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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O 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 O 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 .

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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O 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 O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O 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 .

(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 O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 .

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 O O O O O O O O O O O O O O O O O O O O O O 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 .

(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 O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 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 .

(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 O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 .

(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 O O O O O O O O O O O O O O O O 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 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-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

(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 O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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

(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 O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O 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 .

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(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 O O O O O O O O O O O O O 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 .

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(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 O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O 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 .

(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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 O O O O O O O O O O O O O O O O O O O O O O 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 .

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(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 O O O O O O O O O O O O O O O O O O O O O O O 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 .

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(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 .

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(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 .

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(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 .

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(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 .

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(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.


( B ) CSCs treated with or without DAPT ( 10 μM ) 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 .

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(B) CSCs treated with or without DAPT (10 μM) 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.


( C ) CSCs treated with or without CpG ODN ( 10 μg / ml ) were analysed for interactions between Notch1 and AMPK . 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-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

(C) CSCs treated with or without CpG ODN (10 μg/ml) were analysed for interactions between Notch1 and AMPK. Representative and mean±SEM from 6 patients. Data information: Paired t-test with Bonferroni method, **p < 0.01, ***p < 0.001.


( D ) CSCs transfected with LKB1 shRNA （ right ） or the control ( left ) were treated with CpG ODN ( 10 μg / ml ) and analysed for AMPK phosphorylation . Mean ± SEM from 6 patients . Data information : Paired t - test with Bonferroni method , * * p < 0 . 01 , * * * p < 0 . 001 .

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(D) CSCs transfected with LKB1 shRNA（right）or the control (left) were treated with CpG ODN (10 μg/ml) and analysed for AMPK phosphorylation. Mean±SEM from 6 patients. Data information: Paired t-test with Bonferroni method, **p < 0.01, ***p < 0.001.


( E ) CSCs transfected with TAK1 shRNA or the control were treated with CpG ODN ( 10 μg / ml ) and analysed for AMPK phosphorylation . Mean ± SEM from 6 patients . Data information : Paired t - test with Bonferroni method , * * p < 0 . 01 , * * * p < 0 . 001 .

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(E) CSCs transfected with TAK1 shRNA or the control were treated with CpG ODN (10 μg/ml) and analysed for AMPK phosphorylation. Mean±SEM from 6 patients. Data information: Paired t-test with Bonferroni method, **p < 0.01, ***p < 0.001.


( F ) CSCs transfected with LAMTOR1 shRNA or the control were treated with CpG ODN ( 10 μg / ml ) and analysed for AMPK phosphorylation . Mean ± SEM from 6 patients . Data information : Paired t - test with Bonferroni method , * * p < 0 . 01 , * * * p < 0 . 001 .

(F) CSCs transfected with LAMTOR1 shRNA or the control were treated with CpG ODN (10 μg/ml) and analysed for AMPK phosphorylation. Mean±SEM from 6 patients. Data information: Paired t-test with Bonferroni method, **p < 0.01, ***p < 0.001.


( G ) CSCs transfected with AXIN1 shRNA or the control were treated with CpG ODN ( 10 μg / ml ) and analysed for AMPK phosphorylation . Mean ± SEM from 6 patients . Data information : Paired t - test with Bonferroni method , * * p < 0 . 01 , * * * p < 0 . 001 .

(G) CSCs transfected with AXIN1 shRNA or the control were treated with CpG ODN (10 μg/ml) and analysed for AMPK phosphorylation. Mean±SEM from 6 patients. Data information: Paired t-test with Bonferroni method, **p < 0.01, ***p < 0.001.


( A ) NSCLC PDX models treated with or without chemotherapy for the first 2 weeks were analyzed for tumor growth at the indicated time . Mean ± SEM from 6 experiments . Data information : Paired t - test with Bonferroni method . * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 .

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(A) NSCLC PDX models treated with or without chemotherapy for the first 2 weeks were analyzed for tumor growth at the indicated time. Mean±SEM from 6 experiments. Data information: Paired t-test with Bonferroni method. **p < 0.01, ***p < 0.001, ****p < 0.0001.


( B ) Frequencies of CD133 - expressing CSCs from NSCLC xenografts treated with or without chemotherapy were determined after 4 weeks . Mean ± SEM from 6 experiments . Data information : Paired t - test with Bonferroni method . * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 .

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(B) Frequencies of CD133-expressing CSCs from NSCLC xenografts treated with or without chemotherapy were determined after 4 weeks. Mean±SEM from 6 experiments. Data information: Paired t-test with Bonferroni method. **p < 0.01, ***p < 0.001, ****p < 0.0001.


( C - E ) Flow cytometric analyses of protein expressions of TLR9 , activated Notch1 , and phosphor - AMPKα in cancer cells from NSCLC xenografts treated with or without chemotherapy after 4 weeks . Mean ± SEM from 6 experiments . Data information : Paired t - test with Bonferroni method . * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 .

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(C-E) Flow cytometric analyses of protein expressions of TLR9, activated Notch1, and phosphor-AMPKα in cancer cells from NSCLC xenografts treated with or without chemotherapy after 4 weeks. Mean±SEM from 6 experiments. Data information: Paired t-test with Bonferroni method. **p < 0.01, ***p < 0.001, ****p < 0.0001.


( F ) NSCLC PDX models were treated with chemotherapy alone or together with the indicated inhibitors , respectively , for the first 2 weeks . Tumor volume was determined at the indicated time . Mean ± SEM from 6 experiments . ( G ) Frequencies of CD133 - expressing CSCs from NSCLC xenografts treated with chemotherapy alone or with the indicated inhibitors after 4 weeks . Mean ± SEM from 6 experiments . Data information : ANOVA test with Turkey ' s method . * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 .

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(F) NSCLC PDX models were treated with chemotherapy alone or together with the indicated inhibitors, respectively, for the first 2 weeks. Tumor volume was determined at the indicated time. Mean±SEM from 6 experiments. (G) Frequencies of CD133-expressing CSCs from NSCLC xenografts treated with chemotherapy alone or with the indicated inhibitors after 4 weeks. Mean±SEM from 6 experiments. Data information: ANOVA test with Turkey's method. **p < 0.01, ***p < 0.001, ****p < 0.0001.


C - F ( C , E ) Immunofluorescence labelling with an antibody that recognizes both full - length and cleaved Xkr8 in developing ( P0 , P8 ) and adult S1 cortex and hippocampus . ( D , F ) Immunofluorescence labelling with an antibody recognizing full - length uncleaved Xkr8 in developing ( P0 , P8 ) and adult S1 cortex and hippocampus . Loss of Xkr8 immunosignal in double transgenic Xkr8flx / flx ; Emx1 : : Cre animals ( Xkr8 cKO ) is observed with both Xkr8 antibodies ( C - F ) .

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C-F (C, E) Immunofluorescence labelling with an antibody that recognizes both full-length and cleaved Xkr8 in developing (P0, P8) and adult S1 cortex and hippocampus. (D, F) Immunofluorescence labelling with an antibody recognizing full-length uncleaved Xkr8 in developing (P0, P8) and adult S1 cortex and hippocampus. Loss of Xkr8 immunosignal in double transgenic Xkr8flx/flx;Emx1::Cre animals (Xkr8 cKO) is observed with both Xkr8 antibodies (C-F).


A , B Organotypic slices of Xkr8 WT and cKO hippocampus with Thy1 : : GFP neurons ( green ) were labelled with PtdSer - binding fluorescently tagged Annexin V ( magenta ) at 16 - 19 DIV . The fluorescence of bound Annexin V was quantified on ImageJ by measuring the integrated density of fluorescent structures within the same volume for each slice and expressing the value as fluorescence units ( f . u . ; two - tailed Student ' s t - test , each dot represents an individual slice preparation , n = 4 ) .

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A, B Organotypic slices of Xkr8 WT and cKO hippocampus with Thy1::GFP neurons (green) were labelled with PtdSer-binding fluorescently tagged Annexin V (magenta) at 16-19 DIV. The fluorescence of bound Annexin V was quantified on ImageJ by measuring the integrated density of fluorescent structures within the same volume for each slice and expressing the value as fluorescence units (f.u.; two-tailed Student's t-test, each dot represents an individual slice preparation, n = 4).


C Immunofluorescence labelling of active caspase - 3 in developing brain from P8 to P28 in Xkr8 WT and Xkr8 cKO brains was quantified and compared by nested design and mixed model ANOVA ( F ( 2 , 30 ) = 0 . 86 , p = 0 . 433 ) , each dot represents an individual mouse ( n = 6 per age group )

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C Immunofluorescence labelling of active caspase-3 in developing brain from P8 to P28 in Xkr8 WT and Xkr8 cKO brains was quantified and compared by nested design and mixed model ANOVA (F(2,30) = 0.86, p = 0.433), each dot represents an individual mouse (n = 6 per age group)


A - C The size and density of axonal boutons on Thy1 : : GFP axons ( green ) in Xkr8 WT and cKO animals ( nested design and mixed model ANOVA , each dot represents an individual mouse , n = 6 per age and genotype group ) .

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A-C The size and density of axonal boutons on Thy1::GFP axons (green) in Xkr8 WT and cKO animals (nested design and mixed model ANOVA, each dot represents an individual mouse, n = 6 per age and genotype group).


D , E Dendritic spine morphology ( D ) and density ( E ) of Thy1 : : GFP + CA1 neurons in Xkr8 WT and cKO animals from P8 to P28 ( nested design and mixed model ANOVA , each dot represents an individual mouse , n = 5 - 6 per age and genotype group ) .

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D, E Dendritic spine morphology (D) and density (E) of Thy1::GFP+ CA1 neurons in Xkr8 WT and cKO animals from P8 to P28 (nested design and mixed model ANOVA, each dot represents an individual mouse, n = 5-6 per age and genotype group).


J - L The density of cortical ( vGluT1 + , J ) and thalamic ( vGluT2 + , K ) and the ratio of vGluT1 + and vGluT2 + inputs ( L ) in Xkr8 WT and cKO brains ( nested design and mixed model ANOVA , each dot represents an individual mouse , n = 6 per age and genotype group ) .

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J-L The density of cortical (vGluT1+, J) and thalamic (vGluT2+, K) and the ratio of vGluT1+ and vGluT2+ inputs (L) in Xkr8 WT and cKO brains (nested design and mixed model ANOVA, each dot represents an individual mouse, n = 6 per age and genotype group).


A - F The immunolabelling of internalized pan - axonal material ( A , B ) , pre - synaptic vGluT1 particles ( C , D ) and post - synaptic PSD95 particles ( E , F ) within Iba1 + microglia in P8 cortex of Xkr8 WT and Xkr8 cKO mice was quantified per individual 3D reconstructed microglial cell . The total volume of internalized particles was normalized to microglial cell volume and compared by nested design and mixed model ANOVA , each dot represents an individual mouse ( n = 3 per genotype group ) . Data presented as mean ± SEM ; * * p < 0 . 01 , * * * * p < 0 . 0001 ; scale bar 10 µm , grid cell 10 µm .

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A-F The immunolabelling of internalized pan-axonal material (A, B), pre-synaptic vGluT1 particles (C, D) and post-synaptic PSD95 particles (E, F) within Iba1+ microglia in P8 cortex of Xkr8 WT and Xkr8 cKO mice was quantified per individual 3D reconstructed microglial cell. The total volume of internalized particles was normalized to microglial cell volume and compared by nested design and mixed model ANOVA, each dot represents an individual mouse (n = 3 per genotype group). Data presented as mean ± SEM; ** p < 0.01, **** p < 0.0001; scale bar 10 µm, grid cell 10 µm.


A , B Corticospinal axons of the axonal bundles of pyramidal tracts in medulla of P8 and P28 Xkr8 WT and cKO mice were visualized by Palmgren staining and quantified per each bundle , delineated by a dashed line ( Mann - Whitney test , n = 6 mice per genotype , the data are presented as median and quartiles ) .

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A, B Corticospinal axons of the axonal bundles of pyramidal tracts in medulla of P8 and P28 Xkr8 WT and cKO mice were visualized by Palmgren staining and quantified per each bundle, delineated by a dashed line (Mann-Whitney test, n = 6 mice per genotype, the data are presented as median and quartiles).


To label callosal projections in vivo , cholera toxin subunit B ( CTB ) was injected stereotactically into corpus callosum of Xkr8 WT and cKO brains at P30 - 32 . Back - labelled neurons were quantified in L4 of primary ( S1 ) and secondary ( S2 ) somatosensory cortex .

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To label callosal projections in vivo, cholera toxin subunit B (CTB) was injected stereotactically into corpus callosum of Xkr8 WT and cKO brains at P30-32. Back-labelled neurons were quantified in L4 of primary (S1) and secondary (S2) somatosensory cortex.


Xkr8 cKO neurons had increased mean sEPSC amplitude Data were analyzed by two - tailed Student ' s t - test ; n = 7 - 14 neurons per genotype .

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Xkr8 cKO neurons had increased mean sEPSC amplitude Data were analyzed by two-tailed Student's t-test ; n = 7-14 neurons per genotype.


F - H Evoked excitatory postsynaptic currents ( eEPSC ) of Xkr8 cKO neurons showed a trend to decrease ( p = 0 . 08 ) ( F , G ) and the ratio of eEPSC and sEPSC amplitudes was lower in Xkr8 knockout neurons ( H ) . two - way repeated measures ANOVA ( H ) ; n = 7 - 14 neurons per genotype .

F-H Evoked excitatory postsynaptic currents (eEPSC) of Xkr8 cKO neurons showed a trend to decrease (p = 0.08) (F, G) and the ratio of eEPSC and sEPSC amplitudes was lower in Xkr8 knockout neurons (H). two-way repeated measures ANOVA (H); n = 7-14 neurons per genotype.


A Brain regions with significantly increased functional connectivity in Xkr8 cKO mice compared to Xkr8 WT mice as identified by seed - based mapping of rsfMRI networks ( t - test , p < 0 . 05 FWE cluster - corrected , with cluster - defining threshold of t24 > 2 . 07 , p < 0 . 05 ; Colored regions represent the areas of the brain exhibiting increased functional connectivity in Xkr8 cKO compared to Xkr8 WT brains ; color scale represents the level of functional correlation ; abbreviations specify anatomical designations of identified regions . ACA : anterior cingulate cortex , AI : agranular insular area , AUD : auditory areas MOp : primary motor area , MOs : secondary motor area , PL : prelimbic area , SSp : primary somatosensory area , STR : striatum , STRd : striatum dorsal region , SUB : subiculum , TH : thalamus , VIS : visual areas , VISC : visceral area .

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A Brain regions with significantly increased functional connectivity in Xkr8 cKO mice compared to Xkr8 WT mice as identified by seed-based mapping of rsfMRI networks (t-test, p < 0.05 FWE cluster-corrected, with cluster-defining threshold of t24 > 2.07, p < 0.05; Colored regions represent the areas of the brain exhibiting increased functional connectivity in Xkr8 cKO compared to Xkr8 WT brains; color scale represents the level of functional correlation; abbreviations specify anatomical designations of identified regions. ACA: anterior cingulate cortex, AI: agranular insular area, AUD: auditory areas MOp: primary motor area, MOs: secondary motor area, PL: prelimbic area, SSp: primary somatosensory area, STR: striatum, STRd: striatum dorsal region, SUB: subiculum, TH: thalamus, VIS: visual areas, VISC: visceral area.


( B ) Quantification by micro - CT of mean tumor volumes in KP mice derived from 50 randomly - selected tumors at 8 , 12 , 16 , and 20 weeks post - infection with control sgTom , sgNf1 . 1 , sgNf1 . 2 , or sgNf1 . 3 ( n = 18 mice , 21 mice , 21 mice , and 20 mice , respectively ) .

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(B) Quantification by micro-CT of mean tumor volumes in KP mice derived from 50 randomly-selected tumors at 8, 12, 16, and 20 weeks post-infection with control sgTom, sgNf1.1, sgNf1.2, or sgNf1.3 (n = 18 mice, 21 mice, 21 mice, and 20 mice, respectively).


( C ) Quantification by micro - CT of total tumor volume per mouse in KP mice after infection with control sgTom , sgNf1 . 1 , sgNf1 . 2 , or sgNf1 . 3 at 8 , 12 , 16 , and 20 weeks post - infection .

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(C) Quantification by micro-CT of total tumor volume per mouse in KP mice after infection with control sgTom, sgNf1.1, sgNf1.2, or sgNf1.3 at 8, 12, 16, and 20 weeks post-infection.


( D ) Depictions and quantitation of total tumor burden ( total tumor area / total lung area ) in KP mice after infection with control sgTom , sgNf1 . 1 , sgNf1 . 2 , or sgNf1 . 3 at 21 weeks after infection . For boxplots , whiskers indicate the minimum and maximum values , the upper and lower perimeters represent the first and third quartiles , the midline represents the median value , and the x symbol represents the mean .

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(D) Depictions and quantitation of total tumor burden (total tumor area/total lung area) in KP mice after infection with control sgTom, sgNf1.1, sgNf1.2, or sgNf1.3 at 21 weeks after infection. For boxplots, whiskers indicate the minimum and maximum values, the upper and lower perimeters represent the first and third quartiles, the midline represents the median value, and the x symbol represents the mean.


( E ) Distribution of histological tumor grades in KP mice after infection with control sgTom , sgNf1 . 1 , sgNf1 . 2 , or sgNf1 . 3 ( n = 50 tumors each ) at 21 weeks after infection . For bar charts , data presented as means with error bars representing standard deviations ( SDs ) .

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(E) Distribution of histological tumor grades in KP mice after infection with control sgTom, sgNf1.1, sgNf1.2, or sgNf1.3 (n = 50 tumors each) at 21 weeks after infection. For bar charts, data presented as means with error bars representing standard deviations (SDs).


( F ) Assessment of the mitotic index of tumor cells by phosphorylated - histone H3 ( pHH3 ) - positive nuclei density in KP mice LUAD tumors at 21 weeks after infection with control sgTom , sgNf1 . 1 , sgNf1 . 2 , or sgNf1 . 3 ( n = 50 tumors each ) . For boxplots , whiskers indicate the minimum and maximum values , the upper and lower perimeters represent the first and third quartiles , the midline represents the median value , and the x symbol represents the mean .

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(F) Assessment of the mitotic index of tumor cells by phosphorylated-histone H3 (pHH3)-positive nuclei density in KP mice LUAD tumors at 21 weeks after infection with control sgTom, sgNf1.1, sgNf1.2, or sgNf1.3 (n = 50 tumors each). For boxplots, whiskers indicate the minimum and maximum values, the upper and lower perimeters represent the first and third quartiles, the midline represents the median value, and the x symbol represents the mean.


( G ) Nf1 mRNA expression in LUAD tumor sections 21 weeks after infection with control sgTom , sgNf1 . 1 , sgNf1 . 2 , or sgNf1 . 3 . For boxplots , whiskers indicate the minimum and maximum values , the upper and lower perimeters represent the first and third quartiles , the midline represents the median value , and the x symbol represents the mean .

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(G) Nf1 mRNA expression in LUAD tumor sections 21 weeks after infection with control sgTom, sgNf1.1, sgNf1.2, or sgNf1.3. For boxplots, whiskers indicate the minimum and maximum values, the upper and lower perimeters represent the first and third quartiles, the midline represents the median value, and the x symbol represents the mean.


( H ) Representative hematoxylin and eosin ( H & E ) and p - Fak1 immunohistochemical ( IHC ) staining of LUAD tumor sections 21 weeks after infection with control sgTom ( grade 1 depicted ) , sgNf1 . 1 ( grade 3 depicted ) , sgNf1 . 2 ( grade 3 depicted ) , or sgNf1 . 3 ( grade 3 depicted ) ( n = 50 tumors each ) . H & E scale bars ( low - magnification top row = 100 μm , high - magnification bottom row = 250 μm ) ; p - Fak1 IHC scale bars ( low - magnification top row = 250 μm , high - magnification bottom row = 500 μm ) .

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(H) Representative hematoxylin and eosin (H&E) and p-Fak1 immunohistochemical (IHC) staining of LUAD tumor sections 21 weeks after infection with control sgTom (grade 1 depicted), sgNf1.1 (grade 3 depicted), sgNf1.2 (grade 3 depicted), or sgNf1.3 (grade 3 depicted) (n = 50 tumors each). H&E scale bars (low-magnification top row = 100 μm, high-magnification bottom row = 250 μm); p-Fak1 IHC scale bars (low-magnification top row = 250 μm, high-magnification bottom row = 500 μm).


( I ) Quantification of p - Fak1 IHC signals in LUAD tumor sections 21 weeks after infection with control sgTom , sgNf1 . 1 , sgNf1 . 2 , or sgNf1 . 3 . For boxplots , whiskers indicate the minimum and maximum values , the upper and lower perimeters represent the first and third quartiles , the midline represents the median value , and the x symbol represents the mean .

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(I) Quantification of p-Fak1 IHC signals in LUAD tumor sections 21 weeks after infection with control sgTom, sgNf1.1, sgNf1.2, or sgNf1.3. For boxplots, whiskers indicate the minimum and maximum values, the upper and lower perimeters represent the first and third quartiles, the midline represents the median value, and the x symbol represents the mean.


( J ) Quantification of p - Fak1 IHC signals in sgNf1 . 3 LUAD tumor sections analyzed by tumor grade . For boxplots , whiskers indicate the minimum and maximum values , the upper and lower perimeters represent the first and third quartiles , the midline represents the median value , and the x symbol represents the mean .

(J) Quantification of p-Fak1 IHC signals in sgNf1.3 LUAD tumor sections analyzed by tumor grade. For boxplots, whiskers indicate the minimum and maximum values, the upper and lower perimeters represent the first and third quartiles, the midline represents the median value, and the x symbol represents the mean.


( K ) Quantification of Psat1 mRNA expression in LUAD tumor sections 21 weeks after infection with control sgTom , sgNf1 . 1 , sgNf1 . 2 , or sgNf1 . 3 . For boxplots , whiskers indicate the minimum and maximum values , the upper and lower perimeters represent the first and third quartiles , the midline represents the median value , and the x symbol represents the mean .

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(K) Quantification of Psat1 mRNA expression in LUAD tumor sections 21 weeks after infection with control sgTom, sgNf1.1, sgNf1.2, or sgNf1.3. For boxplots, whiskers indicate the minimum and maximum values, the upper and lower perimeters represent the first and third quartiles, the midline represents the median value, and the x symbol represents the mean.


( L ) Quantification of Psat1 mRNA expression in sgNf1 . 3 LUAD tumor sections analyzed by tumor grade . For boxplots , whiskers indicate the minimum and maximum values , the upper and lower perimeters represent the first and third quartiles , the midline represents the median value , and the x symbol represents the mean .

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(L) Quantification of Psat1 mRNA expression in sgNf1.3 LUAD tumor sections analyzed by tumor grade. For boxplots, whiskers indicate the minimum and maximum values, the upper and lower perimeters represent the first and third quartiles, the midline represents the median value, and the x symbol represents the mean.


( A ) Western blotting analysis of p53 expression in various p53 / TP53 - WT cell lines incubated with the DNA - intercalating agent doxorubicin ( DOXO , 0 . 2 µg / ml for 6 hours ) to induce p53 stabilization ( n = 3 biological replicates ) .

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(A) Western blotting analysis of p53 expression in various p53/TP53-WT cell lines incubated with the DNA-intercalating agent doxorubicin (DOXO, 0.2 µg/ml for 6 hours) to induce p53 stabilization (n = 3 biological replicates).


( B , C ) Western blotting analysis confirming NF1 upregulation and p - FAK1 downregulation in ( B ) DOXO - treated PDKN1 cells and ( C ) DOXO - treated PDKN2 cells transduced with PGK - NF1 ( n = 3 biological replicates ) .

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(B, C) Western blotting analysis confirming NF1 upregulation and p-FAK1 downregulation in (B) DOXO-treated PDKN1 cells and (C) DOXO-treated PDKN2 cells transduced with PGK-NF1 (n = 3 biological replicates).


( D ) Western blotting analysis confirming NF1 downregulation and p - FAK1 upregulation in DOXO - treated SW1573 cells transduced with sgNf1 . 3 ( n = 3 biological replicates ) .

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(D) Western blotting analysis confirming NF1 downregulation and p-FAK1 upregulation in DOXO-treated SW1573 cells transduced with sgNf1.3 (n = 3 biological replicates).


( E , F ) Western blotting analysis confirming Nf1 downregulation and p - Fak1 upregulation in ( E ) DOXO - treated LKR10 and ( F ) DOXO - treated LKR13 clones transduced with sgNf1 . 3 ( n = 3 biological replicates ) .

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(E, F) Western blotting analysis confirming Nf1 downregulation and p-Fak1 upregulation in (E) DOXO-treated LKR10 and (F) DOXO-treated LKR13 clones transduced with sgNf1.3 (n = 3 biological replicates).


( G ) FAK1 target gene expression in DOXO - treated PDKN1 cells transduced with PGK - control or PGK - NF1 ( n = 3 biological replicates ) . For bar charts , data presented as means with error bars representing standard deviations ( SDs ) .

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(G) FAK1 target gene expression in DOXO-treated PDKN1 cells transduced with PGK-control or PGK-NF1 (n = 3 biological replicates). For bar charts, data presented as means with error bars representing standard deviations (SDs).


( H , I ) Subcutaneous tumor volumes of DOXO - treated PDKN1 and PDKN2 cells transduced with PGK - control or PGK - NF1 ( n = 18 each ) .

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(H, I) Subcutaneous tumor volumes of DOXO-treated PDKN1 and PDKN2 cells transduced with PGK-control or PGK-NF1 (n=18 each).


( J - L ) Subcutaneous tumor volumes of ( J ) SW1573 , ( K ) LKR10 , and ( L ) LKR13 cells transfected with sgTom or sgNf1 . 3 ( n = 18 each ) .

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(J-L) Subcutaneous tumor volumes of (J) SW1573, (K) LKR10, and (L) LKR13 cells transfected with sgTom or sgNf1.3 (n=18 each).


( M ) Depictions and quantitation of KrasG12D / + ; p53 + / + ( K - only ) autochthonous tumor burden ( total tumor area / total lung area ) in pSECC - sgTom ( n = 18 mice ) or pSECC - sgNf1 . 3 mice ( n = 21 mice ) . For boxplots , whiskers indicate the minimum and maximum values , the upper and lower perimeters represent the first and third quartiles , the midline represents the median value , and the x symbol represents the mean .

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(M) Depictions and quantitation of KrasG12D/+; p53+/+ (K-only) autochthonous tumor burden (total tumor area/total lung area) in pSECC-sgTom (n = 18 mice) or pSECC-sgNf1.3 mice (n = 21 mice). For boxplots, whiskers indicate the minimum and maximum values, the upper and lower perimeters represent the first and third quartiles, the midline represents the median value, and the x symbol represents the mean.


( N ) Analysis of tumor grades in K - only autochthonous tumors derived from pSECC - sgTom ( n = 18 mice ) or pSECC - sgNf1 . 3 mice ( n = 21 mice ) . For bar charts , data presented as means with error bars representing standard deviations ( SDs ) .

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(N) Analysis of tumor grades in K-only autochthonous tumors derived from pSECC-sgTom (n = 18 mice) or pSECC-sgNf1.3 mice (n = 21 mice). For bar charts, data presented as means with error bars representing standard deviations (SDs).


( O ) Quantification of Ki - 67 - positive nuclei per mm2 in K - only autochthonous tumors derived from pSECC - sgTom ( n = 18 mice ) or pSECC - sgNf1 . 3 mice ( n = 21 mice ) . For boxplots , whiskers indicate the minimum and maximum values , the upper and lower perimeters represent the first and third quartiles , the midline represents the median value , and the x symbol represents the mean .

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(O) Quantification of Ki-67-positive nuclei per mm2 in K-only autochthonous tumors derived from pSECC-sgTom (n = 18 mice) or pSECC-sgNf1.3 mice (n = 21 mice). For boxplots, whiskers indicate the minimum and maximum values, the upper and lower perimeters represent the first and third quartiles, the midline represents the median value, and the x symbol represents the mean.


( A ) Glucose consumption and lactate excretion in KP and KPΔNF1 clones normalized by cell count ( n = 4 biological replicates ) . For bar charts , data presented as means with error bars representing standard deviations ( SDs ) .

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(A) Glucose consumption and lactate excretion in KP and KPΔNF1 clones normalized by cell count (n = 4 biological replicates). For bar charts, data presented as means with error bars representing standard deviations (SDs).


( B ) Glutamine consumption in KP and KPΔNF1 clones normalized by cell count ( n = 4 biological replicates ) . For bar charts , data presented as means with error bars representing standard deviations ( SDs ) .

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(B) Glutamine consumption in KP and KPΔNF1 clones normalized by cell count (n = 4 biological replicates). For bar charts, data presented as means with error bars representing standard deviations (SDs).


( C ) Relative viability of KP and KPΔNF1 cells after 72 h of GPNA treatment assayed by cell - titer glo ( relative luminescent units ) ( n = 4 biological replicates ) .

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(C) Relative viability of KP and KPΔNF1 cells after 72 h of GPNA treatment assayed by cell-titer glo (relative luminescent units) (n = 4 biological replicates).


( D ) Cumulative population doublings of KP and KPΔNF1 cells cultured with 2 . 0 or 0 . 5 mM glutamine ( n = 4 biological replicates ) .

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(D) Cumulative population doublings of KP and KPΔNF1 cells cultured with 2.0 or 0.5 mM glutamine (n = 4 biological replicates).


( E ) Two patient - derived KRAS ; NF1 - mutant LUAD cell lines ( PDKN1 , PDKN2 ) , as well as the control patient - derived KRAS - mutant / NF1 - WT LUAD cell line ( PDK ) , were passaged for 14 population doublings prior to collection . The heatmap displays the most significantly upregulated genes in the PDKN1 and PDKN2 cell lines ( relative to the PDK control ) , with the degree of upregulation depicted by color as indicated in the legend .

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(E) Two patient-derived KRAS;NF1-mutant LUAD cell lines (PDKN1, PDKN2), as well as the control patient-derived KRAS-mutant/NF1-WT LUAD cell line (PDK), were passaged for 14 population doublings prior to collection. The heatmap displays the most significantly upregulated genes in the PDKN1 and PDKN2 cell lines (relative to the PDK control), with the degree of upregulation depicted by color as indicated in the legend.


Quantification of the expression of the six key altered genes in cultured PDKN1 cells ( passaged for 14 population doublings ) vs . their respective original patient tumor samples using qPCR . For bar charts , data presented as means with error bars representing standard deviations ( SDs ) .

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Quantification of the expression of the six key altered genes in cultured PDKN1 cells (passaged for 14 population doublings) vs. their respective original patient tumor samples using qPCR. For bar charts, data presented as means with error bars representing standard deviations (SDs).


Quantification of the expression of the six key altered genes in cultured PDKN2 cells ( passaged for 14 population doublings ) vs . their respective original patient tumor samples using qPCR . For bar charts , data presented as means with error bars representing standard deviations ( SDs ) .

Quantification of the expression of the six key altered genes in cultured PDKN2 cells (passaged for 14 population doublings) vs. their respective original patient tumor samples using qPCR. For bar charts, data presented as means with error bars representing standard deviations (SDs).


( H ) Western blotting analysis of Psat1 expression in KP and KPΔNF1 cells infected with sgTom or sgPsat1 following selection . GAPDH was used as a loading control .

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(H) Western blotting analysis of Psat1 expression in KP and KPΔNF1 cells infected with sgTom or sgPsat1 following selection. GAPDH was used as a loading control.


( I ) Cumulative population doublings of KP and KPΔNF1 cells after transduction with sgTom - or sgPsat1 - containing vectors ( n = 4 biological replicates ) .

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(I) Cumulative population doublings of KP and KPΔNF1 cells after transduction with sgTom- or sgPsat1-containing vectors (n = 4 biological replicates).


( J - L ) Cumulative population doublings of patient - derived KRAS - mutant LUAD cell lines that are either ( J , K ) NF1 - mutant ( PDKN1 and PDKN2 ) or ( L ) NF1 - WT ( PDK ) after selection with sgTom - or sgPsat1 - containing vectors ( n = 4 biological replicates ) .

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(J-L) Cumulative population doublings of patient-derived KRAS-mutant LUAD cell lines that are either (J, K) NF1-mutant (PDKN1 and PDKN2) or (L) NF1-WT (PDK) after selection with sgTom- or sgPsat1-containing vectors (n = 4 biological replicates).


Relative viability of KP and KPΔNF1 clones after CB - 839 treatment ( IC50 : 18 nM ) for 72 h assayed via CellTiter - Glo ( relative luminescence units ) . All data points are relative to vehicle - treated controls ( n = 4 biological replicates ) .

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Relative viability of KP and KPΔNF1 clones after CB-839 treatment (IC50: 18 nM) for 72 h assayed via CellTiter-Glo (relative luminescence units). All data points are relative to vehicle-treated controls (n = 4 biological replicates).


Relative viability of KP and KPΔNF1 clones after AOA treatment ( IC50 : 6 µM ) for 72 h assayed via CellTiter - Glo ( relative luminescence units ) . All data points are relative to vehicle - treated controls ( n = 4 biological replicates ) .

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Relative viability of KP and KPΔNF1 clones after AOA treatment (IC50: 6 µM) for 72 h assayed via CellTiter-Glo (relative luminescence units). All data points are relative to vehicle-treated controls (n = 4 biological replicates).


( D ) Cumulative population doublings of KP and KPΔNF1 cells incubated with vehicle , 18 nM CB - 839 , or 6 µM AOA after 6 d in culture ( n = 4 biological replicates ) . For bar charts , data presented as means with error bars representing standard deviations ( SDs ) .

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(D) Cumulative population doublings of KP and KPΔNF1 cells incubated with vehicle, 18 nM CB-839, or 6 µM AOA after 6 d in culture (n = 4 biological replicates). For bar charts, data presented as means with error bars representing standard deviations (SDs).


( E ) Relative viability of the indicated human KRAS - mutant lung cancer cell lines by trypan blue exclusion assay . Cells were incubated with vehicle , 18 nM CB - 839 , or 6 µM AOA for 72 h ( n = 4 biological replicates ) . All data points are normalized to vehicle - treated cell lines . For bar charts , data presented as means with error bars representing standard deviations ( SDs ) .

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(E) Relative viability of the indicated human KRAS-mutant lung cancer cell lines by trypan blue exclusion assay. Cells were incubated with vehicle, 18 nM CB-839, or 6 µM AOA for 72 h (n = 4 biological replicates). All data points are normalized to vehicle-treated cell lines. For bar charts, data presented as means with error bars representing standard deviations (SDs).


Quantification by micro - CT of mean tumor volumes in ( B ) CB - 839 - , or vehicle - treated KP mice derived from 50 randomly - selected tumors at 8 , 12 , 16 , and 20 weeks post - infection with control sgTom or sgNf1 . 3 ( n = 21 mice and 21 mice , respectively ) . Vehicle - treated KP mice are the same control group for ( B )

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Quantification by micro-CT of mean tumor volumes in (B) CB-839-, or vehicle-treated KP mice derived from 50 randomly-selected tumors at 8, 12, 16, and 20 weeks post-infection with control sgTom or sgNf1.3 (n = 21 mice and 21 mice, respectively). Vehicle-treated KP mice are the same control group for (B)


Quantification by micro - CT of mean tumor volumes in ( C ) AOA - , or vehicle - treated KP mice derived from 50 randomly - selected tumors at 8 , 12 , 16 , and 20 weeks post - infection with control sgTom or sgNf1 . 3 ( n = 21 mice and 21 mice , respectively ) . Vehicle - treated KP mice are the same control group for ( C ) .

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Quantification by micro-CT of mean tumor volumes in (C) AOA-, or vehicle-treated KP mice derived from 50 randomly-selected tumors at 8, 12, 16, and 20 weeks post-infection with control sgTom or sgNf1.3 (n = 21 mice and 21 mice, respectively). Vehicle-treated KP mice are the same control group for (C).


Quantification by micro - CT of total tumor volume per mouse in ( D ) CB - 839 - , or vehicle - treated KP mice after infection with control sgTom or sgNf1 . 3 at 8 , 12 , 16 , and 20 weeks post - infection . Vehicle - treated KP mice are the same control group for ( D )

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Quantification by micro-CT of total tumor volume per mouse in (D) CB-839-, or vehicle-treated KP mice after infection with control sgTom or sgNf1.3 at 8, 12, 16, and 20 weeks post-infection. Vehicle-treated KP mice are the same control group for (D)


E ) Quantification by micro - CT of total tumor volume per mouse in , ( E ) AOA - , or vehicle - treated KP mice after infection with control sgTom or sgNf1 . 3 at 8 , 12 , 16 , and 20 weeks post - infection . Vehicle - treated KP mice are the same control group for ( E ) .

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E) Quantification by micro-CT of total tumor volume per mouse in , (E) AOA-, or vehicle-treated KP mice after infection with control sgTom or sgNf1.3 at 8, 12, 16, and 20 weeks post-infection. Vehicle-treated KP mice are the same control group for (E).


Relative responses of KP mice tumors treated with ( F ) CB - 839 , or vehicle starting from week 8 post - infection and measured until week 20 . Relative response = average tumor volume with treatment / average tumor volume with vehicle .

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Relative responses of KP mice tumors treated with (F) CB-839, or vehicle starting from week 8 post-infection and measured until week 20. Relative response = average tumor volume with treatment / average tumor volume with vehicle.


Relative responses of KP mice tumors treated with ( G ) AOA , or vehicle starting from week 8 post - infection and measured until week 20 . Relative response = average tumor volume with treatment / average tumor volume with vehicle .

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Relative responses of KP mice tumors treated with (G) AOA, or vehicle starting from week 8 post-infection and measured until week 20. Relative response = average tumor volume with treatment / average tumor volume with vehicle.


( H ) Depictions and quantitation of total tumor burden ( total tumor area / total lung area ) in CB - 839 - , AOA - , or vehicle - treated KP mice after infection with control sgTom or sgNf1 . 3 at 20 weeks post - infection . Vehicle - treated KP mice are the same control group in both boxplots . For boxplots , whiskers indicate the minimum and maximum values , the upper and lower perimeters represent the first and third quartiles , the midline represents the median value , and the x symbol represents the mean .

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(H) Depictions and quantitation of total tumor burden (total tumor area/total lung area) in CB-839-, AOA-, or vehicle-treated KP mice after infection with control sgTom or sgNf1.3 at 20 weeks post-infection. Vehicle-treated KP mice are the same control group in both boxplots. For boxplots, whiskers indicate the minimum and maximum values, the upper and lower perimeters represent the first and third quartiles, the midline represents the median value, and the x symbol represents the mean.


Relative responses of subcutaneous KP and KPΔNF1 tumors treated with vehicle , ( B ) CB - 839 , starting from day 12 and measured until day 27 ( n = 6 tumors per cell line per treatment ) Relative response = average tumor volume with treatment / average tumor volume with vehicle .

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Relative responses of subcutaneous KP and KPΔNF1 tumors treated with vehicle, (B) CB-839, starting from day 12 and measured until day 27 (n = 6 tumors per cell line per treatment) Relative response = average tumor volume with treatment / average tumor volume with vehicle.


) Relative responses of subcutaneous KP and KPΔNF1 tumors treated with vehicle , C ) AOA starting from day 12 and measured until day 27 ( n = 6 tumors per cell line per treatment ) Relative response = average tumor volume with treatment / average tumor volume with vehicle .

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) Relative responses of subcutaneous KP and KPΔNF1 tumors treated with vehicle, C) AOA starting from day 12 and measured until day 27 (n = 6 tumors per cell line per treatment) Relative response = average tumor volume with treatment / average tumor volume with vehicle.


Relative responses of subcutaneous KP and KPΔNF1 tumors treated with vehicle , CB - 839 , or AOA ( D ) the final masses of these tumors . or boxplots , whiskers indicate the minimum and maximum values , the upper and lower perimeters represent the first and third quartiles , the midline represents the median value , and the x symbol represents the mean .

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Relative responses of subcutaneous KP and KPΔNF1 tumors treated with vehicle, CB-839, or AOA (D) the final masses of these tumors. or boxplots, whiskers indicate the minimum and maximum values, the upper and lower perimeters represent the first and third quartiles, the midline represents the median value, and the x symbol represents the mean.


Relative responses of orthotopic KP and KPΔNF1 tumors treated with vehicle , ( E ) CB - 839 starting from day 12 and measured until day 27 ( n = 24 mice per cell line per treatment ) . Relative response = average tumor volume with treatment / average tumor volume with vehicle . Quantification of tumor growth by photon flux luminescence after orthotopic transplantation with KP or KPΔNF1 cells transduced with a luciferase vector .

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Relative responses of orthotopic KP and KPΔNF1 tumors treated with vehicle, (E) CB-839 starting from day 12 and measured until day 27 (n = 24 mice per cell line per treatment). Relative response = average tumor volume with treatment / average tumor volume with vehicle. Quantification of tumor growth by photon flux luminescence after orthotopic transplantation with KP or KPΔNF1 cells transduced with a luciferase vector.


Relative responses of orthotopic KP and KPΔNF1 tumors treated with vehicle , ( F ) AOA starting from day 12 and measured until day 27 ( n = 24 mice per cell line per treatment ) . Relative response = average tumor volume with treatment / average tumor volume with vehicle . Quantification of tumor growth by photon flux luminescence after orthotopic transplantation with KP or KPΔNF1 cells transduced with a luciferase vector .

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Relative responses of orthotopic KP and KPΔNF1 tumors treated with vehicle, (F) AOA starting from day 12 and measured until day 27 (n = 24 mice per cell line per treatment). Relative response = average tumor volume with treatment / average tumor volume with vehicle. Quantification of tumor growth by photon flux luminescence after orthotopic transplantation with KP or KPΔNF1 cells transduced with a luciferase vector.


Relative responses of subcutaneous KP - ix tumors harboring doxycycline ( DOX ) - inducible gain - of - function ( GOF ) - Fak1 cDNA treated with vehicle , ( G ) CB - 839 , with or without DOX / PIP2 ( n = 36 mice per treatment group ) . Relative response = average tumor volume with treatment / average tumor volume with vehicle .

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Relative responses of subcutaneous KP-ix tumors harboring doxycycline (DOX)-inducible gain-of-function (GOF)-Fak1 cDNA treated with vehicle, (G) CB-839, with or without DOX/PIP2 (n = 36 mice per treatment group). Relative response = average tumor volume with treatment / average tumor volume with vehicle.


Relative responses of subcutaneous KP - ix tumors harboring doxycycline ( DOX ) - inducible gain - of - function ( GOF ) - Fak1 cDNA treated with vehicle ( H ) AOA with or without DOX / PIP2 ( n = 36 mice per treatment group ) . Relative response = average tumor volume with treatment / average tumor volume with vehicle .

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Relative responses of subcutaneous KP-ix tumors harboring doxycycline (DOX)-inducible gain-of-function (GOF)-Fak1 cDNA treated with vehicle (H) AOA with or without DOX/PIP2 (n = 36 mice per treatment group). Relative response = average tumor volume with treatment / average tumor volume with vehicle.


Relative responses of subcutaneous patient - derived NF1 - mutant ( PDKN1 and PDKN2 ) and NF1 - WT ( PDK ) LUAD tumors treated with vehicle , ( I ) CB - 839 starting from day 12 and measured until day 30 ( n = 6 tumors per cell line per treatment ) . Relative response = average tumor volume with treatment / average tumor volume with vehicle .

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Relative responses of subcutaneous patient-derived NF1-mutant (PDKN1 and PDKN2) and NF1-WT (PDK) LUAD tumors treated with vehicle, (I) CB-839 starting from day 12 and measured until day 30 (n = 6 tumors per cell line per treatment). Relative response = average tumor volume with treatment / average tumor volume with vehicle.


Relative responses of subcutaneous patient - derived NF1 - mutant ( PDKN1 and PDKN2 ) and NF1 - WT ( PDK ) LUAD tumors treated with vehicle ( J ) AOA starting from day 12 and measured until day 30 ( n = 6 tumors per cell line per treatment ) . Relative response = average tumor volume with treatment / average tumor volume with vehicle .

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Relative responses of subcutaneous patient-derived NF1-mutant (PDKN1 and PDKN2) and NF1-WT (PDK) LUAD tumors treated with vehicle (J) AOA starting from day 12 and measured until day 30 (n = 6 tumors per cell line per treatment). Relative response = average tumor volume with treatment / average tumor volume with vehicle.


Relative responses of orthotopic patient - derived NF1 - mutant ( PDKN1 and PDKN2 ) and NF1 - WT ( PDK ) LUAD tumors treated with vehicle , ( K ) CB - 839 starting from day 12 and measured until day 27 ( n = 12 mice per cell line per treatment ) . Relative response = average tumor volume with treatment / average tumor volume with vehicle . Quantification of tumor growth by photon flux luminescence after orthotopic transplantation with KP or KPΔNF1 cells transduced with a luciferase vector .

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

Relative responses of orthotopic patient-derived NF1-mutant (PDKN1 and PDKN2) and NF1-WT (PDK) LUAD tumors treated with vehicle, (K) CB-839 starting from day 12 and measured until day 27 (n = 12 mice per cell line per treatment). Relative response = average tumor volume with treatment / average tumor volume with vehicle. Quantification of tumor growth by photon flux luminescence after orthotopic transplantation with KP or KPΔNF1 cells transduced with a luciferase vector.


Relative responses of orthotopic patient - derived NF1 - mutant ( PDKN1 and PDKN2 ) and NF1 - WT ( PDK ) LUAD tumors treated with vehicle , ( L ) AOA starting from day 12 and measured until day 27 ( n = 12 mice per cell line per treatment ) . Relative response = average tumor volume with treatment / average tumor volume with vehicle . Quantification of tumor growth by photon flux luminescence after orthotopic transplantation with KP or KPΔNF1 cells transduced with a luciferase vector .

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Relative responses of orthotopic patient-derived NF1-mutant (PDKN1 and PDKN2) and NF1-WT (PDK) LUAD tumors treated with vehicle, (L) AOA starting from day 12 and measured until day 27 (n = 12 mice per cell line per treatment). Relative response = average tumor volume with treatment / average tumor volume with vehicle. Quantification of tumor growth by photon flux luminescence after orthotopic transplantation with KP or KPΔNF1 cells transduced with a luciferase vector.


A Indicated tail - anchored ( TA ) proteins were in vitro translated in rabbit reticulocyte lysate ( RRL ) using RNAs containing a stop codon . Translations were carried out in the presence of 2 μM HisTrx or HisTrx - SGTA ( HT - SGTA ) , followed by incubation with HisPur Cobalt resin Bound proteins were eluted at high imidazole concentration , samples resolved by SDS - PAGE and results visualised by phosphorimaging . Sec61β 3R carries three Arg residues within Sec61β transmembrane domain ( TMD ) which abolish its hydrophobic character and was used as a negative control for SGTA binding . A schematic representation of a TA - protein is also shown . Open circles indicate TA - proteins selectively bound by HisTrx - SGTA . RAMP4 - stress - associated endoplasmic reticulum protein 1 ; Syb2 - synaptobrevin 2 ; Synt5 - syntaxin 5 . B As for ( A ) but total translation products ( input ) were resolved by SDS - PAGE and results visualised by phosphorimaging . When total products are analysed high amounts of haemoglobin in the RRL distort the migration of Sec61β and its variant .

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A Indicated tail-anchored (TA) proteins were in vitro translated in rabbit reticulocyte lysate (RRL) using RNAs containing a stop codon. Translations were carried out in the presence of 2 μM HisTrx or HisTrx-SGTA (HT-SGTA), followed by incubation with HisPur Cobalt resin Bound proteins were eluted at high imidazole concentration, samples resolved by SDS-PAGE and results visualised by phosphorimaging. Sec61β 3R carries three Arg residues within Sec61β transmembrane domain (TMD) which abolish its hydrophobic character and was used as a negative control for SGTA binding. A schematic representation of a TA-protein is also shown. Open circles indicate TA-proteins selectively bound by HisTrx-SGTA. RAMP4 - stress-associated endoplasmic reticulum protein 1; Syb2 - synaptobrevin 2; Synt5 - syntaxin 5. B As for (A) but total translation products (input) were resolved by SDS-PAGE and results visualised by phosphorimaging. When total products are analysed high amounts of haemoglobin in the RRL distort the migration of Sec61β and its variant.


RNAs coding for C99 variants and containing a stop codon introduced either after the complete coding sequence or before the predicted TMD were used . E As for ( D ) but total translation products were subjected to immunoprecipation with either anti - C99 antibody ( for the full - length variants ) or with anti - β - amyloid antibody ( for products lacking the TMD ) . A filled square indicates inefficiently translated APP - C99 - TMD immunoprecipitated with anti - β - amyloid antibody .

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RNAs coding for C99 variants and containing a stop codon introduced either after the complete coding sequence or before the predicted TMD were used. E As for (D) but total translation products were subjected to immunoprecipation with either anti-C99 antibody (for the full-length variants) or with anti-β-amyloid antibody (for products lacking the TMD). A filled square indicates inefficiently translated APP-C99-TMD immunoprecipitated with anti-β-amyloid antibody.


A Full - length polypeptides were translated in the presence of 2 μM HisTrx or HisTrx - SGTA using RNAs lacking a stop codon and either stabilised at the ribosome with cycloheximide ( CHX ) or released with puromycin ( puro ) . Reactions were incubated with HisPur Cobalt resin , bound proteins eluted at high imidazole concentration , resolved by SDS - PAGE and visualised by phosphorimaging Filled circles indicate slower migrating species of radiolabelled products selectively enriched in CHX - treated samples .

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A Full-length polypeptides were translated in the presence of 2 μM HisTrx or HisTrx-SGTA using RNAs lacking a stop codon and either stabilised at the ribosome with cycloheximide (CHX) or released with puromycin (puro). Reactions were incubated with HisPur Cobalt resin, bound proteins eluted at high imidazole concentration, resolved by SDS-PAGE and visualised by phosphorimaging Filled circles indicate slower migrating species of radiolabelled products selectively enriched in CHX-treated samples.


total translation reactions were treated with RNaseA and immunoprecipitated with anti - C99

total translation reactions were treated with RNaseA and immunoprecipitated with anti-C99 antibody