±SEM with 50 cells from 5 patients. Data information: Paired t-test with Bonferroni method, **p < 0.01, ***p < 0.001.


( C ) Lysosomal content of mtDNA in CSCs and non - CSCs was determined with qPCR . Mean ± SEM from 6 patients . Data information : Paired t - test with Bonferroni method , * * p < 0 . 01 , * * * p < 0 . 001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) Lysosomal content of mtDNA in CSCs and non-CSCs was determined with qPCR. Mean±SEM from 6 patients. Data information: Paired t-test with Bonferroni method, **p < 0.01, ***p < 0.001.


( D ) CSCs were treated with Mdivi - 1 ( 50 μM ) and analysed for lysosomal mtDNA . Mean ± SEM from 6 patients . Data information : Paired t - test with Bonferroni method , * * p < 0 . 01 , * * * p < 0 . 001 .

O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(D) CSCs were treated with Mdivi-1 (50 μM) and analysed for lysosomal mtDNA. Mean±SEM from 6 patients. Data information: Paired t-test with Bonferroni method, **p < 0.01, ***p < 0.001.


( E ) CSCs with or without transfection of PINK1 shRNA were analysed for lysosomal mtDNA . Mean ± SEM from 6 patients . Data information : Paired t - test with Bonferroni method , * * p < 0 . 01 , * * * p < 0 . 001 .

O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(E) CSCs with or without transfection of PINK1 shRNA were analysed for lysosomal mtDNA. Mean±SEM from 6 patients. Data information: Paired t-test with Bonferroni method, **p < 0.01, ***p < 0.001.


( F - G ) Protein expressions of TLR9 and MyD88 in CSCs and non - CSCs . Representative and mean ± SEM from 6 patients . Data information : Paired t - test with Bonferroni method , * * p < 0 . 01 , * * * p < 0 . 001 .

O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(F-G) Protein expressions of TLR9 and MyD88 in CSCs and non-CSCs. Representative and mean±SEM from 6 patients. Data information: Paired t-test with Bonferroni method, **p < 0.01, ***p < 0.001.


( H ) TLR9 proteins were immunoprecipitated from CSCs and non - CSCs and analyzed for the binding load of mtDNA . Mean ± SEM from 6 patients . Data information : Paired t - test with Bonferroni method , * * p < 0 . 01 , * * * p < 0 . 001 .

O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(H) TLR9 proteins were immunoprecipitated from CSCs and non-CSCs and analyzed for the binding load of mtDNA. Mean±SEM from 6 patients. Data information: Paired t-test with Bonferroni method, **p < 0.01, ***p < 0.001.


( I ) CSCs were transfected with mtDNA and analysed for expressions of TLR9 plus MyD88 . Mean ± SEM from 6 patients . Data information : Paired t - test with Bonferroni method , * * p < 0 . 01 , * * * p < 0 . 001 .

O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(I) CSCs were transfected with mtDNA and analysed for expressions of TLR9 plus MyD88. Mean±SEM from 6 patients. Data information: Paired t-test with Bonferroni method, **p < 0.01, ***p < 0.001.


( J - K ) CSCs treated with EtBr ( 50 ng / ml ) were analysed for lysosomal mtDNA , TLR9 , and MyD88 . Mean ± SEM from 6 patients . Data information : Paired t - test with Bonferroni method , * * p < 0 . 01 , * * * p < 0 . 001 .

O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O B-MEASURED_VAR O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(J-K) CSCs treated with EtBr (50 ng/ml) were analysed for lysosomal mtDNA, TLR9, and MyD88. Mean±SEM from 6 patients. Data information: Paired t-test with Bonferroni method, **p < 0.01, ***p < 0.001.


( L ) CSCs transfected with DNase II expression vector or the control were analyzed for lysosomal DNA plus MyD88 protein . Mean ± SEM from 6 patients . ( M ) CSCs transfected with DNase II shRNA or the control were analyzed for lysosomal DNA plus MyD88 protein . Mean ± SEM from 6 patients . Data information : Paired t - test with Bonferroni method , * * p < 0 . 01 , * * * p < 0 . 001 .

O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(L) CSCs transfected with DNase II expression vector or the control were analyzed for lysosomal DNA plus MyD88 protein. Mean±SEM from 6 patients. (M) CSCs transfected with DNase II shRNA or the control were analyzed for lysosomal DNA plus MyD88 protein. Mean±SEM from 6 patients. Data information: Paired t-test with Bonferroni method, **p < 0.01, ***p < 0.001.


( A ) CSCs were stimulated with CpG ODN ( 10 μg / ml ) or control and analysed for sphere formation and tumorgenicity . Mean ± SEM from 6 experiments . Data information : Paired t - test with Bonferroni method , * * p < 0 . 01 , * * * p < 0 . 001 .

O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) CSCs were stimulated with CpG ODN (10 μg/ml) or control and analysed for sphere formation and tumorgenicity. Mean±SEM from 6 experiments. Data information: Paired t-test with Bonferroni method, **p < 0.01, ***p < 0.001.


CSCs transfected with mtDNA were analyzed for sphere formation Mean ± SEM from 6 experiments . Data information : Paired t - test with Bonferroni method , * * p < 0 . 01 , * * * p < 0 . 001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

CSCs transfected with mtDNA were analyzed for sphere formation Mean±SEM from 6 experiments. Data information: Paired t-test with Bonferroni method, **p < 0.01, ***p < 0.001.


CSCs transfected with mtDNA were analyzed for tumorgenicity . Mean ± SEM from 6 experiments . Data information : Paired t - test with Bonferroni method , * * p < 0 . 01 , * * * p < 0 . 001 .

CSCs transfected with mtDNA were analyzed for tumorgenicity. Mean±SEM from 6 experiments. Data information: Paired t-test with Bonferroni method, **p < 0.01, ***p < 0.001.


( D ) CSCs were transfected with 2 different TLR9 shRNAs or the control , and analysed for sphere formation and tumorgenicity . Mean ± SEM from 6 experiments . Data information : Paired t - test with Bonferroni method , * * p < 0 . 01 , * * * p < 0 . 001 .

O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(D) CSCs were transfected with 2 different TLR9 shRNAs or the control, and analysed for sphere formation and tumorgenicity. Mean±SEM from 6 experiments. Data information: Paired t-test with Bonferroni method, **p < 0.01, ***p < 0.001.


( E - F ) CSCs transfected with DNase II expression vector , DNase II shRNA , or the control was analyzed for sphere formation . Mean ± SEM from 6 patients . Data information : Paired t - test with Bonferroni method , * * p < 0 . 01 , * * * p < 0 . 001 .

O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(E-F) CSCs transfected with DNase II expression vector, DNase II shRNA, or the control was analyzed for sphere formation. Mean±SEM from 6 patients. Data information: Paired t-test with Bonferroni method, **p < 0.01, ***p < 0.001.


( G ) CpG promoted tumor growth of PDX chimeras . Mean ± SEM from 6 mice . Data information : Paired t - test with Bonferroni method , * * p < 0 . 01 , * * * p < 0 . 001 .

O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(G) CpG promoted tumor growth of PDX chimeras. Mean±SEM from 6 mice. Data information: Paired t-test with Bonferroni method, **p < 0.01, ***p < 0.001.


( H ) TLR9 inhibitor chloroquine abrogated the effect of CpG on tumor growth in PDX chimeras . Mean ± SEM from 6 mice . Data information : Paired t - test with Bonferroni method , * * p < 0 . 01 , * * * p < 0 . 001 .

O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(H) TLR9 inhibitor chloroquine abrogated the effect of CpG on tumor growth in PDX chimeras. Mean±SEM from 6 mice. Data information: Paired t-test with Bonferroni method, **p < 0.01, ***p < 0.001.


( I ) CpG treatment increased the frequency of CD133 - expressing CSCs in PDX chimeras . Mean ± SEM from 6 mice . Data information : Paired t - test with Bonferroni method , * * p < 0 . 01 , * * * p < 0 . 001 .

O O O B-CONTROLLED_VAR O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(I) CpG treatment increased the frequency of CD133-expressing CSCs in PDX chimeras. Mean±SEM from 6 mice. Data information: Paired t-test with Bonferroni method, **p < 0.01, ***p < 0.001.


( J ) Chloroquine impeded the function of CpG in elevating CD133 - expressing CSCs of PDX chimeras . Mean ± SEM from 6 mice . Data information : Paired t - test with Bonferroni method , * * p < 0 . 01 , * * * p < 0 . 001 .

O O O B-CONTROLLED_VAR O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(J) Chloroquine impeded the function of CpG in elevating CD133-expressing CSCs of PDX chimeras. Mean±SEM from 6 mice. Data information: Paired t-test with Bonferroni method, **p < 0.01, ***p < 0.001.


( A ) CSCs transfected with TLR9 shRNA or control were analysed for protein expressions of activated Notch1 plus HES1 . Mean ± SEM from 6 patients . Data information : Paired t - test with Bonferroni method , * * p < 0 . 01 , * * * p < 0 . 001 .

O O O O O O B-CONTROLLED_VAR O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) CSCs transfected with TLR9 shRNA or control were analysed for protein expressions of activated Notch1 plus HES1. Mean±SEM from 6 patients. Data information: Paired t-test with Bonferroni method, **p < 0.01, ***p < 0.001.


( B ) CSCs treated with or without EtBr ( 50 ng / ml ) were analyzed for activated Notch1 . Mean ± SEM from 6 patients . Data information : Paired t - test with Bonferroni method , * * p < 0 . 01 , * * * p < 0 . 001 .

O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(B) CSCs treated with or without EtBr (50 ng/ml) were analyzed for activated Notch1. Mean±SEM from 6 patients. Data information: Paired t-test with Bonferroni method, **p < 0.01, ***p < 0.001.


( C ) CSCs transfected with DNase II expression vector or control were analyzed for activated Notch1 expression . Mean ± SEM from 6 patients . Data information : Paired t - test with Bonferroni method , * * p < 0 . 01 , * * * p < 0 . 001 .

O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) CSCs transfected with DNase II expression vector or control were analyzed for activated Notch1 expression. Mean±SEM from 6 patients. Data information: Paired t-test with Bonferroni method, **p < 0.01, ***p < 0.001.


( D ) CSCs with or without CpG treatment were analysed for interaction between TLR9 and Notch1 . Representative and mean ± SEM from 6 patients . Data information : Paired t - test with Bonferroni method , * * p < 0 . 01 , * * * p < 0 . 001 .

O O O O O O O B-CONTROLLED_VAR O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(D) CSCs with or without CpG treatment were analysed for interaction between TLR9 and Notch1. Representative and mean±SEM from 6 patients. Data information: Paired t-test with Bonferroni method, **p < 0.01, ***p < 0.001.


( E ) CSCs were treated with CpG ODN ( 10 μg / ml ) in the presence or absence of DAPT ( 10 μM ) , and analyzed for sphere formation plus tumorgenicity . Mean ± SEM from 6 experiments . Data information : Paired t - test with Bonferroni method , * * p < 0 . 01 , * * * p < 0 . 001 .

O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(E) CSCs were treated with CpG ODN (10 μg/ml) in the presence or absence of DAPT (10 μM), and analyzed for sphere formation plus tumorgenicity. Mean±SEM from 6 experiments. Data information: Paired t-test with Bonferroni method, **p < 0.01, ***p < 0.001.


( F ) CSCs were transfected with 2 different Notch1 shRNAs or control , treated with CpG ODN ( 10 μg / ml ) , and analyzed for sphere formation plus tumorgenicity . Mean ± SEM from 6 experiments . Data information : Paired t - test with Bonferroni method , * * p < 0 . 01 , * * * p < 0 . 001 .

O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(F) CSCs were transfected with 2 different Notch1 shRNAs or control, treated with CpG ODN (10 μg/ml), and analyzed for sphere formation plus tumorgenicity. Mean±SEM from 6 experiments. Data information: Paired t-test with Bonferroni method, **p < 0.01, ***p < 0.001.


PDX chimeras treated by CpG with or without DAPT were analysed for tumor growth Mean ± SEM from 6 mice . Data information : Paired t - test with Bonferroni method , * * p < 0 . 01 , * * * p < 0 . 001 .

O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

PDX chimeras treated by CpG with or without DAPT were analysed for tumor growth Mean±SEM from 6 mice. Data information: Paired t-test with Bonferroni method, **p < 0.01, ***p < 0.001.


PDX chimeras treated by CpG with or without DAPT were analysed for CD133 - expressing CSCs . Mean ± SEM from 6 mice . Data information : Paired t - test with Bonferroni method , * * p < 0 . 01 , * * * p < 0 . 001 .

O O O O O O O O B-CONTROLLED_VAR O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

PDX chimeras treated by CpG with or without DAPT were analysed for CD133-expressing CSCs. Mean±SEM from 6 mice. Data information: Paired t-test with Bonferroni method, **p < 0.01, ***p < 0.001.


( A ) CSCs and non - CSCs were analyzed for mitochondrial metabolism using seahorse assay . Mean ± SEM from 6 patients . Data information : Paired t - test with Bonferroni method ( A , * * p < 0 . 01 , * * * p < 0 . 001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) CSCs and non-CSCs were analyzed for mitochondrial metabolism using seahorse assay. Mean±SEM from 6 patients. Data information: Paired t-test with Bonferroni method (A, **p < 0.01, ***p < 0.001.


( B ) CSCs were treated with 2 - DG ( 10 mM ) or Malonate ( 10 mM ) , and assayed for sphere formation plus tumorgenicity . Mean ± SEM from 6 patients . Data information : ANOVA test with Turkey ' s method ( B ) . * * p < 0 . 01 , * * * p < 0 . 001 .

O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(B) CSCs were treated with 2-DG (10 mM) or Malonate (10 mM), and assayed for sphere formation plus tumorgenicity. Mean±SEM from 6 patients. Data information: ANOVA test with Turkey's method (B). **p < 0.01, ***p < 0.001.


( C ) CSCs treated with DAPT ( 10 μM ) were analysed for mitochondrial OCR . Mean ± SEM from 6 patients . Data information : Paired t - test with Bonferroni method * * p < 0 . 01 , * * * p < 0 . 001 .

O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) CSCs treated with DAPT (10 μM) were analysed for mitochondrial OCR. Mean±SEM from 6 patients. Data information: Paired t-test with Bonferroni method **p < 0.01, ***p < 0.001.


( D ) CSCs transfected with Notch1 shRNA or control shRNA were analysed for mitochondrial OCR . Mean ± SEM from 6 patients . Data information : Paired t - test with Bonferroni method * * p < 0 . 01 , * * * p < 0 . 001 .

(D) CSCs transfected with Notch1 shRNA or control shRNA were analysed for mitochondrial OCR. Mean±SEM from 6 patients. Data information: Paired t-test with Bonferroni method **p < 0.01, ***p < 0.001.


( E ) CSCs treated with EtBr ( 50 ng / ml ) were analysed for mitochondrial OCR . Mean ± SEM from 6 patients . Data information : Paired t - test with Bonferroni method * * p < 0 . 01 , * * * p < 0 . 001 .

O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(E) CSCs treated with EtBr (50 ng/ml) were analysed for mitochondrial OCR. Mean±SEM from 6 patients. Data information: Paired t-test with Bonferroni method **p < 0.01, ***p < 0.001.


( F ) CSCs transfected with DNase II expression vector or control were analysed for mitochondrial OCR . Mean ± SEM from 6 patients . Data information : Paired t - test with Bonferroni method * * p < 0 . 01 , * * * p < 0 . 001 .

O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(F) CSCs transfected with DNase II expression vector or control were analysed for mitochondrial OCR. Mean±SEM from 6 patients. Data information: Paired t-test with Bonferroni method **p < 0.01, ***p < 0.001.


( G ) CSCs treated with chloroquine ( 50 μM ) were analyzed for mitochondrial OCR . Mean ± SEM from 6 patients . Data information : Paired t - test with Bonferroni method * * p < 0 . 01 , * * * p < 0 . 001 .

(G) CSCs treated with chloroquine (50 μM) were analyzed for mitochondrial OCR. Mean±SEM from 6 patients. Data information: Paired t-test with Bonferroni method **p < 0.01, ***p < 0.001.


( A ) CSCs stimulated with or without CpG ODN ( 10 μg / ml ) were analyzed for expression of phosphor - AMPKα . Representative and mean ± SEM from 6 patients . Data information : Paired t - test with Bonferroni method , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 .

O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) CSCs stimulated with or without CpG ODN (10 μg/ml) were analyzed for expression of phosphor-AMPKα. Representative and mean±SEM from 6 patients. Data information: Paired t-test with Bonferroni method , **p < 0.01, ***p < 0.001, ****p < 0.0001.


( B ) CSCs were stimulated with CpG ODN ( 10 μg / ml ) in the presence or absence of DAPT ( 10 μM ) , and analyzed for phosphor - AMPKα . Mean ± SEM from 6 patients . Data information : Paired t - test with Bonferroni method , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 .

O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(B) CSCs were stimulated with CpG ODN (10 μg/ml) in the presence or absence of DAPT (10 μM), and analyzed for phosphor-AMPKα. Mean±SEM from 6 patients. Data information: Paired t-test with Bonferroni method , **p < 0.01, ***p < 0.001, ****p < 0.0001.


( C ) CSCs transfected with Notch1 shRNA or control were stimulated with CpG ODN ( 10 μg / ml ) and analyzed for phosphor - AMPKα . Mean ± SEM from 6 patients . Data information : Paired t - test with Bonferroni method , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 .

O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) CSCs transfected with Notch1 shRNA or control were stimulated with CpG ODN (10 μg/ml) and analyzed for phosphor-AMPKα. Mean±SEM from 6 patients. Data information: Paired t-test with Bonferroni method , **p < 0.01, ***p < 0.001, ****p < 0.0001.


( D ) CSCs were treated with DAPT ( 10 μM ) in the presence or absence of A769662 ( 10 μM ) , and analysed for mitochondrial OCR . Ctrl represents CSCs without any treatment . Mean ± SEM from 6 patients . Data information : ANOVA with Turkey method ( D ) , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 .

O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(D) CSCs were treated with DAPT (10 μM) in the presence or absence of A769662 (10 μM), and analysed for mitochondrial OCR. Ctrl represents CSCs without any treatment. Mean±SEM from 6 patients. Data information: ANOVA with Turkey method (D), **p < 0.01, ***p < 0.001, ****p < 0.0001.


( E ) CSCs were stimulated with or without CpG ODN ( 10 μg / ml ) and analyzed for mitochondrial OCR . Mean ± SEM from 6 patients . Data information : Paired t - test with Bonferroni method , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 .

O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(E) CSCs were stimulated with or without CpG ODN (10 μg/ml) and analyzed for mitochondrial OCR. Mean±SEM from 6 patients. Data information: Paired t-test with Bonferroni method , **p < 0.01, ***p < 0.001, ****p < 0.0001.


( F ) CSCs were stimulated with CpG ODN ( 10 μg / ml ) in the presence or absence of Compound C ( 10 μM ) and analysed for mitochondrial OCR . Mean ± SEM from 6 patients . Data information : Paired t - test with Bonferroni method , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 .

O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(F) CSCs were stimulated with CpG ODN (10 μg/ml) in the presence or absence of Compound C (10 μM) and analysed for mitochondrial OCR. Mean±SEM from 6 patients. Data information: Paired t-test with Bonferroni method , **p < 0.01, ***p < 0.001, ****p < 0.0001.


( G ) CSCs were transfected with 2 different AMPKα shRNA or the control , stimulated with CpG ODN ( 10 μg / ml ) , and analysed for mitochondrial OCR . Mean ± SEM from 6 patients . Data information : Paired t - test with Bonferroni method , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 .

O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(G) CSCs were transfected with 2 different AMPKα shRNA or the control, stimulated with CpG ODN (10 μg/ml), and analysed for mitochondrial OCR. Mean±SEM from 6 patients. Data information: Paired t-test with Bonferroni method , **p < 0.01, ***p < 0.001, ****p < 0.0001.


( A ) CSCs transfected with or without Notch1 ICD were analyzed for AMP / ATP ratio plus LKB1 phosphorylation . Mean ± SEM from 6 patients . Data information : Paired t - test with Bonferroni method , * * p < 0 . 01 , * * * p < 0 . 001 .

O O O O O O O O B-CONTROLLED_VAR O O O O B-MEASURED_VAR O B-MEASURED_VAR O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) CSCs transfected with or without Notch1 ICD were analyzed for AMP/ATP ratio plus LKB1 phosphorylation. Mean±SEM from 6 patients. Data information: Paired t-test with Bonferroni method, **p < 0.01, ***p < 0.001.