C Chromatin immunoprecipitation ( ChIP ) assays to detect NIPPED - A binding at E - box , transcription start site ( TSS ) and gene body of tim , Pdp1ε and per using w1118 flies ( n = 3 ) . Data information : Error bars represent SEM . Two - way ANOVA , significant effect between NIPPED - A and IgG were found for tim E - box ( P < 0 . 001 ) , Pdp1ε E - box ( P < 0 . 001 ) , per E - box ( P < 0 . 001 ) , tim TSS ( P < 0 . 001 ) , Pdp1ε TSS ( P < 0 . 001 ) , per TSS ( P < 0 . 001 ) , tim gene body ( P < 0 . 001 ) , Pdp1ε gene body and per gene body ( P < 0 . 001 ) . Student ' s t - test , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 . G4 , GAL4 ; U , UAS .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Chromatin immunoprecipitation (ChIP) assays to detect NIPPED-A binding at E-box, transcription start site (TSS) and gene body of tim, Pdp1ε and per using w1118 flies (n=3). Data information: Error bars represent SEM. Two-way ANOVA, significant effect between NIPPED-A and IgG were found for tim E-box (P<0.001), Pdp1ε E-box (P<0.001), per E-box (P<0.001), tim TSS (P<0.001), Pdp1ε TSS (P<0.001), per TSS (P<0.001), tim gene body (P<0.001), Pdp1ε gene body and per gene body (P<0.001). Student's t-test, *P < 0.05, **P < 0.01, ***P < 0.001. G4, GAL4; U, UAS.


A Plots of relative pre - mRNA abundance of tim ( n = 5 ) and Pdp1ε ( n = 3 ) determined by qRT - PCR in the whole head extracts of Nipped - A RNAi ( timG4 / + ; Udcr2 / UNipped - ARNAi - 1 ) and control ( timG4 / + ; Udcr2 / + ) flies during DD1 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Plots of relative pre-mRNA abundance of tim (n=5) and Pdp1ε (n=3) determined by qRT-PCR in the whole head extracts of Nipped-A RNAi (timG4/+;Udcr2/UNipped-ARNAi-1) and control (timG4/+;Udcr2/+) flies during DD1.


B ChIP assays to detect ub - H2B binding at E - box , TSS and gene body of tim , Pdp1ε , per and clk in Nipped - A RNAi ( timG4 / + ; Udcr2 / UNipped - ARNAi - 1 ) and control ( timG4 / + ; Udcr2 / + ) ( n ≥ 3 ) . Data information : Student ' s t - test , * P < 0 . 05 . Two - way ANOVA , significant effect between ub - H2B and IgG were found for all genomic regions tested ( P < 0 . 001 ) . Significant effect of genotypes were found for tim E - box ( P < 0 . 05 ) , Pdp1ε E - box ( P < 0 . 01 ) , tim TSS ( P < 0 . 01 ) and Pdp1ε TSS ( P < 0 . 001 ) .

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 O O O O 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 O O O O O O O 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 ChIP assays to detect ub-H2B binding at E-box, TSS and gene body of tim, Pdp1ε, per and clk in Nipped-A RNAi (timG4/+;Udcr2/UNipped-ARNAi-1) and control (timG4/+;Udcr2/+) (n≥3). Data information: Student's t-test, *P < 0.05. Two-way ANOVA, significant effect between ub-H2B and IgG were found for all genomic regions tested (P<0.001). Significant effect of genotypes were found for tim E-box (P<0.05), Pdp1ε E-box (P<0.01), tim TSS (P<0.01) and Pdp1ε TSS (P<0.001).


A , B The period ( A ) and power ( B ) of DD locomotor rhythms of flies with not knocked down and controls . Digits on the bar are the number of flies tested . Percentage of rhythmicity is indicated above the bars . Statistical difference is measured using one - way ANOVA , P < 0 . 001 , Tukey ' s multiple comparison test , * * P < 0 . 01 , * * * / # # # / + + + / $ $ $ P < 0 . 001 , * compared with the G4 control , # compared with the UAS control , + compared with the Nipped - A RNAi flies , $ compared with the not overexpression flies . White bar indicates UAS or GAL4 controls . Dashed bar indicates flies with genetically manipulated not . Gray bar indicates flies with Nipped - A knocked down . Checked bar indicates flies with both genetically manipulated not and Nipped - A knocked down .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 The period (A) and power (B) of DD locomotor rhythms of flies with not knocked down and controls. Digits on the bar are the number of flies tested. Percentage of rhythmicity is indicated above the bars. Statistical difference is measured using one-way ANOVA, P<0.001, Tukey's multiple comparison test, **P<0.01, ***/###/+++/$$$P<0.001, * compared with the G4 control, # compared with the UAS control, + compared with the Nipped-A RNAi flies, $ compared with the not overexpression flies. White bar indicates UAS or GAL4 controls. Dashed bar indicates flies with genetically manipulated not. Gray bar indicates flies with Nipped-A knocked down. Checked bar indicates flies with both genetically manipulated not and Nipped-A knocked down.


C Plots of relative mRNA abundance vs CT for clock genes determined by qRT - PCR in whole head extracts of not RNAi ( timG4 / + ; Udcr2 / UnotRNAi ) and control ( timG4 / + ; Udcr2 / + ) collected on DD1 ( n = 5 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Plots of relative mRNA abundance vs CT for clock genes determined by qRT-PCR in whole head extracts of not RNAi (timG4/+;Udcr2/UnotRNAi) and control (timG4/+;Udcr2/+) collected on DD1 (n=5).


D ChIP assays to detect ub - H2B binding at E - box , TSS and gene body of tim and Pdp1ε in not RNAi ( timG4 / + ; Udcr2 / UnotRNAi ) and control ( timG4 / + ; Udcr2 / + ) ( n = 3 ) . Student ' s t - test , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 . Statistical difference is measured using two - way ANOVA , P < 0 . 001 , significant effect of genotypes were found for tim E - box ( P < 0 . 01 ) , Pdp1ε E - box ( P < 0 . 05 ) , tim TSS ( P < 0 . 05 ) and Pdp1ε TSS ( P < 0 . 001 ) and tim gene body ( P < 0 . 01 ) . G4 , GAL4 ; U , UAS .

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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 ChIP assays to detect ub-H2B binding at E-box, TSS and gene body of tim and Pdp1ε in not RNAi (timG4/+;Udcr2/UnotRNAi) and control (timG4/+;Udcr2/+) (n=3). Student's t-test, *P < 0.05, **P < 0.01, ***P < 0.001. Statistical difference is measured using two-way ANOVA, P<0.001, significant effect of genotypes were found for tim E-box (P<0.01), Pdp1ε E-box (P<0.05), tim TSS (P<0.05) and Pdp1ε TSS (P<0.001) and tim gene body (P<0.01). G4, GAL4; U, UAS.


E , F The period of DD locomotor rhythm of flies when knocking down Nipped - A and over - expressing not . Digits on the bar are the number of flies tested . Percentage of rhythmicity is indicated above the bars . Statistical difference is measured using one - way ANOVA , P < 0 . 001 , Tukey ' s multiple comparison test , * * P < 0 . 01 , * * * / # # # / + + + / $ $ $ P < 0 . 001 , * compared with the G4 control , # compared with the UAS control , + compared with the Nipped - A RNAi flies , $ compared with the not overexpression flies . White bar indicates UAS or GAL4 controls . Dashed bar indicates flies with genetically manipulated not . Gray bar indicates flies with Nipped - A knocked down . Checked bar indicates flies with both genetically manipulated not and Nipped - A knocked down .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 The period of DD locomotor rhythm of flies when knocking down Nipped-A and over-expressing not. Digits on the bar are the number of flies tested. Percentage of rhythmicity is indicated above the bars. Statistical difference is measured using one-way ANOVA, P<0.001, Tukey's multiple comparison test, **P<0.01, ***/###/+++/$$$P<0.001, * compared with the G4 control, # compared with the UAS control, + compared with the Nipped-A RNAi flies, $ compared with the not overexpression flies. White bar indicates UAS or GAL4 controls. Dashed bar indicates flies with genetically manipulated not. Gray bar indicates flies with Nipped-A knocked down. Checked bar indicates flies with both genetically manipulated not and Nipped-A knocked down.


G The period of DD locomotor rhythm of flies co - expressing Nipped - A RNAi and not RNAi . Digits on the bar are the number of flies tested . Percentage of rhythmicity is indicated above the bars . Statistical difference is measured using one - way ANOVA , P < 0 . 001 , Tukey ' s multiple comparison test , * * P < 0 . 01 , * * * / # # # / + + + / $ $ $ P < 0 . 001 , * compared with the G4 control , # compared with the UAS control , + compared with the Nipped - A RNAi flies , $ compared with the not overexpression flies . White bar indicates UAS or GAL4 controls . Dashed bar indicates flies with genetically manipulated not . Gray bar indicates flies with Nipped - A knocked down . Checked bar indicates flies with both genetically manipulated not and Nipped - A knocked down .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 The period of DD locomotor rhythm of flies co-expressing Nipped-A RNAi and not RNAi. Digits on the bar are the number of flies tested. Percentage of rhythmicity is indicated above the bars. Statistical difference is measured using one-way ANOVA, P<0.001, Tukey's multiple comparison test, **P<0.01, ***/###/+++/$$$P<0.001, * compared with the G4 control, # compared with the UAS control, + compared with the Nipped-A RNAi flies, $ compared with the not overexpression flies. White bar indicates UAS or GAL4 controls. Dashed bar indicates flies with genetically manipulated not. Gray bar indicates flies with Nipped-A knocked down. Checked bar indicates flies with both genetically manipulated not and Nipped-A knocked down.


A : Volcano plot showing significantly regulated genes between control and ATP - treated msEGCs .

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

A: Volcano plot showing significantly regulated genes between control and ATP-treated msEGCs.


G - I : qPCR analysis of indicated gliosis genes in ATP - treated EGCs .

G-I: qPCR analysis of indicated gliosis genes in ATP-treated EGCs.


J : qPCR analysis of IL - 6 in msEGCs that were treated for 6h with ATP .

J: qPCR analysis of IL-6 in msEGCs that were treated for 6h with ATP.


K : IL - 6 protein levels in supernatants from msEGCs collected after 24h treatment with ATP .

K: IL-6 protein levels in supernatants from msEGCs collected after 24h treatment with ATP.


A : Effect of P2 receptor antagonism on ATP induced IL - 6 release . Cells were treated with P2 - antagonist suramin ( 1 , 10 and 100µM ) alone or together with ATP ( 100µM ) for 24h .

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 B-CONTROLLED_VAR O O O O O O

A: Effect of P2 receptor antagonism on ATP induced IL-6 release. Cells were treated with P2-antagonist suramin (1, 10 and 100µM) alone or together with ATP (100µM) for 24h.


B : ATP induced IL - 6 release in msEGCs measured by ELISA . Cells were treated with the indicated concentrations of ATP - and ATPɣS for 24h .

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-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O

B: ATP induced IL-6 release in msEGCs measured by ELISA. Cells were treated with the indicated concentrations of ATP-and ATPɣS for 24h.


C : Effects of P2X antagonists on ATP induced IL - 6 release . Cells were treated for 24h alone or together with ATP ( 100µM ) in absence or presence of P2X2 , P2X4 and P2X7 - antagonists PSB - 1011 , 5 - BD - BD and A740003 , respectively .

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

C: Effects of P2X antagonists on ATP induced IL-6 release. Cells were treated for 24h alone or together with ATP (100µM) in absence or presence of P2X2, P2X4 and P2X7-antagonists PSB-1011, 5-BD-BD and A740003, respectively.


D : P2X2 antagonism of ATP induced mRNA expression of IL - 6 , GFAP and RCAN by qPCR in msEGCs . Cells were treated with the P2X2 - antagonist PSB - 1011 ( 20µM ) alone or together with ATP ( 10µM ) for 6h .

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

D: P2X2 antagonism of ATP induced mRNA expression of IL-6, GFAP and RCAN by qPCR in msEGCs. Cells were treated with the P2X2-antagonist PSB-1011 (20µM) alone or together with ATP (10µM) for 6h.


E : Representative confocal images of P2X2 ( green ) and GFAP ( violet ) positive msEGCs in vivo and in vitro . White arrows mark double positive ( white ) cells . Scale bar 50µm .

O O O O O O O O O O O O O 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: Representative confocal images of P2X2 (green) and GFAP (violet) positive msEGCs in vivo and in vitro. White arrows mark double positive (white) cells. Scale bar 50µm.


F : P2X2 - siRNA reduces P2X2 - mRNA and dampens the gliosis gene expression after ATPɣS ( 100µM ) treatment for 6h .

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

F: P2X2-siRNA reduces P2X2-mRNA and dampens the gliosis gene expression after ATPɣS (100µM) treatment for 6h.


G : P2X2 - siRNA reduces IL - 6 release after ATPɣS treatment ( 10µM , 100µM ) for 6h .

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

G: P2X2-siRNA reduces IL-6 release after ATPɣS treatment (10µM, 100µM) for 6h.


A : ATP measurement at the indicated postoperative time points in peritoneal lavages of mice that underwent intestinal manipulation ( IM ) or were naïve .

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

A: ATP measurement at the indicated postoperative time points in peritoneal lavages of mice that underwent intestinal manipulation (IM) or were naïve.


B : PCA plot of gene expression from POI mice at different disease stages and naïve mice ; n = 3 for each group .

O 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: PCA plot of gene expression from POI mice at different disease stages and naïve mice; n=3 for each group.


F - H : qPCR analysis of indicated gliosis marker in mice that underwent IM ( n = 7 ) .

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

F-H: qPCR analysis of indicated gliosis marker in mice that underwent IM (n=7).


I : Histological analysis of EGC proliferation in vivo . Representative confocal images and quantification show Sox10 ( green ) and Ki67 ( violet ) positive EGCs ( white arrows ) in the small bowel muscularis externa at naïve , IM6h and 24h . Scale bar 50µm .

O O O O O O O O O O O O O O O O O O O O O O O O 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: Histological analysis of EGC proliferation in vivo. Representative confocal images and quantification show Sox10 (green) and Ki67 (violet) positive EGCs (white arrows) in the small bowel muscularis externa at naïve, IM6h and 24h. Scale bar 50µm.


K - M : qPCR analysis of indicated gliosis markers in td + glia cells from naïve mice and mice that underwent IM ( n = 5 - 7 , POI mice ) .

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

K-M: qPCR analysis of indicated gliosis markers in td+ glia cells from naïve mice and mice that underwent IM (n=5-7, POI mice).


Postoperative gene expression analyses of indicated gliosis marker in the ME .

Postoperative gene expression analyses of indicated gliosis marker in the ME.


E : Histological analysis of EGC proliferation by quantification of Ki67 ( violet ) and Sox10 positive EGCs at IM24h . Mice were treated as seen in ( A ) .

O O O O O O O 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: Histological analysis of EGC proliferation by quantification of Ki67 (violet) and Sox10 positive EGCs at IM24h. Mice were treated as seen in (A).


F : Histological counting of infiltrating myeloperoxidase ( MPO ) - positive leukocytes in the postoperative ME ( n = 6 - 8 ) .

F: Histological counting of infiltrating myeloperoxidase (MPO)-positive leukocytes in the postoperative ME (n=6-8).


G : FACS analysis of infiltrating cells in the ME of mice treated with ambroxol or vehicle . CD45 , Ly6C and CX3CR1 were used to distinguish resident macrophages ( CD45 + / Ly6C - / CX3CR1 + ) , infiltrating monocytes ( CD45 + / Ly6C + / CX3CR1 - ) and infiltrated monocyte - derived macrophages ( CD45 + / Ly6C + / CX3CR1 + ) .

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

G: FACS analysis of infiltrating cells in the ME of mice treated with ambroxol or vehicle. CD45, Ly6C and CX3CR1 were used to distinguish resident macrophages (CD45+/Ly6C-/CX3CR1+), infiltrating monocytes (CD45+/Ly6C+/CX3CR1-) and infiltrated monocyte-derived macrophages (CD45+/Ly6C+/CX3CR1+).


H : Postoperative in vivo GI transit measurement in mice treated with ambroxol or vehicle .

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

H: Postoperative in vivo GI transit measurement in mice treated with ambroxol or vehicle.


E : IL - 6 protein release in hEGC cultures upon stimulation with ATP ( 200µM ) or ATPɣS ( 100µM ) after 6h and 24h treatment ( n = 4 - 6 , hEGCs ) .

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 O O O O O O O O

E: IL-6 protein release in hEGC cultures upon stimulation with ATP (200µM) or ATPɣS (100µM) after 6h and 24h treatment (n=4-6, hEGCs).


F : IL - 6 protein release in hEGC cultures upon stimulation with NTPDase inhibitor ARL67156 at indicated concentrations ; n = 4 - 6 , hEGCs .

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

F: IL-6 protein release in hEGC cultures upon stimulation with NTPDase inhibitor ARL67156 at indicated concentrations; n=4-6, hEGCs.


G : Immunofluorescence microscopy revealed P2X2 expression ( green ) in a majority of s100β + ( violet ) hEGCs in intact myenteric ganglia of the human colon . White arrows mark double positive glia cells and green arrows mark P2X2 positive neurons . Scale bar , 50µm .

O O O O O O O O O O O O O O O O O O O O O O O O O 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: Immunofluorescence microscopy revealed P2X2 expression (green) in a majority of s100β+ (violet) hEGCs in intact myenteric ganglia of the human colon. White arrows mark double positive glia cells and green arrows mark P2X2 positive neurons. Scale bar, 50µm.


H : Effect of the P2X2 antagonism on ATP - induced IL - 6 release . ELISA measurement of IL - 6 in hEGCs upon treatment with P2X2 - antagonist PSB - 1011 ( 20µM ) alone or together with ATP ( 200µM ) treated for 24h ( n = 6 , hEGCs ) .

O O O O O O 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

H: Effect of the P2X2 antagonism on ATP-induced IL-6 release. ELISA measurement of IL-6 in hEGCs upon treatment with P2X2-antagonist PSB-1011 (20µM) alone or together with ATP (200µM) treated for 24h (n=6, hEGCs).


I : PSB - 1011 ( 20μM ) treatment inhibited ATP - triggered calcium responses in HEK cells transfected with P2X2 . Data are represented as ΔF / F0 + SEM ; n = 102 HEK cells . J : The P2X2 receptor antagonist PSB - 0711 ( 20μM ) nearly abolished the ATP - triggered calcium response in HEK cells transfected with P2X2 . n = 219 HEK cells .

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 B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_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

I: PSB-1011 (20μM) treatment inhibited ATP-triggered calcium responses in HEK cells transfected with P2X2. Data are represented as ΔF/F0 +SEM; n=102 HEK cells. J: The P2X2 receptor antagonist PSB-0711 (20μM) nearly abolished the ATP-triggered calcium response in HEK cells transfected with P2X2. n=219 HEK cells.


K : Ambroxol blocks ATP - induced IL - 6 release in hEGCs ; Protein release measurement by ELISA of IL - 6 in hEGCs . Cells were treated with ATP ( 200 µM ) alone or together with ambroxol ( 20 µM ) for 24h ; n = 6 , hEGCs .

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

K: Ambroxol blocks ATP-induced IL-6 release in hEGCs; Protein release measurement by ELISA of IL-6 in hEGCs. Cells were treated with ATP (200 µM) alone or together with ambroxol (20 µM) for 24h; n=6, hEGCs.


L - N : qPCR analysis of several gliosis and ATP - target genes in the mechanically manipulated surgical specimens incubated in the presence or absence of ambroxol ( 20µM ) . ( n = 7 , 4 human patients ) .

O O O 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

L-N: qPCR analysis of several gliosis and ATP-target genes in the mechanically manipulated surgical specimens incubated in the presence or absence of ambroxol (20µM). (n=7, 4 human patients).


A . Ovaries from control heterozygous Rpp3018 . 2 flies ( left ) , homozygous Rpp3018 . 2 or transheterozygous Rpp3018 . 2 / Rpp30PE flies ( middle ) and homozygous Rpp3018 . 2 carrying a copy of ubiRpp30GFP transgene ( right ) . Scale bar , 100µm .

A. Ovaries from control heterozygous Rpp3018.2 flies (left), homozygous Rpp3018.2 or transheterozygous Rpp3018.2/Rpp30PE flies (middle) and homozygous Rpp3018.2 carrying a copy of ubiRpp30GFP transgene (right). Scale bar, 100µm.


D . Ovaries were dissected , lysed and protein extracts analyzed by Western Blot using anti - Rpp30 , anti - GFP or anti - tubulin antibodies . Orange arrows indicate overexpressed and endogenous Rpp30 signals . Genotypes are numbered as 1 , 2 , 3 .

O O O O O O O O O O O O O O O O O O O O O O O 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. Ovaries were dissected, lysed and protein extracts analyzed by Western Blot using anti-Rpp30, anti- GFP or anti-tubulin antibodies. Orange arrows indicate overexpressed and endogenous Rpp30 signals. Genotypes are numbered as 1,2,3.


E . Rpp30 - dependent early oogenesis arrest is restored by expression of Rpp30GFP transgene in two different genetic backgrounds : Left . Homozygous Rpp3018 . 2 ovariole expressing ubiquitous ubiRpp30GFP ( st , stage ) . Right . Transheterozygous Rpp3018 . 2 / Rpp30PE ovarioles overexpressing germline specific nanosGal4 , UASRpp30GFP . Note follicular somatic cells with no Rpp30GFP expression ( arrowhead ) . Magnifications show nuclear Rpp30 localization , Rpp30 perinuclear foci ( arrows ) , and Orb ( a specific oocyte marker ) . Scale bar , 10µm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Rpp30-dependent early oogenesis arrest is restored by expression of Rpp30GFP transgene in two different genetic backgrounds: Left. Homozygous Rpp3018.2 ovariole expressing ubiquitous ubiRpp30GFP (st, stage). Right. Transheterozygous Rpp3018.2/Rpp30PE ovarioles overexpressing germline specific nanosGal4,UASRpp30GFP. Note follicular somatic cells with no Rpp30GFP expression (arrowhead). Magnifications show nuclear Rpp30 localization, Rpp30 perinuclear foci (arrows), and Orb (a specific oocyte marker). Scale bar, 10µm.


F . flp / FRT clones mutant for Rpp3018 . 2specifically in the germline ( star ) and / or the follicular cells ( arrows ) are detected by the absence of GFP . Magnifications show mutant oocytes with Orb mislocalization next to the karyosome ( dotted circle ) instead of being localized to the posterior , as seen in stage 3 ( st . 3 ) wild - type clones . Scale bar , 10µm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. flp/FRT clones mutant for Rpp3018.2specifically in the germline (star) and/or the follicular cells (arrows) are detected by the absence of GFP. Magnifications show mutant oocytes with Orb mislocalization next to the karyosome (dotted circle) instead of being localized to the posterior, as seen in stage 3 (st.3) wild-type clones. Scale bar, 10µm.


Figure 2 . Rpp30 is required for pre - tRNA processing . Whole fly RNA extracts were used to study tRNA processing by Northern Blot . Top box : the three different probes used are depicted : 5 ' and 3 ' probes ( red and green , respectively ) can detect full pre - tRNAs and tRNAs intermediates , but will not detect mature tRNAs . An Internal Probe ( IP , blue ) corresponding to the anti - codon region was used to detect all non - mature and mature tRNAs . The genotypes used in this experiment are numbered at the bottom ( 1 - 6 ) . Northern Blot panels : tRNA - his 5 ' probe ( top ) , tRNA - his 3 ' probe ( middle ) and tRNA - his IP ( bottom ) . U6 probe was used as loading control .

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 O O O O O O O O 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 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 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

Figure 2. Rpp30 is required for pre-tRNA processing.Whole fly RNA extracts were used to study tRNA processing by Northern Blot. Top box : the three different probes used are depicted : 5' and 3' probes (red and green, respectively) can detect full pre-tRNAs and tRNAs intermediates, but will not detect mature tRNAs. An Internal Probe (IP, blue) corresponding to the anti-codon region was used to detect all non-mature and mature tRNAs. The genotypes used in this experiment are numbered at the bottom (1-6). Northern Blot panels: tRNA-his 5' probe (top), tRNA-his 3' probe (middle) and tRNA-his IP (bottom). U6 probe was used as loading control.


A . Rpp3018 . 2 homozygous early oogenesis arrest is partially rescued by p53 mutation . Scale bar , 100μm .

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A. Rpp3018.2 homozygous early oogenesis arrest is partially rescued by p53 mutation. Scale bar,100μm.


B . Early oogenesis arrest found in Rpp3018 . 2 homozygous ovaries is rescued by chk2 mutation ( Rpp3018 . 2 , mnkp6 and Rpp3018 . 2 / Rpp30PE , mnkp6 ) . Scale bar , 100μm . Star : example of one rescued stage 9 egg chamber . Arrow : example of non - rescued egg chambers .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Early oogenesis arrest found in Rpp3018.2 homozygous ovaries is rescued by chk2 mutation (Rpp3018.2 , mnkp6 and Rpp3018.2/ Rpp30PE, mnkp6). Scale bar, 100μm. Star: example of one rescued stage 9 egg chamber. Arrow: example of non-rescued egg chambers.


C . Germline clones mutant for Rpp3018 . 2 were immunostained for PCNA ( red ) . DAPI is in blue . Magnifications : PCNA signal in a control or mutant nurse cell . Scale bar , 10μm .

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C. Germline clones mutant for Rpp3018.2 were immunostained for PCNA (red). DAPI is in blue. Magnifications: PCNA signal in a control or mutant nurse cell. Scale bar, 10μm.


D . Germline clones mutant for Rpp3018 . 2 were immunostained for Brf ( pol - III ) ( red ) . DAPI is in blue . A Z - projection of Brf staining is shown . The arrow points Brf aggregates in a mutant chamber . Scale bar , 10μm .

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D. Germline clones mutant for Rpp3018.2 were immunostained for Brf (pol-III) (red). DAPI is in blue. A Z-projection of Brf staining is shown. The arrow points Brf aggregates in a mutant chamber. Scale bar, 10μm.


E . Early oogenesis arrest found in Rpp3018 . 2 homozygous ovaries is partially rescued by claspin mutation ( Rpp3018 . 2 ; claspinEP / TM3 ) . Scale bar , 100μm .

O O O O O O O O O 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. Early oogenesis arrest found in Rpp3018.2 homozygous ovaries is partially rescued by claspin mutation (Rpp3018.2; claspinEP/TM3). Scale bar, 100μm.


G . Top panel . Early oogenesis arrest found in Rpp30182 . / PEtransheterozygous ovaries is rescued by sqd - S - HAoverexpression . Scale bars : 100µm . Low panel . Rpp3018 . 2 / PE ; sqd - S - HA / sqd - S - HA ovaries were dissected and stained for Orb ( green ) , HA ( squid , red ) and DAPI ( blue ) . Scale bar , 10µm .

G. Top panel. Early oogenesis arrest found in Rpp30182./PEtransheterozygous ovaries is rescued by sqd-S-HAoverexpression. Scale bars: 100µm. Low panel. Rpp3018.2/PE ; sqd-S-HA/sqd-S-HA ovaries were dissected and stained for Orb (green), HA (squid, red) and DAPI (blue). Scale bar, 10µm.


. D . Nuage specific markers ( Aub , Ago3 and Mael ) are shown in stage 3 wild - type and germline mutant clones for Rpp3018 . 2 . Scale bar , 10µm .

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. D. Nuage specific markers (Aub, Ago3 and Mael) are shown in stage 3 wild-type and germline mutant clones for Rpp3018.2. Scale bar, 10µm.


E - H . Double mutant ovaries for Rpp30 and chk2 ( Rpp3018 . 2 , mnkp6 ) rescued the specific and general piRNA populations , nuage morphology and the characteristic ping - pong signal . Scale bar , 10µm .

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E-H. Double mutant ovaries for Rpp30 and chk2 (Rpp3018.2, mnkp6) rescued the specific and general piRNA populations, nuage morphology and the characteristic ping-pong signal. Scale bar, 10µm.


D . Testes from control heterozygous , transheterozygous Rpp3018 . 2 / Rpp30PE or aubHN / QC42 were fixed and stained for Stellate crystal formation . Z - projections are shown .

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D. Testes from control heterozygous, transheterozygous Rpp3018.2/Rpp30PE or aubHN/QC42 were fixed and stained for Stellate crystal formation. Z-projections are shown.


( C ) Enrichment of proteins detected by mass spectrometry on a pool of TATA - box promoters over control DNA

(C) Enrichment of proteins detected by mass spectrometry on a pool of TATA-box promoters over control DNA sequences


( D ) over a pool of DRE promoters . 3 biological replicates were performed for each promoter and control pool , significance measured with a Limma p - value < 0 . 05 .

(D) over a pool of DRE promoters. 3 biological replicates were performed for each promoter and control pool, significance measured with a Limma p-value<0.05.


D . Western blots of the DNA - bound fraction eluted off the beads in a DNA - affinity purification assay . Super core promoter 1 ( SCP1 ) was used a positive control to bind TFIIA - β - FLAG ( top panel ) . DRE promoter pools of varying lengths were assayed for their ability to bind TFIIA . Sonicated salmon sperm DNA was used as competitor DNA and titrated from 100ng to 1 . 6ug per reaction . Histone H3 was used as an abundant non - specific DNA interacting protein for loading control . From top to bottom we have used promoter fragments ranging at 121bp , 350bp and 1kb in length .

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 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 O O O O O O 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 blots of the DNA-bound fraction eluted off the beads in a DNA-affinity purification assay. Super core promoter 1 (SCP1) was used a positive control to bind TFIIA-β-FLAG (top panel). DRE promoter pools of varying lengths were assayed for their ability to bind TFIIA. Sonicated salmon sperm DNA was used as competitor DNA and titrated from 100ng to 1.6ug per reaction. Histone H3 was used as an abundant non-specific DNA interacting protein for loading control. From top to bottom we have used promoter fragments ranging at 121bp, 350bp and 1kb in length.


E . ChIP - seq signal of GTFs and select housekeeping promoter binders from Drosophila S2 cells and embryos normalized to nascent transcription level as measured by PRO - seq at the respective promoter types .

O O O O O O O O O O O O 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. ChIP-seq signal of GTFs and select housekeeping promoter binders from Drosophila S2 cells and embryos normalized to nascent transcription level as measured by PRO-seq at the respective promoter types.


B . Western blot on FLAG tagged TBP and DREF 0 , 1 and 3 hours after auxin addition showing protein degradation .

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B. Western blot on FLAG tagged TBP and DREF 0,1 and 3 hours after auxin addition showing protein degradation.


E . Fisher ' s exact test for motif enrichment in TBP and DREF downregulated promoters compared to all expressed promoters . Log2 of the Odds ratio displayed . The dashed line are set at a value of 1 and - 1 .

O O O O O O O O O O O O O O O O O O O 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. Fisher's exact test for motif enrichment in TBP and DREF downregulated promoters compared to all expressed promoters. Log2 of the Odds ratio displayed. The dashed line are set at a value of 1 and -1.


E . ChIP - seq coverage ( input normalized ) of TBP and TRF2 at TBP and / or TRF2 dependent promoters , and all other active promoters ( ' unchanged ' ) . Orange : TBP dependent promoters ( i . e . promoters downregulated upon TBP depletion ) ; blue : TRF2 dependent promoters ; purple : promoters downregulated when both , TBP and Trf2 are depleted ( orange , blue and purple are not mutually exclusive sets of genes ) ; white : unaffected promoters . Boxes represent the upper and lower quartiles , with the middle band at the median . The whiskers represent the upper and lower 5th percentiles . Data was taken from two merged biological replicates .

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E. ChIP-seq coverage (input normalized) of TBP and TRF2 at TBP and/or TRF2 dependent promoters, and all other active promoters ('unchanged'). Orange: TBP dependent promoters (i.e. promoters downregulated upon TBP depletion); blue: TRF2 dependent promoters; purple: promoters downregulated when both, TBP and Trf2 are depleted (orange, blue and purple are not mutually exclusive sets of genes); white: unaffected promoters. Boxes represent the upper and lower quartiles, with the middle band at the median. The whiskers represent the upper and lower 5th percentiles. Data was taken from two merged biological replicates.


F . Western blot of anti - FLAG antibody of TBP - AID , Trf2 - AID and a double tagged TBP - AID + Trf2 - AID cell lines from a multi - day time course of auxin treatment showing prolonged depletion .

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F. Western blot of anti-FLAG antibody of TBP-AID, Trf2-AID and a double tagged TBP-AID + Trf2-AID cell lines from a multi-day time course of auxin treatment showing prolonged depletion.


J . qPCR on an auxin treatment time course of TBP and Trf2 dependent genes upon individual depletion of TBP or Trf2 and a double depletion of both . Error bars represent the standard deviation across three biological replicates .

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

J. qPCR on an auxin treatment time course of TBP and Trf2 dependent genes upon individual depletion of TBP or Trf2 and a double depletion of both. Error bars represent the standard deviation across three biological replicates.


C . PRO - seq signal at all expressed promoters , represented according to their motif content in box plots . Boxes represent the upper and lower quartiles , with the middle band at the median . The whiskers represent the upper and lower 5th percentiles across two biological replicates .

O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. PRO-seq signal at all expressed promoters, represented according to their motif content in box plots. Boxes represent the upper and lower quartiles, with the middle band at the median. The whiskers represent the upper and lower 5th percentiles across two biological replicates.


E . Luciferase assay in which Gal4 DNA binding domain fusion proteins were recruited to 4xUAS sites upstream of a minimal housekeeping Rps12 promoter . Measurements are normalized to Renilla luciferase ( transfection control ) and GFP . * denotes proteins activating a housekeeping promoter with a log2FC > 1 . 5 and p - value < 0 . 05 , two - tailed student ' s t - test . Error bars represent standard deviation across four biological replices .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Luciferase assay in which Gal4 DNA binding domain fusion proteins were recruited to 4xUAS sites upstream of a minimal housekeeping Rps12 promoter. Measurements are normalized to Renilla luciferase (transfection control) and GFP. * denotes proteins activating a housekeeping promoter with a log2FC>1.5 and p-value<0.05, two-tailed student's t-test. Error bars represent standard deviation across four biological replices.


A . Distribution of CAGE signal from mixed D . mel embryos ( 0 - 24h ) centered on the location of promoter DNA motif sequence set at position 0 across the 6 main promoter types investigated in this study .

A. Distribution of CAGE signal from mixed D. mel embryos (0-24h) centered on the location of promoter DNA motif sequence set at position 0 across the 6 main promoter types investigated in this study.


D . Box plot of the percent of STAP - seq signal ( i . e . percent of initiation ) originating at the dominant TSS at core promoters ( CPs ; N = 1266 ) and random regions ( N = 639 ) that are activated to similar extent by both GFZF and MED25 recruitment . Cofactor recruitment STAP - seq data from ( Haberle et al , 2019 ) , three independent biological replicates merged . * P ≤ 0 . 01 ; Wilcoxon rank sum test .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Box plot of the percent of STAP-seq signal (i.e. percent of initiation) originating at the dominant TSS at core promoters (CPs; N = 1266) and random regions (N = 639) that are activated to similar extent by both GFZF and MED25 recruitment. Cofactor recruitment STAP-seq data from (Haberle et al, 2019), three independent biological replicates merged. *P≤0.01; Wilcoxon rank sum test.


( A - C ) Measurement of autophagy during nutrient starvation in HT29 cells stably expressing YFP - LC3 . ( A ) Increased LC3 puncta per cell following β ‐ catenin knockdown with siRNA compared with a non ‐ targeting ( NT ) control siRNA . Columns show autophagosome numbers ( mean ± s . e . m . , n > 200 cells in > 20 fields of view per experiment of three independent treatments ) assessed under normal and starved ( 2 h ) conditions with β ‐ catenin or non ‐ targeting ( NT ) siRNA . Representative images ( B ) of YFP - LC3 puncta ( green ) and DAPI nuclei staining ( blue ) are shown . ( C ) Upper panel : western blotting showed increased LC3 ‐ II expression in HT29 cells with β ‐ catenin knockdown compared to control cells after 2 h starvation . Lower panel : quantification of the LC3 ‐ II / β ‐ actin ratio by densitometry ( mean ± s . e . m . of four independent treatments , * P = 0 . 011 in nutrient conditions , * P = 0 . 035 under starvation ) .

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(A-C) Measurement of autophagy during nutrient starvation in HT29 cells stably expressing YFP-LC3. (A) Increased LC3 puncta per cell following β‐catenin knockdown with siRNA compared with a non‐targeting (NT) control siRNA. Columns show autophagosome numbers (mean±s.e.m., n>200 cells in >20 fields of view per experiment of three independent treatments) assessed under normal and starved (2 h) conditions with β‐catenin or non‐targeting (NT) siRNA. Representative images (B) of YFP-LC3 puncta (green) and DAPI nuclei staining (blue) are shown. (C) Upper panel: western blotting showed increased LC3‐II expression in HT29 cells with β‐catenin knockdown compared to control cells after 2 h starvation. Lower panel: quantification of the LC3‐II/β‐actin ratio by densitometry (mean±s.e.m. of four independent treatments, *P=0.011 in nutrient conditions, *P=0.035 under starvation).


( D and E ) HCT116 β ‐ cateninWT / − cells overexpressing a control or β ‐ cateninS33Y plasmid nutrient starved for 24 h . ( D ) Immunostaining of LC3 ( green ) with DAPI nuclei staining ( blue ) showed decreased LC3 puncta with β ‐ cateninS33Y overexpression . ( E ) By western blotting , LC3 ‐ II decreased with β ‐ cateninS33Y overexpression ( upper panel ) . Lower panel : quantification of the LC3 ‐ II / β ‐ actin ratio by densitometry ( mean ± s . e . m . of four independent treatments , * P = 0 . 015 ) .

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(D and E) HCT116 β‐cateninWT/− cells overexpressing a control or β‐cateninS33Y plasmid nutrient starved for 24 h. (D) Immunostaining of LC3 (green) with DAPI nuclei staining (blue) showed decreased LC3 puncta with β‐cateninS33Y overexpression. (E) By western blotting, LC3‐II decreased with β‐cateninS33Y overexpression (upper panel). Lower panel: quantification of the LC3‐II/β‐actin ratio by densitometry (mean±s.e.m. of four independent treatments, *P=0.015).


( 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 .

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(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 O O O O O O 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 O O O O 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) 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 .

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(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 O O O O O O O O O O O O O O O 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 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 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 ) .

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(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 ) .

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(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 .

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(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 .

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(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 .

(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 .

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(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 .

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(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 .

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(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 ) .

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(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 .

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(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 .

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(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 ) .

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(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 ) .

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(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 ) .

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(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 .

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(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 .

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(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 ) .

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(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 ) .

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(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 ) .

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(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 ) .

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(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 .

(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 ) .

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(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 .

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(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 .

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(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 .

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(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 .

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(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 .

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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 .

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(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 .

(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 .

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(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 ) .

(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 .

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(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 .

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(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 .

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(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 .

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(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 .

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(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 .

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(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 .

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[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 .

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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 .

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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 .

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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 ) .

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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 .

(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 .

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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 ) .

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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 ) ;

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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 ) .

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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 ) .

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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 .

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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 ) .

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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 .

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(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 ) .

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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 .

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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 .

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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 .

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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 .

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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 .

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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 .

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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.