Figure 1A Detection of the activation of IRE1α , PERK and ATF6 UPR branches by immunoblotting in control ( shCtrl ) and Fam20C stable knockdown ( shFAM20C ) HepG2 cells treated with or without 5 μM Tg for 30 min . ATF6 - FL , full - length ATF6 ; ATF6 - N , N - terminal cleavage product of ATF6 ; Asterisk , unspecific background bands . Fam20C knockdown was verified by protein immunoblotting of Concanavalin A - Sepharose ( Con A ) - enriched culture medium . Ponceau staining was shown as a loading control . B ( Left ) Detection of spliced XBP1 ( S ) and unspliced XBP1 ( U ) mRNA in shCtrl and shFAM20C HepG2 cells treated with or without 5 μM Tg for 1 h . ( Right ) Quantification of XBP1 mRNA splicing levels . Data information : data were shown as mean ± SEM of three independent experiments . * p < 0 . 05 , * * p < 0 . 01 ( two - tailed Student ' s t - test ) . C ( Left ) Detection of XBP1 mRNA splicing in shFAM20C HepG2 cells expressing RNAi - resistant codon - altered Fam20C wild - type ( WT ) or its inactive D478A mutant ( DA ) treated with or without 5 μM Tg for 1 h . Fam20C expression levels were shown by protein immunoblotting . ( Right ) Quantification of XBP1 mRNA splicing levels . Data information : data were shown as mean ± SEM of three independent experiments . * p < 0 . 05 , * * p < 0 . 01 ( two - tailed Student ' s t - test ) . ( Left ) Detection of XBP1 mRNA splicing in shCtrl and shFAM20C HepG2 cells ( D ) treated with or without 2 μg / ml Tm for 8 h . ( Right ) Quantification of XBP1 mRNA splicing levels . Data information : data were shown as mean ± SEM of three independent experiments . * p < 0 . 05 , * * p < 0 . 01 ( two - tailed Student ' s t - test ) . Detection of XBP1 mRNA splicing in shFAM20C HepG2 cells expressing Fam20C WT or DA ( E ) treated with or without 2 μg / ml Tm for 8 h . ( Right ) Quantification of XBP1 mRNA splicing levels . Data information : data were shown as mean ± SEM of three independent experiments . * p < 0 . 05 , * * p < 0 . 01 ( two - tailed Student ' s t - test ) . F Volcano plot depicting the log2 of fold change versus - log10 ( p - value ) for Fam20C - interacting proteins in Tg - treated HepG2 cells . The red dots represent proteins showing statistically significant increase of binding with Fam20C ( log2 fold change ≥ 1 ; - log10 ( p - value ) ≥ 1 . 3 ) ; the blue dots represent proteins showing statistically significant decrease of binding with Fam20C ( log2 fold change ≤ - 1 ; - log10 ( p - value ) ≥ 1 . 3 ) ; the green dot is Fam20C . G HepG2 cells expressing FLAG - tagged Fam20C were treated with or without 5 μM Tg for 30 min . FLAG - immunoprecipitates were analyzed by PDI immunoblotting .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1A Detection of the activation of IRE1α, PERK and ATF6 UPR branches by immunoblotting in control (shCtrl) and Fam20C stable knockdown (shFAM20C) HepG2 cells treated with or without 5 μM Tg for 30 min. ATF6-FL, full-length ATF6; ATF6-N, N-terminal cleavage product of ATF6; Asterisk, unspecific background bands. Fam20C knockdown was verified by protein immunoblotting of Concanavalin A-Sepharose (Con A)-enriched culture medium. Ponceau staining was shown as a loading control.B (Left) Detection of spliced XBP1 (S) and unspliced XBP1 (U) mRNA in shCtrl and shFAM20C HepG2 cells treated with or without 5 μM Tg for 1 h. (Right) Quantification of XBP1 mRNA splicing levels. Data information: data were shown as mean ± SEM of three independent experiments. *p < 0.05, **p < 0.01 (two-tailed Student's t-test).C (Left) Detection of XBP1 mRNA splicing in shFAM20C HepG2 cells expressing RNAi-resistant codon-altered Fam20C wild-type (WT) or its inactive D478A mutant (DA) treated with or without 5 μM Tg for 1 h. Fam20C expression levels were shown by protein immunoblotting. (Right) Quantification of XBP1 mRNA splicing levels. Data information: data were shown as mean ± SEM of three independent experiments. *p < 0.05, **p < 0.01 (two-tailed Student's t-test).(Left) Detection of XBP1 mRNA splicing in shCtrl and shFAM20C HepG2 cells (D) treated with or without 2 μg/ml Tm for 8 h. (Right) Quantification of XBP1 mRNA splicing levels. Data information: data were shown as mean ± SEM of three independent experiments. *p < 0.05, **p < 0.01 (two-tailed Student's t-test).Detection of XBP1 mRNA splicing in shFAM20C HepG2 cells expressing Fam20C WT or DA (E) treated with or without 2 μg/ml Tm for 8 h. (Right) Quantification of XBP1 mRNA splicing levels. Data information: data were shown as mean ± SEM of three independent experiments. *p < 0.05, **p < 0.01 (two-tailed Student's t-test).F Volcano plot depicting the log2 of fold change versus - log10 (p-value) for Fam20C-interacting proteins in Tg-treated HepG2 cells. The red dots represent proteins showing statistically significant increase of binding with Fam20C (log2 fold change ≥ 1; - log10 (p-value) ≥ 1.3); the blue dots represent proteins showing statistically significant decrease of binding with Fam20C (log2 fold change ≤ - 1; - log10 (p-value) ≥ 1.3); the green dot is Fam20C.G HepG2 cells expressing FLAG-tagged Fam20C were treated with or without 5 μM Tg for 30 min. FLAG-immunoprecipitates were analyzed by PDI immunoblotting.


Figure 2A Representative MS / MS fragmentation spectrum of tryptic phosphorylated PDI peptide ( Ile351 - Lys370 ) depicting PDI Ser357 phosphorylation enriched from HepG2 cells treated with 5 μM Tg for 10 min . C Time - dependent incorporation of phosphate group into PDI WT or S357A catalyzed by recombinant Fam20C . Phosphorylated PDI was determined by anti - pS357 - PDI immunoblotting on both regular and phostag gels . D Phosphorylated PDI catalyzed by Fam20C were analyzed by dual - color immunoblotting with anti - PDI ( red ) and anti - pS357 - PDI ( green ) antibodies . The yellow signal depicts the phosphorylated PDI species . Detection of pS357 - PDI in HepG2 cells treated with 5 μM Tg ( E ) for indicated times . Detection of pS357 - PDI in HepG2 cells treated with 2 . 5 μg / ml Tm ( F ) for indicated times . Detection of pS357 - PDI in HepG2 cells treated with 1 mM DTT ( G ) for indicated times . ( Left ) Detection of p - PDI in WT and PDI KO HepG2 cells ( H ) treated with 5 μM Tg for indicated times by protein immunoblotting on both regular and phostag gels . ( Right ) Quantification of p - PDI percentage based on band intensities on phostag gels . Data information : Data were shown as mean ± SEM of three ( H ) independent experiments . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ( one - way ANOVA , the post hoc Tukey ' s HSD test ) . ( Left ) Detection of p - PDI in WT and Fam20C KO HeLa cells ( I ) treated with 5 μM Tg for indicated times by protein immunoblotting on both regular and phostag gels . ( Right ) Quantification of p - PDI percentage based on band intensities on phostag gels . Data information : Data were shown as mean ± SEM of four ( I ) independent experiments . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ( one - way ANOVA , the post hoc Tukey ' s HSD test ) .

O B-PANEL_START O O O O O O O O O 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-PANEL_START O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2A Representative MS/MS fragmentation spectrum of tryptic phosphorylated PDI peptide (Ile351-Lys370) depicting PDI Ser357 phosphorylation enriched from HepG2 cells treated with 5 μM Tg for 10 min.C Time-dependent incorporation of phosphate group into PDI WT or S357A catalyzed by recombinant Fam20C. Phosphorylated PDI was determined by anti-pS357-PDI immunoblotting on both regular and phostag gels.D Phosphorylated PDI catalyzed by Fam20C were analyzed by dual-color immunoblotting with anti-PDI (red) and anti-pS357-PDI (green) antibodies. The yellow signal depicts the phosphorylated PDI species.Detection of pS357-PDI in HepG2 cells treated with 5 μM Tg (E) for indicated times.Detection of pS357-PDI in HepG2 cells treated with 2.5 μg/ml Tm (F) for indicated times.Detection of pS357-PDI in HepG2 cells treated with 1 mM DTT (G) for indicated times.(Left) Detection of p-PDI in WT and PDI KO HepG2 cells (H) treated with 5 μM Tg for indicated times by protein immunoblotting on both regular and phostag gels. (Right) Quantification of p-PDI percentage based on band intensities on phostag gels. Data information: Data were shown as mean ± SEM of three (H) independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001 (one-way ANOVA, the post hoc Tukey's HSD test).(Left) Detection of p-PDI in WT and Fam20C KO HeLa cells (I) treated with 5 μM Tg for indicated times by protein immunoblotting on both regular and phostag gels. (Right) Quantification of p-PDI percentage based on band intensities on phostag gels. Data information: Data were shown as mean ± SEM of four (I) independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001 (one-way ANOVA, the post hoc Tukey's HSD test).


Figure 3A Fluorescent photomicrographs of HepG2 cells co - transfected with PDI - mEmerald - KDEL ( green ) and Fam20C - mApple ( red ) . The cells were treated with DMSO ( Upper ) or 5 μM Tg ( Middle ) for 30 min . After Tg treatment , the cells were washed with PBS and then returned to culture medium for 60 min ( Lower ) . GM130 was immunostained ( blue ) as a cis - Golgi marker . Scale bar = 10 μm . B Pearson correlation coefficient calculated Data were shown as mean ± SEM of N > 80 cells from four independent experiments . * * * p < 0 . 001 ( one - way ANOVA , the post hoc Tukey ' s HSD test ) . C Detection of pS357 - PDI and UPR signaling in HepG2 cells after Tg treatment and washout for indicated times . D HepG2 cells were treated with 25 ng / μl CHX for indicated times and analyzed by immunoblotting for pS357 - PDI and UPR signaling . 5 μM Tg or DMSO was added 30 min before cell harvest . E Fluorescent photomicrographs of HepG2 cells co - transfected with PDI - mEmerald - KDEL ( green ) and Fam20C - mApple ( red ) pretreated with 25 ng / μl CHX for 7 . 5 h , followed by introduction of DMSO ( Upper ) or 5 μM Tg ( Lower ) for 30 min , respectively . GM130 was immunostained ( blue ) as a cis - Golgi marker . Scale bar = 10 μm . F Fluorescent photomicrographs of HepG2 cells co - transfected with PDI - mEmerald - KDEL ( green ) and Fam20C - mApple - KDEL ( red ) treated with DMSO ( Upper ) or 5 μM Tg ( Lower ) for 30 min , respectively . Scale bar = 10 μm . G HepG2 cells were transfected with empty vector ( - ) , FLAG - tagged Fam20C or Fam20C - KDEL followed by introduction without or with 5 μM Tg for 30 min , respectively . PDI phosphorylation was detected by protein immunoblotting on both regular and phostag gels . p - PDI percentage was quantified based on band intensities on phostag gels .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3A Fluorescent photomicrographs of HepG2 cells co-transfected with PDI-mEmerald-KDEL (green) and Fam20C-mApple (red). The cells were treated with DMSO (Upper) or 5 μM Tg (Middle) for 30 min. After Tg treatment, the cells were washed with PBS and then returned to culture medium for 60 min (Lower). GM130 was immunostained (blue) as a cis-Golgi marker. Scale bar = 10 μm.B Pearson correlation coefficient calculated Data were shown as mean ± SEM of N > 80 cells from four independent experiments. ***p < 0.001 (one-way ANOVA, the post hoc Tukey's HSD test).C Detection of pS357-PDI and UPR signaling in HepG2 cells after Tg treatment and washout for indicated times.D HepG2 cells were treated with 25 ng/μl CHX for indicated times and analyzed by immunoblotting for pS357-PDI and UPR signaling. 5 μM Tg or DMSO was added 30 min before cell harvest.E Fluorescent photomicrographs of HepG2 cells co-transfected with PDI-mEmerald-KDEL (green) and Fam20C-mApple (red) pretreated with 25 ng/μl CHX for 7.5 h, followed by introduction of DMSO (Upper) or 5 μM Tg (Lower) for 30 min, respectively. GM130 was immunostained (blue) as a cis-Golgi marker. Scale bar = 10 μm.F Fluorescent photomicrographs of HepG2 cells co-transfected with PDI-mEmerald-KDEL (green) and Fam20C-mApple-KDEL (red) treated with DMSO (Upper) or 5 μM Tg (Lower) for 30 min, respectively. Scale bar = 10 μm.G HepG2 cells were transfected with empty vector (-), FLAG-tagged Fam20C or Fam20C-KDEL followed by introduction without or with 5 μM Tg for 30 min, respectively. PDI phosphorylation was detected by protein immunoblotting on both regular and phostag gels. p-PDI percentage was quantified based on band intensities on phostag gels.


Figure 4B The time course of the distance between the geometrical centers of domains a and a ′ and the angle among domains b , b ′ and a ′ from the simulation of pS357 - PDI ( red ) and PDI ( black ) ( Yang et al . , 2014 ) . C ( Left ) ANS fluorescence spectra of PDI WT and S357E . Controls with ANS alone was shown . A . U . , arbitrary units . ( Right ) Enhancement factor were quantified . Data information : All data were shown as mean ± SEM of three independent experiments . * * p < 0 . 01 , * * * p < 0 . 001 ( two - tailed Student ' s t - test ) . D Limited proteolysis of PDI WT ( Left ) and S357E ( Right ) proteins by proteinase K ( Pro K ) for indicated times . Main digested fragments were indicated on the right margin . Band abb ' x * is a digested product with cleavage after Met356 in the x - linker region ( Wang et al . , 2010 ) . E shCtrl and shFAM20C HepG2 cells were treated without or with 5 μM Tg for 30 min , and in some aliquots of cells Tg was removed for 30 min . Cell lysates were subjected to proteinase K digestion for 3 min and immunoblotting analysis . F HepG2 cells expressing HA - tagged PDI and FLAG - tagged Fam20C with different ratios of plasmid concentrations were subjected to proteinase K digestion for 21 min and immunoblotting analysis . Chaperone activities of PDI WT and S357E ( G , H ) or phosphorylated PDI WT and S357A by Fam20C for indicated times ( I Denatured and reduced GAPDH ( G , I ) and rhodanese ( H , were used as substrates , respectively . The chaperone activity of PDI WT was taken as 100 % . Data information : All data were shown as mean ± SEM of three independent experiments . , * * p < 0 . 01 , * * * p < 0 . 001 ( two - tailed Student ' s t - test ) . In ( I , * * * p < 0 . 001 ( two - way ANOVA , the post hoc Tukey ' s HSD test ) . Chaperone activities of phosphorylated PDI WT and S357A by Fam20C for indicated times J ) . Denatured and reduced rhodanese J ) were used as substrates , respectively . The chaperone activity of PDI WT was taken as 100 % . Data information : All data were shown as mean ± SEM of three independent experiments . * * * p < 0 . 001 ( two - way ANOVA , the post hoc Tukey ' s HSD test ) . Reductase activities of PDI WT and S357E ( K ) measured by insulin reduction assay . The activity of PDI WT was taken as 100 % . Data information : All data were shown as mean ± SEM of three independent experiments . * * p < 0 . 01 , * * * p < 0 . 001 ( two - tailed Student ' s t - test ) . Isomerase activities of PDI WT and S357E ( L ) measured by scrambled RNase A reactivation assay . The activity of PDI WT was taken as 100 % . Data information : All data were shown as mean ± SEM of three independent experiments . , * * p < 0 . 01 , * * * p < 0 . 001 ( two - tailed Student ' s t - test ) . Reductase activities of phosphorylated PDI WT and S357A by Fam20C for indicated times ( M ) measured by insulin reduction assay . The activity of PDI WT was taken as 100 % . Data information : All data were shown as mean ± SEM of three independent experiments . , * * * p < 0 . 001 ( two - way ANOVA , the post hoc Tukey ' s HSD test ) . Isomerase activities of phosphorylated PDI WT and S357A by Fam20C for indicated times ( N ) measured by scrambled RNase A reactivation assay . The activity of PDI WT was taken as 100 % . Data information : All data were shown as mean ± SEM of three independent experiments . , * * * p < 0 . 001 ( two - way ANOVA , the post hoc Tukey ' s HSD test ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4B The time course of the distance between the geometrical centers of domains a and a′ and the angle among domains b, b′ and a′ from the simulation of pS357-PDI (red) and PDI (black) (Yang et al., 2014).C (Left) ANS fluorescence spectra of PDI WT and S357E. Controls with ANS alone was shown. A.U., arbitrary units. (Right) Enhancement factor were quantified. Data information: All data were shown as mean ± SEM of three independent experiments. **p < 0.01, ***p < 0.001 (two-tailed Student's t-test).D Limited proteolysis of PDI WT (Left) and S357E (Right) proteins by proteinase K (Pro K) for indicated times. Main digested fragments were indicated on the right margin. Band abb'x* is a digested product with cleavage after Met356 in the x-linker region (Wang et al., 2010).E shCtrl and shFAM20C HepG2 cells were treated without or with 5 μM Tg for 30 min, and in some aliquots of cells Tg was removed for 30 min. Cell lysates were subjected to proteinase K digestion for 3 min and immunoblotting analysis.F HepG2 cells expressing HA-tagged PDI and FLAG-tagged Fam20C with different ratios of plasmid concentrations were subjected to proteinase K digestion for 21 min and immunoblotting analysis.Chaperone activities of PDI WT and S357E (G, H) or phosphorylated PDI WT and S357A by Fam20C for indicated times (I Denatured and reduced GAPDH (G, I) and rhodanese (H, were used as substrates, respectively. The chaperone activity of PDI WT was taken as 100%. Data information: All data were shown as mean ± SEM of three independent experiments. , **p < 0.01, ***p < 0.001 (two-tailed Student's t-test). In (I , ***p < 0.001 (two-way ANOVA, the post hoc Tukey's HSD test).Chaperone activities of phosphorylated PDI WT and S357A by Fam20C for indicated times J). Denatured and reduced rhodanese J) were used as substrates, respectively. The chaperone activity of PDI WT was taken as 100%. Data information: All data were shown as mean ± SEM of three independent experiments. ***p < 0.001 (two-way ANOVA, the post hoc Tukey's HSD test).Reductase activities of PDI WT and S357E (K) measured by insulin reduction assay. The activity of PDI WT was taken as 100%. Data information: All data were shown as mean ± SEM of three independent experiments. **p < 0.01, ***p < 0.001 (two-tailed Student's t-test).Isomerase activities of PDI WT and S357E (L) measured by scrambled RNase A reactivation assay. The activity of PDI WT was taken as 100%. Data information: All data were shown as mean ± SEM of three independent experiments. , **p < 0.01, ***p < 0.001 (two-tailed Student's t-test).Reductase activities of phosphorylated PDI WT and S357A by Fam20C for indicated times (M) measured by insulin reduction assay. The activity of PDI WT was taken as 100%. Data information: All data were shown as mean ± SEM of three independent experiments. , ***p < 0.001 (two-way ANOVA, the post hoc Tukey's HSD test).Isomerase activities of phosphorylated PDI WT and S357A by Fam20C for indicated times (N) measured by scrambled RNase A reactivation assay. The activity of PDI WT was taken as 100%. Data information: All data were shown as mean ± SEM of three independent experiments. , ***p < 0.001 (two-way ANOVA, the post hoc Tukey's HSD test).


Figure 5B Fluorescent photomicrographs of HepG2 cells expressing AgHaloER ( TMR - labeled , red ) . Endogenous PDI was immunostained ( green ) as an ER marker . Scale bar = 10 μm . C Confocal live cell imaging of HepG2 cells expressing AgHaloER labeled by P1 ( green ) and TMR ( red ) , followed by introduction of DMSO or 5 μM Tg for 30 min . Scale bar = 10 μm . D Confocal live cell imaging of WT and PDI KO HepG2 cells co - transfected with AgHaloER labeled by P1 ( green ) and TMR ( red ) and empty vector ( - ) , PDI WT , S357E or S357A . Scale bar = 10 μm . E Confocal live cell imaging of shCtrl and shFAM20C HepG2 cells expressing AgHaloER labeled by P1 ( green ) and TMR ( red ) . Scale bar = 10 μm . F ( Upper ) Cell viabilities of WT and PDI KO HepG2 cells co - transfected with AgHaloER and empty vector ( - ) , PDI WT , S357E or S357A . Cells were treated by 5 μM Tg for indicated times and visualized by crystal violet staining . ( Lower ) Quantification of surviving fraction of cells . Data were shown as mean ± SEM of three independent experiments . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 versus WT HepG2 ( two - way ANOVA , the post hoc Tukey ' s HSD test ) .

O B-PANEL_START O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5B Fluorescent photomicrographs of HepG2 cells expressing AgHaloER (TMR-labeled, red). Endogenous PDI was immunostained (green) as an ER marker. Scale bar = 10 μm.C Confocal live cell imaging of HepG2 cells expressing AgHaloER labeled by P1 (green) and TMR (red), followed by introduction of DMSO or 5 μM Tg for 30 min. Scale bar = 10 μm.D Confocal live cell imaging of WT and PDI KO HepG2 cells co-transfected with AgHaloER labeled by P1 (green) and TMR (red) and empty vector (-), PDI WT, S357E or S357A. Scale bar = 10 μm.E Confocal live cell imaging of shCtrl and shFAM20C HepG2 cells expressing AgHaloER labeled by P1 (green) and TMR (red). Scale bar = 10 μm.F (Upper) Cell viabilities of WT and PDI KO HepG2 cells co-transfected with AgHaloER and empty vector (-), PDI WT, S357E or S357A. Cells were treated by 5 μM Tg for indicated times and visualized by crystal violet staining. (Lower) Quantification of surviving fraction of cells. Data were shown as mean ± SEM of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001 versus WT HepG2 (two-way ANOVA, the post hoc Tukey's HSD test).


Figure 6A ( Left ) Detection of XBP1 mRNA splicing in WT and PDI KO HepG2 cells treated with or without 5 μM Tg for 1 h . PDI KO was verified by protein immunoblotting . ( Right ) Quantification of XBP1 mRNA splicing levels . Data were shown as mean ± SEM of three independent experiments . * p < 0 . 05 ( two - tailed Student ' s t - test ) . B ( Left ) Detection of XBP1 mRNA splicing in PDI KO HepG2 cells expressing PDI WT or its mutants treated with or without 5 μM Tg for 1 h . PDI expression levels were shown by protein immunoblotting . ( Right ) Quantification of XBP1 mRNA splicing levels . Data were shown as mean ± SEM of three independent experiments . * p < 0 . 05 , * * p < 0 . 01 ( one - way ANOVA , the post hoc Tukey ' s HSD test ) . C Detection of IRE1α , PERK and ATF6 activation by immunoblotting in PDI KO HepG2 cells expressing PDI S537A or S357E treated with 5 μM Tg for 30 min . Asterisk indicates unspecific background band . D HepG2 cells were treated without or with 5 μM Tg for 1 h . Endogenous IRE1α - immunoprecipitates were analyzed by PDI and BiP immunoblotting . E HepG2 cells expressing FLAG - tagged IRE1α were treated without or with 5 μM Tg for 1 h . An aliquot of Tg - treated cells was then washed with culture medium and chased for 4 h . FLAG - immunoprecipitates were analyzed by PDI and BiP immunoblotting . F shCtrl and shFAM20C HepG2 cells were treated without or with 5 μM Tg for 1 h . Endogenous IRE1α - immunoprecipitates were analyzed by PDI and BiP immunoblotting . G Co - immunoprecipitation of FLAG - tagged IRE1α and HA - tagged PDI in HepG2 cells expressing V5 - tagged Fam20C WT or DA . H Co - immunoprecipitation of FLAG - tagged IRE1α , HA - tagged PDI WT or S357A in HepG2 cells expressing V5 - tagged Fam20C . I Co - immunoprecipitation of HA - tagged PDI and FLAG - tagged IRE1αNLD in HepG2 cells . J Recombinant GST - PDI was phosphorylated by Fam20C protein for 2 h , and then used to pulldown His - IRE1αNLD protein , as visualized by Coomassie blue staining . Successful phosphorylation of GST - PDI was verified by pS357 - PDI immunoblotting . K Co - immunoprecipitation of FLAG - tagged IRE1αNLD WT or its mutant C109 / 148 / 332S with HA - tagged PDI ( Left ) or MYC - tagged P5 ( Right ) in HepG2 cells . L Co - immunoprecipitation of FLAG - tagged IRE1α and HA - tagged PDI WT or its binding mutant W128I / L403W in HepG2 cells expressing V5 - tagged Fam20C or not .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O 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-PANEL_START O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O 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-PANEL_START O 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-PANEL_START O 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-PANEL_START O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6A (Left) Detection of XBP1 mRNA splicing in WT and PDI KO HepG2 cells treated with or without 5 μM Tg for 1 h. PDI KO was verified by protein immunoblotting. (Right) Quantification of XBP1 mRNA splicing levels. Data were shown as mean ± SEM of three independent experiments. *p < 0.05 (two-tailed Student's t-test).B (Left) Detection of XBP1 mRNA splicing in PDI KO HepG2 cells expressing PDI WT or its mutants treated with or without 5 μM Tg for 1 h. PDI expression levels were shown by protein immunoblotting. (Right) Quantification of XBP1 mRNA splicing levels. Data were shown as mean ± SEM of three independent experiments. *p < 0.05, **p < 0.01 (one-way ANOVA, the post hoc Tukey's HSD test).C Detection of IRE1α, PERK and ATF6 activation by immunoblotting in PDI KO HepG2 cells expressing PDI S537A or S357E treated with 5 μM Tg for 30 min. Asterisk indicates unspecific background band.D HepG2 cells were treated without or with 5 μM Tg for 1 h. Endogenous IRE1α-immunoprecipitates were analyzed by PDI and BiP immunoblotting.E HepG2 cells expressing FLAG-tagged IRE1α were treated without or with 5 μM Tg for 1 h. An aliquot of Tg-treated cells was then washed with culture medium and chased for 4 h. FLAG-immunoprecipitates were analyzed by PDI and BiP immunoblotting.F shCtrl and shFAM20C HepG2 cells were treated without or with 5 μM Tg for 1 h. Endogenous IRE1α-immunoprecipitates were analyzed by PDI and BiP immunoblotting.G Co-immunoprecipitation of FLAG-tagged IRE1α and HA-tagged PDI in HepG2 cells expressing V5-tagged Fam20C WT or DA.H Co-immunoprecipitation of FLAG-tagged IRE1α, HA-tagged PDI WT or S357A in HepG2 cells expressing V5-tagged Fam20C.I Co-immunoprecipitation of HA-tagged PDI and FLAG-tagged IRE1αNLD in HepG2 cells.J Recombinant GST-PDI was phosphorylated by Fam20C protein for 2 h, and then used to pulldown His-IRE1αNLD protein, as visualized by Coomassie blue staining. Successful phosphorylation of GST-PDI was verified by pS357-PDI immunoblotting.K Co-immunoprecipitation of FLAG-tagged IRE1αNLD WT or its mutant C109/148/332S with HA-tagged PDI (Left) or MYC-tagged P5 (Right) in HepG2 cells.L Co-immunoprecipitation of FLAG-tagged IRE1α and HA-tagged PDI WT or its binding mutant W128I/L403W in HepG2 cells expressing V5-tagged Fam20C or not.


Figure 7B Sanger sequencing results of Pdi + / + ( WT ) and PdiS359A / S359A ( KI ) mice . C ( Left ) Detection of Xbp1 mRNA splicing in mice liver after injection of Tm ( 50 ng / g , i . p . ) for indicated times . Each lane represents independent animals . ( Right ) Quantification of Xbp1 mRNA splicing levels at indicated times . Data information : All data were shown as mean ± SEM from five biological replicates . , * p < 0 . 05 , * * * p < 0 . 001 ( two - tailed Student ' s t - test ) . Detection of Xbp1 mRNA splicing in primary hepatocytes isolated from WT and KI mice treated with 0 . 2 μM Tg for 2 h ( D ) Detection of Xbp1 mRNA splicing in primary hepatocytes isolated from WT and KI mice treated with 0 . 5 μg / ml Tm for 2 h ( E ) . Detection of Bip ( F ) , Pdi ( G ) , Erdj4 ( H ) mRNA levels by real - time PCR in the liver of the same animals after 24 h of Tm injection . Data information : All data were shown as mean ± SEM from five biological replicates . , * p < 0 . 05 , * * * p < 0 . 001 ( two - way ANOVA , the post hoc Tukey ' s HSD test ) . Detection of Edem1 ( I ) , Il6 ( J ) , Chop ( K ) mRNA levels by real - time PCR in the liver of the same animals after 24 h of Tm injection . Data information : All data were shown as mean ± SEM from five biological replicates . , * p < 0 . 05 , * * * p < 0 . 001 ( two - way ANOVA , the post hoc Tukey ' s HSD test ) . L Detection of serum alanine transaminase ( ALT ) levels of the same animals as in ( C ) after 24 h of Tm injection . Data information : All data were shown as mean ± SEM from five biological replicates . , * p < 0 . 05 , * * * p < 0 . 001 ( two - way ANOVA , the post hoc Tukey ' s HSD test ) . M Hematoxylin and eosin staining of liver tissue the same animals as in ( C ) after 24 h of Tm injection ( magnification × 400 ) . Immune cell infiltration was indicated with a black arrow . Scale bar = 50 μm . Representative images were shown from five animals per group analyzed . N ( Left ) TUNEL assay of the same liver as in ( M ) . Scale bar = 40 μm ; Zoom box , scale bar = 5 μm . ( Right ) Quantification of apoptotic cells . 10 random fields were counted for each TUNEL - stained tissue sample . Data information : All data were shown as mean ± SEM from five biological replicates . , * p < 0 . 05 , * * * p < 0 . 001 ( two - tailed Student ' s t - test ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O 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-PANEL_START O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7B Sanger sequencing results of Pdi+/+ (WT) and PdiS359A/S359A (KI) mice.C (Left) Detection of Xbp1 mRNA splicing in mice liver after injection of Tm (50 ng/g, i.p.) for indicated times. Each lane represents independent animals. (Right) Quantification of Xbp1 mRNA splicing levels at indicated times. Data information: All data were shown as mean ± SEM from five biological replicates. , *p < 0.05, ***p < 0.001 (two-tailed Student's t-test).Detection of Xbp1 mRNA splicing in primary hepatocytes isolated from WT and KI mice treated with 0.2 μM Tg for 2 h (D)Detection of Xbp1 mRNA splicing in primary hepatocytes isolated from WT and KI mice treated with 0.5 μg/ml Tm for 2 h (E).Detection of Bip (F), Pdi (G), Erdj4 (H) mRNA levels by real-time PCR in the liver of the same animals after 24 h of Tm injection. Data information: All data were shown as mean ± SEM from five biological replicates. , *p < 0.05, ***p < 0.001 (two-way ANOVA, the post hoc Tukey's HSD test).Detection of Edem1 (I), Il6 (J), Chop (K) mRNA levels by real-time PCR in the liver of the same animals after 24 h of Tm injection. Data information: All data were shown as mean ± SEM from five biological replicates. , *p < 0.05, ***p < 0.001 (two-way ANOVA, the post hoc Tukey's HSD test).L Detection of serum alanine transaminase (ALT) levels of the same animals as in (C) after 24 h of Tm injection. Data information: All data were shown as mean ± SEM from five biological replicates. , *p < 0.05, ***p < 0.001 (two-way ANOVA, the post hoc Tukey's HSD test).M Hematoxylin and eosin staining of liver tissue the same animals as in (C) after 24 h of Tm injection (magnification × 400). Immune cell infiltration was indicated with a black arrow. Scale bar = 50 μm. Representative images were shown from five animals per group analyzed.N (Left) TUNEL assay of the same liver as in (M). Scale bar = 40 μm; Zoom box, scale bar = 5 μm. (Right) Quantification of apoptotic cells. 10 random fields were counted for each TUNEL-stained tissue sample. Data information: All data were shown as mean ± SEM from five biological replicates. , *p < 0.05, ***p < 0.001 (two-tailed Student's t-test).


Figure 1Naïve CD4 + T cells were isolated from the spleen and LNs of WT , Stim2fl / fl Cd4Cre ( S2 ) , Orai1fl / fl Cd4Cre ( O1 ) and Stim1fl / fl Cd4Cre ( S1 ) mice and differentiated into Th1 , Th17 and iTreg cells . ( A ) Fura - 2 loaded cells were stimulated with 0 . 3 μM ionomycin to induce SOCE . Representative Ca2 + traces ( left ) and peak Ca2 + levels ( right ) . Data are from the mean ± SEM of 4 mice per group . ( B ) Induction of colitis in Rag1 - / - mice by adoptive transfer of naive CD4 + T cells from wildtype , Stim2fl / fl Cd4Cre , Orai1fl / fl Cd4Cre and Stim1fl / fl Cd4Cre mice . Weight curves of recipient Rag1 - / - mice following injection of naive CD4 + T cells . Data are from two independent experiments with 8 recipient mice per group . ( C ) H & E stains of colon tissues and histological inflammation scores of Rag1 - / - mice 8 weeks after T cell transfer . Data are from one representative experiment with 5 mice per group . ( D - E ) Representative flow cytometry plots and frequencies of donor CD4 + T cells producing IFNγ , IL - 17A , TNF or expressing Foxp3 that were isolated from the mesenteric LNs of recipient Rag1 - / - mice . Data are from 2 independent experiments with 3 - 8 mice per group .

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Figure 1Naïve CD4+ T cells were isolated from the spleen and LNs of WT, Stim2fl/fl Cd4Cre (S2), Orai1fl/fl Cd4Cre (O1) and Stim1fl/fl Cd4Cre (S1) mice and differentiated into Th1, Th17 and iTreg cells. (A) Fura-2 loaded cells were stimulated with 0.3 μM ionomycin to induce SOCE. Representative Ca2+ traces (left) and peak Ca2+ levels (right). Data are from the mean ± SEM of 4 mice per group.(B) Induction of colitis in Rag1-/- mice by adoptive transfer of naive CD4+ T cells from wildtype, Stim2fl/fl Cd4Cre, Orai1fl/fl Cd4Cre and Stim1fl/fl Cd4Cre mice. Weight curves of recipient Rag1-/- mice following injection of naive CD4+ T cells. Data are from two independent experiments with 8 recipient mice per group.(C) H&E stains of colon tissues and histological inflammation scores of Rag1-/- mice 8 weeks after T cell transfer. Data are from one representative experiment with 5 mice per group.(D-E) Representative flow cytometry plots and frequencies of donor CD4+ T cells producing IFNγ, IL-17A, TNF or expressing Foxp3 that were isolated from the mesenteric LNs of recipient Rag1-/- mice. Data are from 2 independent experiments with 3-8 mice per group.


Figure 2 ( A , B ) Ca2 + influx in human lamina propria CD4 + and CD8 + T cells from 3 IBD patient samples analyzed by flow cytometry . ( A ) Experimental design . ( B ) Ca2 + influx rates in T cells pre - incubated for 4 h in the presence of 15 - 1000 nM BTP2 , which was present until data acquisition . SOCE was induced by stimulation with the sarco - endoplasmic reticulum Ca2 + ATPase ( SERCA ) inhibitor thapsigargin ( TG ) in Ca2 + free buffer followed by the addition of 2 mM Ca2 + . Bar graphs show the mean ± SEM of Ca2 + influx rates after Ca2 + readdition from 1 experiment ( n = 3 IBD patients ) . ( D ) viSNE plots of CD45 + CD3 + LPMCs stimulated with 20 ng / ml PMA and 1 µg / ml ionomycin for 4 hours in the presence of 15 - 1000 nM BTP2 . viSNE plots are colored according to the expression level of IL - 2 ( blue : low , red : high ) and are representative of one CD patient . ( E ) Dose - response curves of the frequencies of cytokine producing CD4 + and CD8 + T cells after PMA / ionomycin stimulation for 4h in the presence of increasing doses of BTP2 . The frequencies of cytokine producing cells were normalized to control samples treated with PMA / ionomycin alone . Bar graphs show the mean ± SEM of 5 CD patient samples . ( F ) Heatmaps representing the median fold change of cytokine and cell surface marker expression in CD45 + CD3 + LPMCs stimulated for 4h with PMA / ionomycin in the presence of BTP2 ; data are normalized to PMA / ionomycin treatment alone . ( G ) Cytokine production by CD19 + HLA - DR + B cells and CD11c + dendritic cells in the presence of increasing doses ( 15 - 1 , 000 nM ) of BTP2 . Left : Dose - response curves showing frequencies of cytokine - producing cells after 4h stimulation with PMA / ionomycin ex vivo . Data are normalized to control samples stimulated with PMA / ionomycin in the absence of BTP2 . Error bars represent SEM obtained from 1 experiment ( n = 5 CD patients ) . Right : heatmaps showing the median fold change of cytokines and surface markers in CD19 + HLA - DR + B cells and CD11c + dendritic cells treated as described for the left panel .

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Figure 2(A,B) Ca2+ influx in human lamina propria CD4+ and CD8+ T cells from 3 IBD patient samples analyzed by flow cytometry. (A) Experimental design. (B) Ca2+ influx rates in T cells pre-incubated for 4 h in the presence of 15-1000 nM BTP2, which was present until data acquisition. SOCE was induced by stimulation with the sarco-endoplasmic reticulum Ca2+ ATPase (SERCA) inhibitor thapsigargin (TG) in Ca2+ free buffer followed by the addition of 2 mM Ca2+. Bar graphs show the mean ± SEM of Ca2+ influx rates after Ca2+ readdition from 1 experiment (n=3 IBD patients).(D) viSNE plots of CD45+CD3+ LPMCs stimulated with 20 ng/ml PMA and 1 µg/ml ionomycin for 4 hours in the presence of 15-1000 nM BTP2. viSNE plots are colored according to the expression level of IL-2 (blue: low, red: high) and are representative of one CD patient.(E) Dose-response curves of the frequencies of cytokine producing CD4+ and CD8+ T cells after PMA/ionomycin stimulation for 4h in the presence of increasing doses of BTP2. The frequencies of cytokine producing cells were normalized to control samples treated with PMA/ionomycin alone. Bar graphs show the mean ± SEM of 5 CD patient samples.(F) Heatmaps representing the median fold change of cytokine and cell surface marker expression in CD45+CD3+ LPMCs stimulated for 4h with PMA/ionomycin in the presence of BTP2; data are normalized to PMA/ionomycin treatment alone.(G) Cytokine production by CD19+HLA-DR+ B cells and CD11c+ dendritic cells in the presence of increasing doses (15-1,000 nM) of BTP2. Left: Dose-response curves showing frequencies of cytokine-producing cells after 4h stimulation with PMA/ionomycin ex vivo. Data are normalized to control samples stimulated with PMA/ionomycin in the absence of BTP2. Error bars represent SEM obtained from 1 experiment (n=5 CD patients). Right: heatmaps showing the median fold change of cytokines and surface markers in CD19+HLA-DR+ B cells and CD11c+ dendritic cells treated as described for the left panel.


Figure 3 ( A ) Experimental setup for mass cytometric assays . Non - inflamed : n = 4 , CD : n = 6 , UC : n = 6 . ( B ) FlowSOM plot of merged FCS files from LPMCs of non - inflamed controls , UC or CD patients after stimulation with PMA / ionomycin ± 1 μM BTP2 for 4h ex vivo . Colors of the t - SNE plot represent 20 clusters of distinct CD45 + LPMC lineages . The heatmap shows the expression levels of 21 markers used for defining cell clusters . ( C ) FlowSOM map of merged FCS files from CD45 + cells of non - inflamed controls , UC and CD patients that were left unstimulated or treated with PMA / ionomycin ± 1 μM BTP2 . Colors indicate altered cell clusters in CD patients vs . controls , UC patients vs . controls and CD vs . UC patients . ( D ) viSNE plots of one exemplary non - inflamed control , UC and CD patient colored by marker expression levels ( blue : low , red : high ) . ( E ) Quantified frequencies ( % ) of each cell subset defined by the cluster analysis . Statistical significance was calculated using the edgeR statistical framework with negative binomial GLM and a false discovery rate adjusted to 10 % using the Benjiamini - Hochberg procedure ( * padj < 0 . 05 , * * padj < 0 . 01 , * * * padj < 0 . 001 ) . Boxes extend from the 25th to 75th percentiles . Whisker plots show the min ( smallest ) and max ( largest ) values . The line in the box denotes the median from 1 experiment ( non - inflamed n = 4 , CD : n = 6 , UC : n = 6 patients ) . ( F ) Heatmaps representing the median fold change of cytokine and surface marker expression in CD45 + LPMCs that were isolated from non - inflamed , UC and CD samples and activated for 4h with PMA / ionomycin ex vivo . Statistical significance was calculated using an unpaired t test with FDR adjustment to 1 % using the Benjamini , Krieger and Yekutieli procedure ; *

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Figure 3(A) Experimental setup for mass cytometric assays. Non-inflamed: n = 4, CD: n =6, UC: n =6. (B) FlowSOM plot of merged FCS files from LPMCs of non-inflamed controls, UC or CD patients after stimulation with PMA/ionomycin ± 1 μM BTP2 for 4h ex vivo. Colors of the t-SNE plot represent 20 clusters of distinct CD45+ LPMC lineages. The heatmap shows the expression levels of 21 markers used for defining cell clusters.(C) FlowSOM map of merged FCS files from CD45+ cells of non-inflamed controls, UC and CD patients that were left unstimulated or treated with PMA/ionomycin ± 1 μM BTP2. Colors indicate altered cell clusters in CD patients vs. controls, UC patients vs. controls and CD vs. UC patients. (D) viSNE plots of one exemplary non-inflamed control, UC and CD patient colored by marker expression levels (blue: low, red: high).(E) Quantified frequencies (%) of each cell subset defined by the cluster analysis. Statistical significance was calculated using the edgeR statistical framework with negative binomial GLM and a false discovery rate adjusted to 10% using the Benjiamini-Hochberg procedure (*padj< 0.05, **padj < 0.01, ***padj< 0.001). Boxes extend from the 25th to 75th percentiles. Whisker plots show the min (smallest) and max (largest) values. The line in the box denotes the median from 1 experiment (non-inflamed n = 4, CD: n =6, UC: n =6 patients).(F) Heatmaps representing the median fold change of cytokine and surface marker expression in CD45+ LPMCs that were isolated from non-inflamed, UC and CD samples and activated for 4h with PMA/ionomycin ex vivo. Statistical significance was calculated using an unpaired t test with FDR adjustment to 1% using the Benjamini, Krieger and Yekutieli procedure; *


Figure 4 ( A ) FlowSOM plot of merged FCS files from samples treated with PMA / ionomycin ± 1 µM BTP2 ( non - inflamed : n = 4 , CD : n = 6 , UC : n = 6 ) . Colors of FlowSOM plot indicate 20 distinct clusters of CD45 + CD3 + LPMCs . Heatmaps show the expression levels of 24 markers used for cluster determination . The table shows the mean ± SEM of frequencies ( % ) for each cell subset defined in non - inflamed controls , UC and CD patients after 4h stimulation with PMA / ionomycin in vitro . Statistical significance was calculated using the edgeR statistical framework with negative binomial GLM and a false discovery rate adjusted to 10 % using the Benjiamini - Hochberg procedure . ( B ) Box plots showing frequencies ( % ) of each cell subset defined by the cluster analysis in non - inflamed , UC and CD samples following stimulation with PMA / ionomycin ± BTP2 for 4h in vitro . Statistical significances were calculated using a one - tailed paired Wilcoxon matched - pairs signed - rank test , * p < 0 . 05 . Boxes range from the 25th to 75th percentiles . Whisker plots show the min ( smallest ) and max ( largest ) values . The line in the box denotes the median obtained from 1 experiment ( non - inflamed : n = 4 , CD : n = 6 , UC : n = 6 patients ) . ( C ) viSNE plots of one exemplary CD patient . Identification of cell subsets ( left ) and expression levels of cytokines after PMA / ionomycin stimulation in the presence or absence of BTP2 ( blue ; low , red : high ) . ( D ) Heatmaps indicating the median fold change expression of cytokines and surface molecules in CD45 + CD3 + LPMCs stimulated with PMA / ionomycin ± BTP2 for 4h in vitro . Values are normalized to stimulated , non - BTP2 treated samples .

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Figure 4(A) FlowSOM plot of merged FCS files from samples treated with PMA/ionomycin ± 1 µM BTP2 (non-inflamed: n = 4, CD: n =6, UC: n =6). Colors of FlowSOM plot indicate 20 distinct clusters of CD45+CD3+ LPMCs. Heatmaps show the expression levels of 24 markers used for cluster determination. The table shows the mean ± SEM of frequencies (%) for each cell subset defined in non-inflamed controls, UC and CD patients after 4h stimulation with PMA/ionomycin in vitro. Statistical significance was calculated using the edgeR statistical framework with negative binomial GLM and a false discovery rate adjusted to 10% using the Benjiamini-Hochberg procedure.(B) Box plots showing frequencies (%) of each cell subset defined by the cluster analysis in non-inflamed, UC and CD samples following stimulation with PMA/ionomycin ± BTP2 for 4h in vitro. Statistical significances were calculated using a one-tailed paired Wilcoxon matched-pairs signed-rank test, *p < 0.05. Boxes range from the 25th to 75th percentiles. Whisker plots show the min (smallest) and max (largest) values. The line in the box denotes the median obtained from 1 experiment (non-inflamed: n = 4, CD: n =6, UC: n =6 patients).(C) viSNE plots of one exemplary CD patient. Identification of cell subsets (left) and expression levels of cytokines after PMA/ionomycin stimulation in the presence or absence of BTP2 (blue; low, red: high).(D) Heatmaps indicating the median fold change expression of cytokines and surface molecules in CD45+CD3+ LPMCs stimulated with PMA/ionomycin ± BTP2 for 4h in vitro. Values are normalized to stimulated, non-BTP2 treated samples.


Figure 5 ( A ) FlowSOM plot of merged FCS files from unstimulated samples or samples treated with PMA / ionomycin ± 1 µM BTP2 ( non - inflamed : n = 4 , CD : n = 6 , UC : n = 6 ) . Colors of FlowSOM plot indicate 20 distinct clusters of CD45 + CD3 - LPMCs . Heatmap clusters showing the expression levels of 34 markers used for cluster determination . The table shows the mean ± SEM of frequencies ( % ) for each cell subset defined in non - Inflamed , UC and CD samples after stimulation with PMA / ionomycin for 4h . Statistical significance was calculated using the edgeR statistical framework with negative binomial GLM and a false discovery rate adjusted to 10 % using the Benjiamini - Hochberg procedure ( B ) Box plots show frequencies ( % ) of each cell subset defined by the cluster analysis in samples of non - inflamed controls , UC or CD patients after stimulation with PMA / ionomycin ± BTP2 for 4h ex vivo . Statistical significances were calculated by one - tailed paired Wilcoxon matched - pairs signed - rank test , * p < 0 . 05 . Boxes range from the 25th to 75th percentiles . Whisker plots show the min ( smallest ) and max ( largest ) values . The line in the box denotes the median obtained from 1 experiment ( non - inflamed : n = 4 , CD : n = 6 , UC : n = 6 patients ) . ( C ) viSNE plots of one exemplary CD patient . Identification of cell subsets ( left ) and expression levels of cell markers after PMA / ionomycin stimulation in the presence or absence of BTP2 ( blue ; low , red : high ) . ( D ) Heatmaps indicating the median fold change expression of cytokines and surface markers in CD45 + CD3 - LPMCs activated with PMA / ionomycin ± BTP2 for 4h in vitro . Values are normalized to stimulated , non - BTP2 treated samples .

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Figure 5(A) FlowSOM plot of merged FCS files from unstimulated samples or samples treated with PMA/ionomycin ± 1 µM BTP2 (non-inflamed: n = 4, CD: n =6, UC: n =6). Colors of FlowSOM plot indicate 20 distinct clusters of CD45+CD3- LPMCs. Heatmap clusters showing the expression levels of 34 markers used for cluster determination. The table shows the mean ± SEM of frequencies (%) for each cell subset defined in non-Inflamed, UC and CD samples after stimulation with PMA/ionomycin for 4h. Statistical significance was calculated using the edgeR statistical framework with negative binomial GLM and a false discovery rate adjusted to 10% using the Benjiamini-Hochberg procedure(B) Box plots show frequencies (%) of each cell subset defined by the cluster analysis in samples of non-inflamed controls, UC or CD patients after stimulation with PMA/ionomycin ± BTP2 for 4h ex vivo. Statistical significances were calculated by one-tailed paired Wilcoxon matched-pairs signed-rank test, *p < 0.05. Boxes range from the 25th to 75th percentiles. Whisker plots show the min (smallest) and max (largest) values. The line in the box denotes the median obtained from 1 experiment (non-inflamed: n = 4, CD: n =6, UC: n =6 patients).(C) viSNE plots of one exemplary CD patient. Identification of cell subsets (left) and expression levels of cell markers after PMA/ionomycin stimulation in the presence or absence of BTP2 (blue; low, red: high).(D) Heatmaps indicating the median fold change expression of cytokines and surface markers in CD45+CD3- LPMCs activated with PMA/ionomycin ± BTP2 for 4h in vitro. Values are normalized to stimulated, non-BTP2 treated samples.


Figure 6 ( A - B ) Adult stem cell - derived intestinal epithelial spheroids of wild - type C57BL / 6 mice were cultured for 4 days in Wnt - free medium to induce differentiation . Initial viability was determined on day 0 . DMSO control ( vehicle ) or 1 μM BTP2 were added to the culture medium and viability was measured every 24h by resazurin viability assay and microscopy . Scale bar depicts 100 µm . ( C ) Transepithelial electrical resistance ( TEER ) was measured on differentiated murine colon monolayers in the presence or absence of 1 µM BTP2 . ( D ) Human colonic spheroids were generated from primary human epithelial crypts obtained from colon resectates of 2 CD patients and cultured for 6 days in Wnt - free medium to induce differentiation into colonic organoids . Initial viability was determined on day 0 . DMSO control or 1 μM BTP2 were added to the culture medium and viability was measured on days 2 , 4 and 6 . ( E ) TEER of differentiated human colon monolayers was analyzed in the presence or absence of 1 µM BTP2 . ( F ) Relative mRNA expression of markers for enterocytes ( ALPI ) , goblet cells ( MUC2 ) , and entero - endocrine cells ( CHGA ) was quantified by RT - PCR in differentiated human colon monolayers after addition of 1 µM BTP2 .

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Figure 6(A-B) Adult stem cell-derived intestinal epithelial spheroids of wild-type C57BL/6 mice were cultured for 4 days in Wnt-free medium to induce differentiation. Initial viability was determined on day 0. DMSO control (vehicle) or 1 μM BTP2 were added to the culture medium and viability was measured every 24h by resazurin viability assay and microscopy. Scale bar depicts 100 µm. (C) Transepithelial electrical resistance (TEER) was measured on differentiated murine colon monolayers in the presence or absence of 1 µM BTP2.(D) Human colonic spheroids were generated from primary human epithelial crypts obtained from colon resectates of 2 CD patients and cultured for 6 days in Wnt-free medium to induce differentiation into colonic organoids. Initial viability was determined on day 0. DMSO control or 1 μM BTP2 were added to the culture medium and viability was measured on days 2, 4 and 6.(E) TEER of differentiated human colon monolayers was analyzed in the presence or absence of 1 µM BTP2.(F) Relative mRNA expression of markers for enterocytes (ALPI), goblet cells (MUC2), and entero-endocrine cells (CHGA) was quantified by RT-PCR in differentiated human colon monolayers after addition of 1 µM BTP2.


Figure 7 ( A - C ) Acute administration of the CRAC channel blocker CM4620 inhibits CRAC currents ( Icrac ) . ( A ) Time course of peak amplitudes of ICRAC before and after application of 3 μM CM4620 to HEK293 cells transfected with ORAI1 and STIM1 . ( B ) Current - voltage ( I - V ) relationship of ICRAC before and after CM4620 treatment . I - Vs were obtained at the time points indicated by the arrowheads in ( A ) . ( C ) Fractional blockade of ICRAC by CM4620 was measured by comparing current amplitudes before and after CM4620 administration ( arrowheads ) using the formula 1 - ( ICM4620 / ICtrl ) . Data are from 8 cells per conditions and shown as the mean ± SEM . ( E ) Relative weight loss of mice treated with vehicle or CM4620 ; the start of treatment is indicated by the white arrow . Data are the mean ± SEM of 9 mice per cohort , statistical analysis by unpaired student ' s t - test , * p < 0 . 05 . ( F ) Concentrations of CM4620 in the serum of mice at day 49 , data are the mean ± SEM . Statistical analysis by unpaired student ' s t - test , * * * p < 0 . 001 . ( G ) Representative H & E staining of the distal colon of Rag1 - / - mice treated with vehicle or CM4620 . Colitis scores of 9 mice per cohort . Each symbol represents one mouse . ( H ) Number of CD11b + Gr - 1 + neutrophils and CD4 + T cells ( normalized to grams of tissue ) in the colon lamina propria ( CLP ) of mice on day 49 quantified by flow cytometry . Each dot represents one mouse . Data are the mean ± SEM of 9 mice per cohort . ( I ) Frequencies of RORγt + Th17 cells and Foxp3 + Treg cells in the CLP of mice treated with CM4620 or vehicle . Each dot represents one mouse . Data are the mean ± SEM of 6 control and 5 CM4620 treated mice . ( J ) Frequencies of IFN - γ , TNF - α and IL - 2 producing CD4 + T cells isolated from the CLP , restimulated ex vivo with PMA and ionomycin for 4 h ( without addition of CM4620 during stimulation in vitro ) and analyzed by flow cytometry . Bar graphs represent the percentages of IFN - γ + , TNF - α + , IL - 2 + cells in CD4 + cells ( top row ) and mean fluorescent intensity ( MFI ) of IFN - γ , TNF - α , IL - 2 on CD4 + cells ( bottom row ) . Each dot represents one mouse . Data are the mean ± SEM of 6 control and 5 CM4620 treated mice .

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Figure 7(A-C) Acute administration of the CRAC channel blocker CM4620 inhibits CRAC currents (Icrac). (A) Time course of peak amplitudes of ICRAC before and after application of 3 μM CM4620 to HEK293 cells transfected with ORAI1 and STIM1. (B) Current-voltage (I-V) relationship of ICRAC before and after CM4620 treatment. I-Vs were obtained at the time points indicated by the arrowheads in (A). (C) Fractional blockade of ICRAC by CM4620 was measured by comparing current amplitudes before and after CM4620 administration (arrowheads) using the formula 1- (ICM4620 / ICtrl). Data are from 8 cells per conditions and shown as the mean ± SEM.(E) Relative weight loss of mice treated with vehicle or CM4620; the start of treatment is indicated by the white arrow. Data are the mean ± SEM of 9 mice per cohort, statistical analysis by unpaired student's t-test , *p<0.05.(F) Concentrations of CM4620 in the serum of mice at day 49, data are the mean ± SEM. Statistical analysis by unpaired student's t-test, ***p<0.001.(G) Representative H&E staining of the distal colon of Rag1-/- mice treated with vehicle or CM4620. Colitis scores of 9 mice per cohort. Each symbol represents one mouse.(H) Number of CD11b+Gr-1+ neutrophils and CD4+ T cells (normalized to grams of tissue) in the colon lamina propria (CLP) of mice on day 49 quantified by flow cytometry. Each dot represents one mouse. Data are the mean ± SEM of 9 mice per cohort.(I) Frequencies of RORγt+ Th17 cells and Foxp3+ Treg cells in the CLP of mice treated with CM4620 or vehicle. Each dot represents one mouse. Data are the mean ± SEM of 6 control and 5 CM4620 treated mice.(J) Frequencies of IFN-γ, TNF-α and IL-2 producing CD4+ T cells isolated from the CLP, restimulated ex vivo with PMA and ionomycin for 4 h (without addition of CM4620 during stimulation in vitro) and analyzed by flow cytometry. Bar graphs represent the percentages of IFN-γ+, TNF-α+, IL-2+ cells in CD4+ cells (top row) and mean fluorescent intensity (MFI) of IFN-γ, TNF-α, IL-2 on CD4+ cells (bottom row). Each dot represents one mouse. Data are the mean ± SEM of 6 control and 5 CM4620 treated mice.


Figure 1Neurosphere assays were performed using mouse E14 . 5 primary neural progenitors transduced with a retroviral vector expressing Ttyh1XTT assays were performed using mouse E14 . 5 primary neural progenitors transduced with a retroviral vector expressing Ttyh1Neurosphere assays were performed using mouse E14 . 5 primary neural progenitors transduced with a short hairpin RNA specific to Ttyh1 ( shTtyh1 ) XTT assays were performed using mouse E14 . 5 primary neural progenitors transduced with a short hairpin RNA specific to Ttyh1 ( shTtyh1 ) Ttyh1 - expressing cells in E15 . 5 embryonic brains that were intraventricularly electroporated at E13 . 5 were immunostained using an anti - GFP primary ( reporter gene ) and Alexa Fluor 488 - conjugated secondary antibodies . GFP immunofluorescence ( green ) was merged with DAPI - counterstained images ( blue ) . VZ , ventricular zone ; SVZ , subventricular zone ; IZ , intermediate zone ; CP , cortical plate . Scale bar : 100 μm . Quantification of ( E ) Double - immunolabeling of E15 . 5 brain sections electroporated in utero with Ttyh1 - expressing plasmid at E13 . 5 using anti - GFP ( green ) and Sox2 ( red ) antibodies , and Alexa Fluor 488 - and 555 - conjugated secondary antibodies . Scale bar : 50 μm . Double - immunolabeling of E15 . 5 brain sections electroporated in utero with Ttyh1 - expressing plasmid at E13 . 5 using anti - GFP ( green ) and Sox2 ( red ) antibodies , and Alexa Fluor 488 - and 555 - conjugated secondary antibodies . Scale bar : 50 μm . Quantification of ( G ) ( n = 3 ) . Immunolabeling of E14 . 5 brain sections electroporated in utero with shTtyh1 alone or in combination with a shTtyh1 - resistant Ttyh1 ( Ttyh1res ) expression vector at E13 . 5 using anti - GFP and Alexa Fluor 488 - conjugated secondary antibodies . The DAPI nuclear counterstain is shown in blue . Scale bar : 100 μm . Quantification of ( I )

O B-PANEL_START O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1Neurosphere assays were performed using mouse E14.5 primary neural progenitors transduced with a retroviral vector expressing Ttyh1XTT assays were performed using mouse E14.5 primary neural progenitors transduced with a retroviral vector expressing Ttyh1Neurosphere assays were performed using mouse E14.5 primary neural progenitors transduced with a short hairpin RNA specific to Ttyh1 (shTtyh1)XTT assays were performed using mouse E14.5 primary neural progenitors transduced with a short hairpin RNA specific to Ttyh1 (shTtyh1)Ttyh1-expressing cells in E15.5 embryonic brains that were intraventricularly electroporated at E13.5 were immunostained using an anti-GFP primary (reporter gene) and Alexa Fluor 488-conjugated secondary antibodies. GFP immunofluorescence (green) was merged with DAPI-counterstained images (blue). VZ, ventricular zone; SVZ, subventricular zone; IZ, intermediate zone; CP, cortical plate. Scale bar: 100 μm. Quantification of (E)Double-immunolabeling of E15.5 brain sections electroporated in utero with Ttyh1-expressing plasmid at E13.5 using anti-GFP (green) and Sox2 (red) antibodies, and Alexa Fluor 488- and 555-conjugated secondary antibodies. Scale bar: 50 μm.Double-immunolabeling of E15.5 brain sections electroporated in utero with Ttyh1-expressing plasmid at E13.5 using anti-GFP (green) and Sox2 (red) antibodies, and Alexa Fluor 488- and 555-conjugated secondary antibodies. Scale bar: 50 μm. Quantification of (G) (n = 3).Immunolabeling of E14.5 brain sections electroporated in utero with shTtyh1 alone or in combination with a shTtyh1-resistant Ttyh1 (Ttyh1res) expression vector at E13.5 using anti-GFP and Alexa Fluor 488-conjugated secondary antibodies. The DAPI nuclear counterstain is shown in blue. Scale bar: 100 μm. Quantification of (I)


Figure 2Two days after E14 . 5 primary neural progenitor cells were transduced with retroviral vectors expressing ( A ) Ttyh1 mRNA expression levels of each indicated Notch target gene were measured by qPCRTwo days after E14 . 5 primary neural progenitor cells were transduced with shTtyh1 , mRNA expression levels of each indicated Notch target gene were measured by qPCREffects of dominant negative MAML1 ( dnMAML1 ) on Ttyh1 - enhanced Notch target gene expression were tested by qPCR using E14 . 5 primary neural progenitors at 2 days posttransduction ( n = 4 ) . The dnMAML1 plasmid was introduced with the Ttyh1 vector into embryonic brains at E13 . 5 and cell localization was analyzed at E15 . 5 by anti - GFP immunostaining . GFP and DAPI signals are shown in green and blue , respectively . VZ , ventricular zone ; SVZ , subventricular zone ; IZ , intermediate zone ; CP , cortical plate . Scale bar : 100 μm . E Quantification of GFP + cell positions in ( D ) ( n = 3 ) . The NICD plasmid was electroporated with the Ttyh1 knockdown vector ( shTtyh1 ) into embryonic brains at E13 . 5 and cell localization was analyzed at E14 . 5 by anti - GFP immunostaining . Scale bar : 100 μm . G Quantification of GFP + cell positions in ( F ) ( n = 3 ) .

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Figure 2Two days after E14.5 primary neural progenitor cells were transduced with retroviral vectors expressing (A) Ttyh1 mRNA expression levels of each indicated Notch target gene were measured by qPCRTwo days after E14.5 primary neural progenitor cells were transduced with shTtyh1, mRNA expression levels of each indicated Notch target gene were measured by qPCREffects of dominant negative MAML1 (dnMAML1) on Ttyh1-enhanced Notch target gene expression were tested by qPCR using E14.5 primary neural progenitors at 2 days posttransduction (n = 4).The dnMAML1 plasmid was introduced with the Ttyh1 vector into embryonic brains at E13.5 and cell localization was analyzed at E15.5 by anti-GFP immunostaining. GFP and DAPI signals are shown in green and blue, respectively. VZ, ventricular zone; SVZ, subventricular zone; IZ, intermediate zone; CP, cortical plate. Scale bar: 100 μm. E Quantification of GFP+ cell positions in (D) (n = 3). The NICD plasmid was electroporated with the Ttyh1 knockdown vector (shTtyh1) into embryonic brains at E13.5 and cell localization was analyzed at E14.5 by anti-GFP immunostaining. Scale bar: 100 μm. G Quantification of GFP+ cell positions in (F) (n = 3).


Figure 3Effects of Ttyh1 expression on the generation of NICD at 2 days posttransduction of E14 . 5 neural progenitor cells were analyzed by Western blotting using an anti - NICD antibody . Quantification of band intensitiesEffects of Ttyh1 knockdown ( C ) on the generation of NICD at 2 days posttransduction of E14 . 5 neural progenitor cells were analyzed by Western blotting using an anti - NICD antibody . ( B , D ) Quantification of band intensitiesTtyh1 - transduced E14 . 5 neural progenitors were incubated with DAPT for 2 days , then cells were harvested and lysates were used for luciferase assay ( n = 3 ) . Effects of Ttyh1 - knockdown on γ - secretase activity was also assessed using E14 . 5 neural progenitor cellsTtyh1 - transduced E14 . 5 neural progenitors were incubated for 2 days in the presence or absence of DAPT , and samples were prepared for Western blotting using anti - NICD antibodyTtyh1 - transduced E14 . 5 neural progenitors were incubated for 2 days in the presence or absence of DAPT , and samples were prepared for qPCR for Notch target genes ( I ) The indicated plasmids were electroporated into E13 . 5 brains , and brain samples were harvested at E15 . 5 for anti - GFP immunolabeling . GFP and DAPI signals are shown in green and blue , respectively . Quantification of GFP + cell localization in ( J ) dnPS1 , dominant negative presenilin 1 ; VZ , ventricular zone ; SVZ , subventricular zone ; IZ , intermediate zone ; CP , cortical plate . Scale bar : 100 μm .

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Figure 3Effects of Ttyh1 expression on the generation of NICD at 2 days posttransduction of E14.5 neural progenitor cells were analyzed by Western blotting using an anti-NICD antibody. Quantification of band intensitiesEffects of Ttyh1 knockdown (C) on the generation of NICD at 2 days posttransduction of E14.5 neural progenitor cells were analyzed by Western blotting using an anti-NICD antibody. (B, D) Quantification of band intensitiesTtyh1-transduced E14.5 neural progenitors were incubated with DAPT for 2 days, then cells were harvested and lysates were used for luciferase assay (n = 3).Effects of Ttyh1-knockdown on γ-secretase activity was also assessed using E14.5 neural progenitor cellsTtyh1-transduced E14.5 neural progenitors were incubated for 2 days in the presence or absence of DAPT, and samples were prepared for Western blotting using anti-NICD antibodyTtyh1-transduced E14.5 neural progenitors were incubated for 2 days in the presence or absence of DAPT, and samples were prepared for qPCR for Notch target genes (I)The indicated plasmids were electroporated into E13.5 brains, and brain samples were harvested at E15.5 for anti-GFP immunolabeling. GFP and DAPI signals are shown in green and blue, respectively. Quantification of GFP+ cell localization in (J) dnPS1, dominant negative presenilin 1; VZ, ventricular zone; SVZ, subventricular zone; IZ, intermediate zone; CP, cortical plate. Scale bar: 100 μm.


Figure 4Effects of the ion selectivity - perturbing R371Q mutation in Ttyh1 E14 . 5 neural progenitor cells were transduced with retroviral vectors expressing each indicated gene , and 2 days posttransduction , cells were harvested for qPCR analyses of Notch target mRNAs ( n = 3 ) . Effects of the ion selectivity - perturbing R371Q mutation in other Ttyh family members on Notch - dependent transcription . E14 . 5 neural progenitor cells were transduced with retroviral vectors expressing each indicated gene , and 2 days posttransduction , cells were harvested for qPCR analyses of Notch target mRNAs ( n = 3 ) . Immunolabeling of E15 . 5 brain sections electroporated in utero with indicated plasmids at E13 . 5 using anti - GFP and Alexa Fluor 488 - conjugated secondary antibodies . GFP immunofluorescence ( green ) was merged with DAPI - counterstained images ( blue ) . ( D ) Quantification of GFP + cell localization in ( C ) ( n = 3 ) . VZ , ventricular zone ; SVZ , subventricular zone ; IZ , intermediate zone ; CP , cortical plate . Scale bar : 100 μm . series of Ttyh1 mutants with deletions in the C - terminal cytoplasmic tail region , and ( F ) their effects on cell distribution assessed by intraventricular in utero electroporation at E13 . 5 followed by immunolabeling at E15 . 5 using anti - GFP antibody . Scale bar : 100 μm .

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Figure 4Effects of the ion selectivity-perturbing R371Q mutation in Ttyh1 E14.5 neural progenitor cells were transduced with retroviral vectors expressing each indicated gene, and 2 days posttransduction, cells were harvested for qPCR analyses of Notch target mRNAs (n = 3).Effects of the ion selectivity-perturbing R371Q mutation in other Ttyh family members on Notch-dependent transcription. E14.5 neural progenitor cells were transduced with retroviral vectors expressing each indicated gene, and 2 days posttransduction, cells were harvested for qPCR analyses of Notch target mRNAs (n = 3).Immunolabeling of E15.5 brain sections electroporated in utero with indicated plasmids at E13.5 using anti-GFP and Alexa Fluor 488-conjugated secondary antibodies. GFP immunofluorescence (green) was merged with DAPI-counterstained images (blue). (D) Quantification of GFP+ cell localization in (C) (n = 3). VZ, ventricular zone; SVZ, subventricular zone; IZ, intermediate zone; CP, cortical plate. Scale bar: 100 μm.series of Ttyh1 mutants with deletions in the C-terminal cytoplasmic tail region , and (F) their effects on cell distribution assessed by intraventricular in utero electroporation at E13.5 followed by immunolabeling at E15.5 using anti-GFP antibody. Scale bar: 100 μm.


Figure 5Representative Western blots showing coimmunoprecipitation ( coIP ) of HA - tagged Rer1 with Myc - tagged Ttyh1 using the HEK293T cell line . WCL , whole - cell lysates . Venus fluorescent protein - based bimolecular fluorescence complementation analysis of Ttyh1 and Rer1 interaction ( green ) in living COS - 7 cells by fluorescence microscopy . Ttyh1 and Rer1 were tagged with N - and C - terminal fragments of the Venus fluorescent protein ( VN and VC ) , respectively . DsRed2 - ER was cotransfected to label the endoplasmic reticulum ( ER , red ) . Scale bar : 25 μm . CoIP assays were performed with anti - Ttyh1 and anti - Rer1 antibodies to show endogenous interaction between Ttyh1 and Rer1 in U373MG cells . Western blot analyses of endogenous Rer1 proteins were performed at 2 days posttransduction using E14 . 5 primary neural progenitor cells transduced with retroviral vectors expressing ( D ) Ttyh1 Quantification of relative band intensitiesD - G Western blot analyses of endogenous Rer1 proteins were performed at 2 days posttransduction using E14 . 5 primary neural progenitor cells transduced with retroviral vectors expressing shTtyh1 . Quantification of relative band intensitiesCell extracts from primary neural progenitor cells transduced with Ttyh1 retroviruses in the presence of DMSO vehicle or a proteasome inhibitor lactacystin ( 10 μM ) , were immunoblotted with an antibody to Rer1 . HEK293T cells were transfected with Ttyh1 - Myc and Rer1 - HA expression plasmids , and after 24 h , cells were treated with cycloheximide ( CHX ) ( 100 μg / ml ) and then harvested at the indicated time intervals . Cell lysates were analyzed by Western blotting using anti - HA antibody . ( J ) Quantification of band intensities in ( I ) ( n = 3 ) . γ - secretase - dependent luciferase activity was measured using primary neural progenitor cells transduced with Ttyh1 with or without Rer1 retroviral vectors at 2 days posttransduction ( n = 3 ) . Effects of Rer1 expression on Ttyh1 activity in vivo were examined by in utero electroporation into E13 . 5 brains and subsequent GFP immunofluorescence of E15 . 5 brain sections . GFP and DAPI signals are shown in green and blue , respectively . ( M ) Distribution of GFP + cells in each brain layer in ( L ) was quantified ( n = 3 ) . VZ , ventricular zone ; SVZ , subventricular zone ; IZ , intermediate zone ; CP , cortical plate . Scale bar : 100 μm .

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Figure 5Representative Western blots showing coimmunoprecipitation (coIP) of HA-tagged Rer1 with Myc-tagged Ttyh1 using the HEK293T cell line. WCL, whole-cell lysates.Venus fluorescent protein-based bimolecular fluorescence complementation analysis of Ttyh1 and Rer1 interaction (green) in living COS-7 cells by fluorescence microscopy. Ttyh1 and Rer1 were tagged with N- and C-terminal fragments of the Venus fluorescent protein (VN and VC), respectively. DsRed2-ER was cotransfected to label the endoplasmic reticulum (ER, red). Scale bar: 25 μm.CoIP assays were performed with anti-Ttyh1 and anti-Rer1 antibodies to show endogenous interaction between Ttyh1 and Rer1 in U373MG cells.Western blot analyses of endogenous Rer1 proteins were performed at 2 days posttransduction using E14.5 primary neural progenitor cells transduced with retroviral vectors expressing (D) Ttyh1 Quantification of relative band intensitiesD-G Western blot analyses of endogenous Rer1 proteins were performed at 2 days posttransduction using E14.5 primary neural progenitor cells transduced with retroviral vectors expressing shTtyh1. Quantification of relative band intensitiesCell extracts from primary neural progenitor cells transduced with Ttyh1 retroviruses in the presence of DMSO vehicle or a proteasome inhibitor lactacystin (10 μM), were immunoblotted with an antibody to Rer1.HEK293T cells were transfected with Ttyh1-Myc and Rer1-HA expression plasmids, and after 24 h, cells were treated with cycloheximide (CHX) (100 μg/ml) and then harvested at the indicated time intervals. Cell lysates were analyzed by Western blotting using anti-HA antibody. (J) Quantification of band intensities in (I) (n = 3).γ-secretase-dependent luciferase activity was measured using primary neural progenitor cells transduced with Ttyh1 with or without Rer1 retroviral vectors at 2 days posttransduction (n = 3).Effects of Rer1 expression on Ttyh1 activity in vivo were examined by in utero electroporation into E13.5 brains and subsequent GFP immunofluorescence of E15.5 brain sections. GFP and DAPI signals are shown in green and blue, respectively. (M) Distribution of GFP+ cells in each brain layer in (L) was quantified (n = 3). VZ, ventricular zone; SVZ, subventricular zone; IZ, intermediate zone; CP, cortical plate. Scale bar: 100 μm.


Figure 1 ( A ) Breast cancer cell lines were sorted using CD44 for luminal MCF - 7 cells ( left panel ) or EpCAM for MDA - MB - 231 cells ( right panel ) . Serial mammosphere assays were performed using 4000 cells for each cell fraction , counting spheres after 3 weeks . Plots show the mean ± SEM of mammospheres formed with 4000 cells from 3 independent experiments performed in triplicate . ( B ) Extreme limiting dilution assay ( ELDA ) of MCF - 7 cells . Sorted cells were serially diluted ( 1250 , 625 , 312 , 156 and 78 cells ) and then seeded in 96 - well plates . After one week , the cells were fixed , stained and counted . The lower limit for well repopulation in CD44 + cells was 312 cells , compared with 615 cells for both unsorted and CD44 - populations . The data obtained were evaluated using the ELDA software . p values that test the single - hit hypothesis using the likelihood ratio test are shown . The curves are depicted in Fig EV1 ( C ) Relative expression of the stemness factors SOX2 and OCT4 and the EMT markers SNAIL , Vimentin and TWIST in MCF - 7 / CD44 + cells . The fold change was calculated using GAPDH as an internal control using the 2 - ΔΔCT method comparing stem or non - stem cells versus the total cell line . The box and whiskers graph shows the range , median and quartile interval values from 3 independent experiments . ( D ) miR - 10b overexpression affecting stemness and EMT factors . Relative quantification of miR - 10b ( relative to RNU6B ) , HOXD10 and RHOC ( relative to GAPDH ) was performed in MCF - 7 / CD44 + fractions compared to non - enriched cells . Box plots show the range , median and quartile interval values from 3 independent experiments . A two - tailed Student ' s t test was used to compare the two groups ( p < 0 . 05 ) . SEM , standard error of the mean ; TLDA , TaqMan Low Density Arrays ; and EMT , epithelial - mesenchymal transition . ( E ) Relative expression of stemness factors SOX2 and OCT4 and EMT markers SNAIL and Vimentin in MDA - MB - 231 / EpCAM + cells . The fold change was calculated using GAPDH as an internal control using the 2 - ΔΔCT method comparing stem or non - stem cells versus the total cells . The box and whiskers graph shows the range , median and quartile interval values from 3 independent experiments . ( F ) Extreme Limiting Dilution Assay ( ELDA ) of MDA - MB - 231 cells . Sorted cells were serially diluted ( 1250 , 625 , 312 , 156 and 78 cells ) and then seeded in 96 - well plates . After one week , the cells were fixed , stained and counted . The lower limit for well repopulation in EpCAM + cells was 156 cells compared with 625 cells for both unsorted and EpCAM - populations . The data obtained were evaluated using the ELDA software . p values that test the single - hit hypothesis using the likelihood ratio test are shown . The curves are depicted in Fig EV1 ( G ) Nine miRNAs are commonly regulated in both CD44 + and EpCam + breast cancer cells . Quantitative RT - PCR of miRNAs was performed on sorted MCF - 7 and MDA - MB - 231 cell populations using TaqMan Low Density Arrays ( TLDA ) . Results were plotted as a heat map comparing CD44 + to CD44 - cells ( MCF - 7 ) and EpCam + to EpCam - cells . The scale is shown below the map .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1(A) Breast cancer cell lines were sorted using CD44 for luminal MCF-7 cells (left panel) or EpCAM for MDA-MB-231 cells (right panel). Serial mammosphere assays were performed using 4000 cells for each cell fraction, counting spheres after 3 weeks. Plots show the mean ± SEM of mammospheres formed with 4000 cells from 3 independent experiments performed in triplicate.(B) Extreme limiting dilution assay (ELDA) of MCF-7 cells. Sorted cells were serially diluted (1250, 625, 312, 156 and 78 cells) and then seeded in 96-well plates. After one week, the cells were fixed, stained and counted. The lower limit for well repopulation in CD44+ cells was 312 cells, compared with 615 cells for both unsorted and CD44- populations. The data obtained were evaluated using the ELDA software. p values that test the single-hit hypothesis using the likelihood ratio test are shown. The curves are depicted in Fig EV1(C) Relative expression of the stemness factors SOX2 and OCT4 and the EMT markers SNAIL, Vimentin and TWIST in MCF-7/CD44+ cells. The fold change was calculated using GAPDH as an internal control using the 2-ΔΔCT method comparing stem or non-stem cells versus the total cell line. The box and whiskers graph shows the range, median and quartile interval values from 3 independent experiments.(D) miR-10b overexpression affecting stemness and EMT factors. Relative quantification of miR-10b (relative to RNU6B), HOXD10 and RHOC (relative to GAPDH) was performed in MCF-7/CD44+ fractions compared to non-enriched cells. Box plots show the range, median and quartile interval values from 3 independent experiments. A two-tailed Student's t test was used to compare the two groups (p < 0.05). SEM, standard error of the mean; TLDA, TaqMan Low Density Arrays; and EMT, epithelial-mesenchymal transition.(E) Relative expression of stemness factors SOX2 and OCT4 and EMT markers SNAIL and Vimentin in MDA-MB-231/EpCAM+ cells. The fold change was calculated using GAPDH as an internal control using the 2-ΔΔCT method comparing stem or non-stem cells versus the total cells. The box and whiskers graph shows the range, median and quartile interval values from 3 independent experiments.(F) Extreme Limiting Dilution Assay (ELDA) of MDA-MB-231 cells. Sorted cells were serially diluted (1250, 625, 312, 156 and 78 cells) and then seeded in 96-well plates. After one week, the cells were fixed, stained and counted. The lower limit for well repopulation in EpCAM+ cells was 156 cells compared with 625 cells for both unsorted and EpCAM- populations. The data obtained were evaluated using the ELDA software. p values that test the single-hit hypothesis using the likelihood ratio test are shown. The curves are depicted in Fig EV1(G) Nine miRNAs are commonly regulated in both CD44+ and EpCam+ breast cancer cells. Quantitative RT-PCR of miRNAs was performed on sorted MCF-7 and MDA-MB-231 cell populations using TaqMan Low Density Arrays (TLDA). Results were plotted as a heat map comparing CD44+ to CD44- cells (MCF-7) and EpCam+ to EpCam- cells. The scale is shown below the map.