A10 cells were transfected with Flag - FMRP then treated with either cycloheximide or its solvent ( DMSO ) for 4 hr prior to fixation . β - catenin ( green ) , Flag ( FMRP , red ) were visualized by immunofluorescence . Nucleus ( blue ) was identified by DNA staining with Hoechst 33342 . Arrows indicate a loss of β - catenin signal from the perinuclear region that was seen in the control following cycloheximide

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

A10 cells were transfected with Flag-FMRP then treated with either cycloheximide or its solvent (DMSO) for 4 hr prior to fixation. β-catenin (green), Flag (FMRP, red) were visualized by immunofluorescence. Nucleus (blue) was identified by DNA staining with Hoechst 33342. Arrows indicate a loss of β-catenin signal from the perinuclear region that was seen in the control following cycloheximide treatment


A10 cells were transfected with Flag - FMRP as above and then pre - treated with rapamycin or solvent ( DMSO ) for 1 hr in serum - free media . Serum was added for 2 hr before the cells were harvested and subjected to coIP analysis using β - catenin antibody . Non - programmed rabbit IgG was used as a control . Existence of Flag - FMRP in the immunocomplex was detected by Flag antibody and β - catenin by GFP

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

A10 cells were transfected with Flag-FMRP as above and then pre-treated with rapamycin or solvent (DMSO) for 1 hr in serum-free media. Serum was added for 2 hr before the cells were harvested and subjected to coIP analysis using β-catenin antibody. Non-programmed rabbit IgG was used as a control. Existence of Flag-FMRP in the immunocomplex was detected by Flag antibody and β-catenin by GFP antibody


A10 cells were transfected with Flag - FMRP followed by rapamycin treatment similar to ( D ) . Cells were fixed and stained for β - catenin ( green ) , Flag ( FMRP , red ) , and nucleus ( blue ) . A loss of β - catenin signal from the perinuclear region that was seen in the control is indicated by the arrows .

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

A10 cells were transfected with Flag-FMRP followed by rapamycin treatment similar to (D). Cells were fixed and stained for β-catenin (green), Flag (FMRP, red), and nucleus (blue). A loss of β-catenin signal from the perinuclear region that was seen in the control is indicated by the arrows.


( Top panel ) Primary VSMCs were serum deprived overnight prior to stimulation with Wnt - 3a ( 100 ng / mL ) or its solvent ( 0 . 1 % BSA in PBS ) for 4 hr . The cells were treated with 0 . 5 µM puromycin for 15 min and harvested for Western blot analysis . Total β - catenin levels are indicated , and actin was used as a loading control . The average puromycin / actin ratio ( n = 3 ) was graphed . Error bars indicate standard deviation . An independent samples t - test was performed ( 2 - tailed ) , with the p - value indicated above the error bar . ( Bottom panel ) Cells were treated similarly to above and fixed and stained for β - catenin ( green ) and nucleus ( blue ) The experiment in ( A ) was repeated using A10

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

(Top panel) Primary VSMCs were serum deprived overnight prior to stimulation with Wnt-3a (100 ng/mL) or its solvent (0.1% BSA in PBS) for 4 hr. The cells were treated with 0.5 µM puromycin for 15 min and harvested for Western blot analysis. Total β-catenin levels are indicated, and actin was used as a loading control. The average puromycin/actin ratio (n=3) was graphed. Error bars indicate standard deviation. An independent samples t-test was performed (2-tailed), with the p-value indicated above the error bar. (Bottom panel) Cells were treated similarly to above and fixed and stained for β-catenin (green) and nucleus (blue) The experiment in (A) was repeated using A10 cells


( i ) HEK 293T cells were serum deprived for 1 hr and then stimulated with Wnt - 3a ( 100 ng / mL ) or its solvent ( 0 . 1 % BSA in PBS ) for 4 hr . The cells were harvested and subjected to an endogenous coIP analysis using either β - catenin or FMRP antibody . The corresponding proteins in the precipitated immunocomplex were detected by Western blot analysis . Non - programed rabbit IgG was used as a control . Efficacy of Wnt - 3a treatment was assessed by inhibition of GSK3 mediated phosphorylation of β - catenin ( S33 / 37 / T41 ) by Wnt - 3a . ( ii ) HEK 293T cells were serum deprived for 1 hr and then stimulated with Wnt - 3a ( 100 ng / mL ) or its solvent ( 0 . 1 % BSA in PBS ) for 4 hr . Cell lysates were then subjected to m7GTP - agarose pull - down analysis . eIF4E and tubulin were used as positive and negative controls , respectively , and phospho - β - catenin ( S33 / 37 / T41 ) Western blots were used as controls for Wnt - 3a stimulation .

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

(i) HEK 293T cells were serum deprived for 1 hr and then stimulated with Wnt-3a (100 ng/mL) or its solvent (0.1% BSA in PBS) for 4 hr. The cells were harvested and subjected to an endogenous coIP analysis using either β-catenin or FMRP antibody. The corresponding proteins in the precipitated immunocomplex were detected by Western blot analysis. Non-programed rabbit IgG was used as a control. Efficacy of Wnt-3a treatment was assessed by inhibition of GSK3 mediated phosphorylation of β-catenin (S33/37/T41) by Wnt-3a. (ii) HEK 293T cells were serum deprived for 1 hr and then stimulated with Wnt-3a (100 ng/mL) or its solvent (0.1% BSA in PBS) for 4 hr. Cell lysates were then subjected to m7GTP-agarose pull-down analysis. eIF4E and tubulin were used as positive and negative controls, respectively, and phospho-β-catenin (S33/37/T41) Western blots were used as controls for Wnt-3a stimulation.


A mRNA expression in FACS - isolated Lin ( Ter119 / CD31 / CD45 ) - Sca1 + progenitor cells from female mouse subcutaneous fat , differentiated in the presence of 1 µM cPGI2 or vehicle for the indicated time , as determined by expression profiling ( n = 3 , E - MTAB - 3693 ) * * * * P = 3 * 10 - 6 ( Day 2 ) , * * * * P = 4 * 10 - 7 ( Day 4 ) , * * * * P = 1 * 10 - 6 ( Day 6 ) in 2x2 ANOVA with Bonferroni posttests ( cPGI2 vs . vehicle )

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

A mRNA expression in FACS-isolated Lin(Ter119/CD31/CD45)-Sca1+ progenitor cells from female mouse subcutaneous fat, differentiated in the presence of 1 µM cPGI2 or vehicle for the indicated time, as determined by expression profiling (n=3, E-MTAB-3693) ****P=3*10-6 (Day 2), ****P=4*10-7 (Day 4), ****P=1*10-6 (Day 6) in 2x2 ANOVA with Bonferroni posttests (cPGI2 vs. vehicle)


B mRNA expression in MACS - isolated Lin - Sca1 + progenitor cells from female mouse subcutaneous fat , differentiated in the presence of 100 nM Rosi or vehicle for the indicated time , as determined by qRT - PCR ( n = 4 ) * * * * P = 1 * 10 - 10 ( Day 1 and 2 ) , * * P = 0 . 001 , in 2x2 ANOVA with Bonferroni posttests ( Rosi vs . vehicle )

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

B mRNA expression in MACS-isolated Lin-Sca1+ progenitor cells from female mouse subcutaneous fat, differentiated in the presence of 100 nM Rosi or vehicle for the indicated time, as determined by qRT-PCR (n=4) ****P=1*10-10 (Day 1 and 2), **P=0.001, in 2x2 ANOVA with Bonferroni posttests (Rosi vs. vehicle)


C mRNA expression in female Lin - Sca1 + cells , differentiated in the presence of 100 nM Rosi or vehicle for 8 days , as determined by qRT - PCR ( n = 3 ) t - test Cited4 - / - vs . Cited4 + / + ( Rosi ) * P = 0 . 013 ( Ucp1 ) , * * P = 0 . 004 ( Cpt1b ) , * P = 0 . 026 ( Dio2 )

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

C mRNA expression in female Lin-Sca1+ cells, differentiated in the presence of 100 nM Rosi or vehicle for 8 days, as determined by qRT-PCR (n=3) t-test Cited4-/- vs. Cited4+/+ (Rosi) *P=0.013 (Ucp1), **P=0.004 (Cpt1b), *P=0.026 (Dio2)


D - F mRNA expression in primary SVF cells from human subcutaneous fat , differentiated in the presence of 100 nM Rosi ( D ) or vehicle ( D - F ) , as determined by qRT - PCR at the indicated time points ( n = 5 patients ) . ♀ / ♂ represent individual data ( D ) * * * * P = 3 * 10 - 5 ( Day 2 ) , * * * * P = 3 * 10 - 6 ( Day 6 ) , * * * * P = 4 * 10 - 9 ( Day 10 ) , * * * * P = 3 * 10 - 7 ( Day 14 ) , in 2x2 ANOVA with Bonferroni posttests ( Rosi vs . vehicle ) . ( E , F ) Pearson correlation coefficient ( r ) and P - value are

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

D-F mRNA expression in primary SVF cells from human subcutaneous fat, differentiated in the presence of 100 nM Rosi (D) or vehicle (D-F), as determined by qRT-PCR at the indicated time points (n=5 patients). ♀/♂ represent individual data (D) ****P=3*10-5 (Day 2), ****P=3*10-6 (Day 6), ****P=4*10-9 (Day 10), ****P=3*10-7 (Day 14), in 2x2 ANOVA with Bonferroni posttests (Rosi vs. vehicle). (E, F) Pearson correlation coefficient (r) and P-value are shown


G mRNA expression in primary SVF cells from human female subcutaneous fat transfected with the indicated siRNA prior to differentiation in the presence of 100 nM Rosi for 9 days , as determined by qRT - PCR ( n = 3 ) * * * P = 0 . 0002 ( CITED4 ) , * * P = 0 . 002 ( UCP1 ) , * P = 0 . 02 ( UCP1 ) , * P = 0 . 035 / 0 . 026 ( PPARG ) , * * * P = 0 . 0006 ( SLC2A4 ) , * * P = 0 . 002 ( ADIPOQ ) in one - way ANOVA with Tukey posttests ( vs . siCtrl )

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

G mRNA expression in primary SVF cells from human female subcutaneous fat transfected with the indicated siRNA prior to differentiation in the presence of 100 nM Rosi for 9 days, as determined by qRT-PCR (n=3) ***P=0.0002 (CITED4), **P=0.002 (UCP1), *P=0.02 (UCP1), *P=0.035/0.026 (PPARG), ***P=0.0006 (SLC2A4), **P=0.002 (ADIPOQ) in one-way ANOVA with Tukey posttests (vs. siCtrl)


A - C Quantitative fluorescence microscopy of LipidTOX - and DAPI - stained female Cited4F / F Lin - Sca1 + progenitor cells transfected with Cre or control mRNA prior to differentiation in the presence of 100 nM Rosi for 8 days ( n = 5 ) . * * P = 0 . 002 in t - test ( Cre vs . Ctrl ) . Scale bar is 10 µm

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A-C Quantitative fluorescence microscopy of LipidTOX- and DAPI-stained female Cited4F/F Lin-Sca1+ progenitor cells transfected with Cre or control mRNA prior to differentiation in the presence of 100 nM Rosi for 8 days (n=5). **P=0.002 in t-test (Cre vs. Ctrl). Scale bar is 10 µm


D , E Ucp1 expression in female Cited4F / F Lin - Sca1 + progenitor cells transfected with Cre or control mRNA prior to differentiation in the presence of 100 nM Rosi for 8 days , as determined by Western blot with VCP as loading control ( n = 3 )

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

D, E Ucp1 expression in female Cited4F/F Lin-Sca1+ progenitor cells transfected with Cre or control mRNA prior to differentiation in the presence of 100 nM Rosi for 8 days, as determined by Western blot with VCP as loading control (n=3)


F Cellular respiration in female Lin - Sca1 + progenitor cells differentiated in the presence of 100 nM Rosi for 8 days . The extracellular oxygen consumption rate ( OCR ) was determined upon injection of the indicated substances and normalized to DNA content . CL : CL - 316243

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

F Cellular respiration in female Lin-Sca1+ progenitor cells differentiated in the presence of 100 nM Rosi for 8 days. The extracellular oxygen consumption rate (OCR) was determined upon injection of the indicated substances and normalized to DNA content. CL: CL-316243


A , B mRNA expression in scWAT of mice fed a diet with 0 . 0075 % Rosi or control diet for 2 . 5 weeks , determined by qRT - PCR t - test Cited4 - / - vs . Cited4 + / + ( Rosi ) , ( A ) n = 5 / 5 / 6 / 6 , * * P = 0 . 003 ( Ucp1 ) , * * P = 0 . 008 ( Cidea ) , * * P = 0 . 007 ( Cyc1 ) , * P = 0 . 048 ( Ndufb3 ) , ( B ) n = 5 / 4 / 5 / 5 , * P = 0 . 022 ( Ucp1 ) , * P = 0 . 037 ( Cidea )

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

A, B mRNA expression in scWAT of mice fed a diet with 0.0075% Rosi or control diet for 2.5 weeks, determined by qRT-PCR t-test Cited4-/- vs. Cited4+/+ (Rosi), (A) n=5/5/6/6, **P=0.003 (Ucp1), **P=0.008 (Cidea), **P=0.007 (Cyc1), *P=0.048 (Ndufb3), (B) n=5/4/5/5, *P=0.022 (Ucp1), *P=0.037 (Cidea)


C , D Ucp1 expression in scWAT of mice treated as in A , as determined by Western blot with VCP as loading control t - test Cited4 - / - vs . Cited4 + / + ( Rosi ) , females : n = 5 / 5 / 6 / 6 , * P = 0 . 011 , males : n = 5 / 4 / 5 /

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

C, D Ucp1 expression in scWAT of mice treated as in A, as determined by Western blot with VCP as loading control t-test Cited4-/- vs. Cited4+/+ (Rosi), females: n=5/5/6/6, *P=0.011, males: n=5/4/5/5


E mRNA expression in scWAT of female mice treated with CL - 316 , 243 ( CL ) ( 1 mg / kg / day via Alzet minipumps ) or vehicle for 10 days ( n = 5 / 7 / 13 , t - test Cited4 - / - vs . Cited4 + / + ( CL ) )

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

E mRNA expression in scWAT of female mice treated with CL-316,243 (CL) (1 mg/kg/day via Alzet minipumps) or vehicle for 10 days (n=5/7/13, t-test Cited4-/- vs. Cited4+/+ (CL))


F mRNA expression in scWAT of female mice exposed to 5 ° C or 23 ° C for 2 weeks ( n = 5 / 10 / 6 , t - test Cited4 - / - vs . Cited4 + / + ( 5 ° C ) )

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

F mRNA expression in scWAT of female mice exposed to 5°C or 23°C for 2 weeks (n=5/10/6, t-test Cited4-/- vs. Cited4+/+ (5°C))


A Oxygen consumption rate of female mice fed a diet with 0 . 0075 % Rosi for 2 . 5 weeks , determined by indirect calorimetry and normalized to body weight ( n = 9 / 10 ) . Grey bars represent the dark phase

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

A Oxygen consumption rate of female mice fed a diet with 0.0075% Rosi for 2.5 weeks, determined by indirect calorimetry and normalized to body weight (n=9/10). Grey bars represent the dark phase


B Oxygen consumption rate of female mice shown in ( A ) adjusted for body weight by ANCOVA . 3 - day averages of VO2 were calculated for each mouse ( n = 9 / 10 ) . * P = 0 . 025 in ANCOVA with Bonferroni posttest ( Cited4 - / - vs . Cited4 + / + )

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

B Oxygen consumption rate of female mice shown in (A) adjusted for body weight by ANCOVA. 3-day averages of VO2 were calculated for each mouse (n=9/10). *P=0.025 in ANCOVA with Bonferroni posttest (Cited4-/- vs. Cited4+/+)


C Oxygen consumption rate of female mice fed a diet with 0 . 0075 % Rosi for 3 . 5 weeks and injected with 1 mg / kg CL - 316 , 243 ( CL ) at the indicated time point , as determined by indirect calorimetry ( n = 9 / 10 ) . * P = 0 . 013 ( - 1 h ) , * P = 0 . 045 ( 3 h ) , * P = 0 . 015 ( 5 h ) , * P = 0 . 016 ( 6 h ) , * P = 0 . 019 ( 8 h ) , * P = 0 . 046 ( 10 h ) in repeated measures 2x2 ANOVA with Holm - Sidak posttests ( Cited4 - / - vs . Cited4 + / + )

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

C Oxygen consumption rate of female mice fed a diet with 0.0075% Rosi for 3.5 weeks and injected with 1 mg/kg CL-316,243 (CL) at the indicated time point, as determined by indirect calorimetry (n=9/10). *P=0.013 (-1 h), *P=0.045 (3 h), *P=0.015 (5 h), *P=0.016 (6 h), *P=0.019 (8 h), *P=0.046 (10 h) in repeated measures 2x2 ANOVA with Holm-Sidak posttests (Cited4-/- vs. Cited4+/+)


D Maximal CL - induced oxygen consumption rate of female mice shown in ( C ) adjusted for body weight by ANCOVA . Averages of VO2 throughout 3 - 8 hours post CL injection were calculated for each mouse ( n = 9 / 10 ) . * P = 0 . 049 in ANCOVA with Bonferroni posttest ( Cited4 - / - vs . Cited4 + / + )

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

D Maximal CL-induced oxygen consumption rate of female mice shown in (C) adjusted for body weight by ANCOVA. Averages of VO2 throughout 3-8 hours post CL injection were calculated for each mouse (n=9/10). *P=0.049 in ANCOVA with Bonferroni posttest (Cited4-/- vs. Cited4+/+)


E Oxygen consumption rate of male mice treated as in ( A ) , determined as in ( C ) ( n = 9 / 10 ) . ANCOVA with Bonferroni posttest ( Cited4 - / - vs . Cited4 + / + )

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

E Oxygen consumption rate of male mice treated as in (A), determined as in (C) (n=9/10). ANCOVA with Bonferroni posttest (Cited4-/- vs. Cited4+/+)


F Oxygen consumption rate of male mice treated as in ( C ) , determined as in ( C ) ( n = 9 / 10 ) . * P = 0 . 044 ( 9 h ) , * P = 0 . 013 ( 12 h ) in repeated measures 2x2 ANOVA with Holm - Sidak posttests ( Cited4 - / - vs . Cited4 + / + )

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

F Oxygen consumption rate of male mice treated as in (C), determined as in (C) (n=9/10). *P=0.044 (9 h), *P=0.013 (12 h) in repeated measures 2x2 ANOVA with Holm-Sidak posttests (Cited4-/- vs. Cited4+/+)


G Maximal CL - induced oxygen consumption rate of male mice shown in ( F ) and calculated as in ( D ) ( n = 9 / 10 ) . ANCOVA with Bonferroni posttest ( Cited4 - / - vs . Cited4 + / + )

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

G Maximal CL-induced oxygen consumption rate of male mice shown in (F) and calculated as in (D) (n=9/10). ANCOVA with Bonferroni posttest (Cited4-/- vs. Cited4+/+)


A , B mRNA expression in scWAT of mice fed a high - fat diet ( HFD ) for 11 weeks followed by 5 weeks of HFD with 0 . 0075 % Rosi ( qRT - PCR ) . t - test Cited4 - / - vs . Cited4 + / + , ( A ) n = 8 / 7 , * P = 0 . 029 ( Ucp1 ) , * * P = 0 . 009 ( Elovl3 ) , * * P = 0 . 006 ( Cpt1b ) , * * P = 0 . 004 ( Cyc1 ) , * P = 0 . 016 ( Ndufb3 ) , * * P = 0 . 003 ( Cd36 ) , * * P = 0 . 009 ( Gyk ) , ( B ) n = 7 / 8 , * P = 0 . 026 ( Pck1 )

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A, B mRNA expression in scWAT of mice fed a high-fat diet (HFD) for 11 weeks followed by 5 weeks of HFD with 0.0075% Rosi (qRT-PCR). t-test Cited4-/- vs. Cited4+/+, (A) n=8/7, *P=0.029 (Ucp1), **P=0.009 (Elovl3), **P=0.006 (Cpt1b), **P=0.004 (Cyc1), *P=0.016 (Ndufb3), **P=0.003 (Cd36), **P=0.009 (Gyk), (B) n=7/8, *P=0.026 (Pck1)


C , D Ucp1 protein expression in scWAT of mice treated as in ( A ) , determined by Western blot with VCP as loading control ( n = 8 / 7 for females and n = 7 / 8 for males ) . Arrow indicates the band specific to Ucp1 ( see Appendix Fig 4F )

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C, D Ucp1 protein expression in scWAT of mice treated as in (A), determined by Western blot with VCP as loading control (n=8/7 for females and n=7/8 for males). Arrow indicates the band specific to Ucp1 (see Appendix Fig 4F)


A Blood glucose during glucose tolerance test ( GTT ) on female mice after 11 weeks of HFD and 3 weeks of HFD + Rosi ( n = 8 ) . * * * P = 0 . 001 , in repeated measures 2x2 ANOVA with Holm - Sidak posttests ( Cited4 - / - vs . Cited4 + / + )

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A Blood glucose during glucose tolerance test (GTT) on female mice after 11 weeks of HFD and 3 weeks of HFD+Rosi (n=8). ***P=0.001, in repeated measures 2x2 ANOVA with Holm-Sidak posttests (Cited4-/- vs. Cited4+/+)


B Fasting serum insulin in female mice after 10 weeks of HFD ( " Before Rosi " ) or 11 weeks of HFD and 2 . 5 weeks of HFD + Rosi ( " After Rosi " ) ( n = 8 ) . * * * P = 0 . 0009 in repeated measures 2x2 ANOVA with Holm - Sidak posttests ( After Rosi vs . Before Rosi ) ; P = 0 . 725 ( Cited4 - / - vs . Cited4 + / + Before Rosi )

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B Fasting serum insulin in female mice after 10 weeks of HFD ("Before Rosi") or 11 weeks of HFD and 2.5 weeks of HFD+Rosi ("After Rosi") (n=8). ***P=0.0009 in repeated measures 2x2 ANOVA with Holm-Sidak posttests (After Rosi vs. Before Rosi); P=0.725 (Cited4-/- vs. Cited4+/+ Before Rosi)


C Blood glucose during GTT on male mice as in ( A ) ( n = 5 / 8 ) . Repeated measures 2x2 ANOVA with Holm - Sidak posttests ( Cited4 - / - vs . Cited4 + / + )

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C Blood glucose during GTT on male mice as in (A) (n=5/8). Repeated measures 2x2 ANOVA with Holm-Sidak posttests (Cited4-/- vs. Cited4+/+)


D Fasting serum insulin in male mice as in ( C ) ( n = 7 / 7 / 8 / 8 ) . * * * P = 0 . 001 ( Cited4 + / + ) , * * * P = 0 . 0008 ( Cited4 - / - ) in repeated measures 2x2 ANOVA with Holm - Sidak posttests ( After Rosi vs . Before Rosi )

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D Fasting serum insulin in male mice as in (C) (n=7/7/8/8). ***P=0.001 (Cited4+/+), ***P=0.0008 (Cited4-/-) in repeated measures 2x2 ANOVA with Holm-Sidak posttests (After Rosi vs. Before Rosi)


E , F Blood glucose during insulin tolerance test ( ITT ) with 1 U insulin per kg body mass on female mice after 12 weeks of HFD ( n = 8 ) ( E ) or 16 weeks of HFD and 4 weeks of HFD + Rosi ( n = 7 ) ( F ) , expressed as % of the 0 - time point value . * * P = 0 . 003 in repeated measures 2x2 ANOVA with Holm - Sidak posttests ( Cited4 - / - vs . Cited4 + / + ) . G Area under the curve ( AUC ) of blood glucose during the ITT ( 0 - 120 min ) in ( E , F ) . 2x2 ANOVA with Holm - Sidak posttests ( After Rosi vs . Before Rosi )

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E, F Blood glucose during insulin tolerance test (ITT) with 1 U insulin per kg body mass on female mice after 12 weeks of HFD (n=8) (E) or 16 weeks of HFD and 4 weeks of HFD+Rosi (n=7) (F), expressed as % of the 0-time point value. **P=0.003 in repeated measures 2x2 ANOVA with Holm-Sidak posttests (Cited4-/- vs. Cited4+/+). G Area under the curve (AUC) of blood glucose during the ITT (0-120 min) in (E,F). 2x2 ANOVA with Holm-Sidak posttests (After Rosi vs. Before Rosi)


H , I Blood glucose during insulin tolerance test ( ITT ) on male mice as in ( E , F ) , n = 8 ( H ) , n = 7 / 8 ( I ) . Repeated measures 2x2 ANOVA with Holm - Sidak posttests ( Cited4 - / - vs . Cited4 + / + ) . J Area under the curve ( AUC ) of blood glucose during the ITT in ( H , J ) . * * P = 0 . 001 , * * * P = 0 . 00006 in 2x2 ANOVA with Holm - Sidak posttests ( After Rosi vs . Before Rosi )

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

H, I Blood glucose during  insulin tolerance test (ITT) on male mice as in (E,F), n=8 (H), n=7/8 (I). Repeated measures 2x2 ANOVA with Holm-Sidak posttests (Cited4-/- vs. Cited4+/+). J Area under the curve (AUC) of blood glucose during the ITT in (H,J). **P=0.001, ***P=0.00006 in 2x2 ANOVA with Holm-Sidak posttests (After Rosi vs. Before Rosi)


K , L Fasting serum non - esterified fatty acids ( NEFA ) in female ( K ) or male ( L ) mice after 10 weeks of HFD ( " Before Rosi " ) or 11 weeks of HFD and 2 . 5 weeks of HFD + Rosi ( " After Rosi " ) , n = 7 / 8 / 8 / 8 ( K ) , n = 6 / 6 / 6 / 8 ( L ) . * * P = 0 . 001 in 2x2 ANOVA with Holm - Sidak posttests ( After Rosi vs . Before Rosi )

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

K, L Fasting serum non-esterified fatty acids (NEFA) in female (K) or male (L) mice after 10 weeks of HFD ("Before Rosi") or 11 weeks of HFD and 2.5 weeks of HFD+Rosi ("After Rosi"), n=7/8/8/8 (K), n=6/6/6/8 (L). **P=0.001 in 2x2 ANOVA with Holm-Sidak posttests (After Rosi vs. Before Rosi)


( B ) Volcano plot showing differentially expressed genes between WT ( WT1 and WT2 ) and patient ( V . 1 ( F1 ) , V . 5 ( F1 ) ) primary dermal fibroblasts . The vertical axis ( y - axis ) shows the - log10 P - value , whereas the horizontal axis ( x - axis ) displays the log2 fold change value . The red dots represent the upregulated transcripts ; the blue dots represent the downregulated transcripts . A total of 172 genes were found significantly dysregulated . TAPT1 , a gene located in the IBD region , appeared among the most significantly downregulated genes in the patients .

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(B) Volcano plot showing differentially expressed genes between WT (WT1 and WT2) and patient (V.1 (F1), V.5 (F1)) primary dermal fibroblasts. The vertical axis (y-axis) shows the -log10 P-value, whereas the horizontal axis (x-axis) displays the log2 fold change value. The red dots represent the upregulated transcripts; the blue dots represent the downregulated transcripts. A total of 172 genes were found significantly dysregulated. TAPT1, a gene located in the IBD region, appeared among the most significantly downregulated genes in the patients.


( B ) qPCR results using specific primers for TAPT1 and TAPT1 - AS1 in 3 WT ( WT1 , WT2 , V . 2 ( F1 ) ) and 3 affected ( V . 1 ( F1 ) , V . 5 ( F1 ) , IV . 1 ( F2 ) ) primary fibroblasts . TAPT1 mRNA is significantly reduced in all patients compared to WTs , whereas TAPT1 - AS1 transcript levels are unaffected . Fold change relative to V . 2 ( F1 ) is plotted as mean ± SD . Asterisks indicate conventional statistical significance ( Student t - test ; n . s . p - value > 0 . 05 , * * * * p - value < 0 . 0001 ) .

O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) qPCR results using specific primers for TAPT1 and TAPT1-AS1 in 3 WT (WT1, WT2 , V.2 (F1)) and 3 affected (V.1 (F1), V.5 (F1), IV.1 (F2)) primary fibroblasts. TAPT1 mRNA is significantly reduced in all patients compared to WTs, whereas TAPT1-AS1 transcript levels are unaffected. Fold change relative to V.2 (F1) is plotted as mean ± SD. Asterisks indicate conventional statistical significance (Student t-test; n.s. p-value > 0.05, **** p-value < 0.0001).


( C ) Western blot analysis of endogenous TAPT1 protein ( ~ 60 kDa ) using whole protein extracts from primary dermal fibroblasts from WT ( WT1 and WT2 ) , heterozygous ( IV . 3 ( F1 ) ) and homozygous ( V . 1 ( F1 ) , V . 5 ( F1 ) and IV . 1 ( F2 ) ) individuals and two different commercial antibodies ( top : Sigma , HPA042567 ; bottom : Sigma , HPA048658 ) . Results show a complete absence of TAPT1 protein in patient samples . GAPDH was used as a loading control .

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 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

(C) Western blot analysis of endogenous TAPT1 protein (~60 kDa) using whole protein extracts from primary dermal fibroblasts from WT (WT1 and WT2), heterozygous (IV.3 (F1)) and homozygous (V.1 (F1), V.5 (F1) and IV.1 (F2)) individuals and two different commercial antibodies (top: Sigma, HPA042567; bottom: Sigma, HPA048658). Results show a complete absence of TAPT1 protein in patient samples. GAPDH was used as a loading control.


( D ) qPCR analysis of TAPT1 expression in 3 WT ( WT1 , WT2 , WT3 ) and 3 affected ( V . 1 ( F1 ) , V . 5 ( F1 ) , IV . 1 ( F2 ) ) primary fibroblasts treated with cycloheximide ( CHX ) . CHX was used to block nonsense mediated decay ( NMD ) . Our results showed a time dependent increase in the level of TAPT1 transcripts in all 3 patient cells while TAPT1 RNA level remained constant in the WT cells . For each graph , fold change relative to non - treated condition is plotted as mean ± SD . Asterisks indicate conventional statistical significance ( Student t - test ; n . s . p - value > 0 . 05 , * * * * p - value < 0 . 0001 ) .

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 B-CONTROLLED_VAR O B-CONTROLLED_VAR 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 B-MEASURED_VAR O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) qPCR analysis of TAPT1 expression in 3 WT (WT1, WT2, WT3) and 3 affected (V.1 (F1), V.5 (F1), IV.1 (F2)) primary fibroblasts treated with cycloheximide (CHX). CHX was used to block nonsense mediated decay (NMD). Our results showed a time dependent increase in the level of TAPT1 transcripts in all 3 patient cells while TAPT1 RNA level remained constant in the WT cells. For each graph, fold change relative to non-treated condition is plotted as mean ± SD. Asterisks indicate conventional statistical significance (Student t-test; n.s. p-value > 0.05, **** p-value < 0.0001).


( B ) Western blot analyses for TAPT1 ( ~ 60 kDa ) using cytosolic , Mito / ER / Golgi and nuclear protein extracts from primary dermal fibroblasts of two WTs ( WT1 and WT2 ) and two patients ( V . 5 ( F1 ) and IV . 1 ( F2 ) ) . TAPT1 protein is highly enriched in the Mito / ER / Golgi fraction , and to a lower extent in the nuclear fraction . GAPDH , TGN46 and BiP served as a cytosolic , Golgi network and ER markers , respectively . Adenylate Kinase ( AK2 ) was used as a mitochondrial marker . Laminin A / C was used as a nuclear marker .

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(B) Western blot analyses for TAPT1 (~60 kDa) using cytosolic, Mito/ER/Golgi and nuclear protein extracts from primary dermal fibroblasts of two WTs (WT1 and WT2) and two patients (V.5 (F1) and IV.1 (F2)). TAPT1 protein is highly enriched in the Mito/ER/Golgi fraction, and to a lower extent in the nuclear fraction. GAPDH, TGN46 and BiP served as a cytosolic, Golgi network and ER markers, respectively. Adenylate Kinase (AK2) was used as a mitochondrial marker. Laminin A/C was used as a nuclear marker.


( C ) Immunofluorescence staining of mitochondria using anti - TOM20 ( green ) , ER using anti - CANX ( red ) and Golgi using anti - GLG1 ( red ) in primary dermal fibroblasts from WT1 and V . 5 ( F1 ) . Similar staining patterns are observed with the three antibodies in both cell lines . Scale bar represents 10 µm .

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(C) Immunofluorescence staining of mitochondria using anti-TOM20 (green), ER using anti-CANX (red) and Golgi using anti-GLG1 (red) in primary dermal fibroblasts from WT1 and V.5 (F1). Similar staining patterns are observed with the three antibodies in both cell lines. Scale bar represents 10 µm.


( D ) qPCR analysis of a panel of canonical ER stress markers shows no significant differences in 3 patients ( - / - ) ( V . I ( F1 ) , V . 5 ( F1 ) and IV . 1 ( F2 ) ) primary dermal fibroblasts compared to WTs ( + / + ) ( WT1 , WT2 and WT3 ) cells . Fold change relative to WT is plotted as mean ± SD . Statistical significance was tested by Student t - test ( n . s . p - value > 0 . 05 ) .

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(D) qPCR analysis of a panel of canonical ER stress markers shows no significant differences in 3 patients (-/-) (V.I (F1), V.5 (F1) and IV.1 (F2)) primary dermal fibroblasts compared to WTs (+/+) (WT1, WT2 and WT3) cells. Fold change relative to WT is plotted as mean ± SD. Statistical significance was tested by Student t-test (n.s. p-value > 0.05).


( E ) CMV cell infection assay on 2 patient ( V . 1 ( F1 ) and V . 5 ( F1 ) ) and 3 WT ( WT1 , WT4 and WT5 ) primary dermal fibroblast cell lines , using β - galactosidase activity as a readout . MRC5 cell line was used as a positive control . All of the cells were infected by the HCMV strain RC256 at a MOI = 0 . 1 . Data are shown as mean ± SD of three technical replicates . Statistical significance was tested by Student t - test ( n . s . p - value > 0 . 05 ) .

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(E) CMV cell infection assay on 2 patient (V.1 (F1) and V.5 (F1)) and 3 WT (WT1, WT4 and WT5) primary dermal fibroblast cell lines, using β-galactosidase activity as a readout. MRC5 cell line was used as a positive control. All of the cells were infected by the HCMV strain RC256 at a MOI=0.1. Data are shown as mean ± SD of three technical replicates. Statistical significance was tested by Student t-test (n.s. p-value > 0.05).


( A ) Volcano plot showing differentially expressed genes as determined by RNA - seq in patient primary dermal fibroblasts ( V . 1 ( F1 ) , V . 5 ( F1 ) ) compared to WT ( WT1 and WT2 ) cells . The y - axis shows the - log10 P - value , whereas the x - axis displays the log2 fold change value . The red dots represent 75 significantly upregulated genes , and the blue dots represent 97 significantly downregulated genes .

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(A) Volcano plot showing differentially expressed genes as determined by RNA-seq in patient primary dermal fibroblasts (V.1 (F1), V.5 (F1)) compared to WT (WT1 and WT2) cells. The y-axis shows the -log10 P-value, whereas the x-axis displays the log2 fold change value. The red dots represent 75 significantly upregulated genes, and the blue dots represent 97 significantly downregulated genes.


( B ) qPCR validation test for 3 top dysregulated genes ( RARRES2 , ZIC1 , and ZIC4 ) detected by RNA - seq . The analysis was performed on RNA samples independent from those sent for RNA - seq for 2 WTs ( WT1 and WT2 ) and 2 patients ( V . 5 ( F1 ) and IV . 1 ( F2 ) ) . Fold change relative to WT1 is plotted as mean ± SD of three technical replicates . Asterisks indicate statistical significance ( Student t - test ; * * * p - value < 0 . 001 , * * * * p - value < 0 . 0001 ) .

O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) qPCR validation test for 3 top dysregulated genes (RARRES2, ZIC1, and ZIC4) detected by RNA-seq. The analysis was performed on RNA samples independent from those sent for RNA-seq for 2 WTs (WT1 and WT2) and 2 patients (V.5 (F1) and IV.1 (F2)). Fold change relative to WT1 is plotted as mean ± SD of three technical replicates. Asterisks indicate statistical significance (Student t-test; *** p-value < 0.001, **** p-value < 0.0001).


( C ) Volcano plot showing genes with an altered occupancy of transcriptionally engaged Pol II in patients ( V . 1 ( F1 ) , V . 5 ( F1 ) ) compared to WT ( WT1 and WT2 ) primary fibroblast cells using SI - NET - seq . The y - axis shows the - Log10 P - value , whereas the x - axis indicates the log2 fold change value for the Pol II occupancy . The Pol II density is increased in 222 genes ( red dots ) , and decreased in 95 genes ( blue dots ) . The yellow dot represents TAPT1 .

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(C) Volcano plot showing genes with an altered occupancy of transcriptionally engaged Pol II in patients (V.1 (F1), V.5 (F1)) compared to WT (WT1 and WT2) primary fibroblast cells using SI-NET-seq. The y-axis shows the -Log10 P-value, whereas the x-axis indicates the log2 fold change value for the Pol II occupancy. The Pol II density is increased in 222 genes (red dots), and decreased in 95 genes (blue dots). The yellow dot represents TAPT1.


The VPR and DPR ( right ) were dissected from E10 . 5 Pdx1 - GFP embryos ( left ) . Scale bar : 500 µm . DPR , dorsal Pdx1 - expressing region ; VPR , ventral Pdx1 - expressing region ; Duo , duodenum bud .

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The VPR and DPR (right) were dissected from E10.5 Pdx1-GFP embryos (left). Scale bar: 500 µm. DPR, dorsal Pdx1-expressing region; VPR, ventral Pdx1-expressing region; Duo, duodenum bud.


FACS gating for sorting GFPlow and GFPhigh cells from the VPR and DPR .

FACS gating for sorting GFPlow and GFPhigh cells from the VPR and DPR.


The ratios of GFPhigh cells to GFP + cells in the VPR and DPR . The data show the mean ± SEM ; n = number of independent biological replicates , unpaired t - test . * * * p - value < 0 . 001 .

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The ratios of GFPhigh cells to GFP+ cells in the VPR and DPR. The data show the mean ± SEM; n = number of independent biological replicates, unpaired t-test. ***p-value < 0.001.


Hierarchical clustering of 815 genes correlating with the first two PCs identified three groups of cell types and four major distinct clusters ( a - d ) of genes ( p < 1 × 10 - 9 , cell cycle - related genes are excluded ) . Each column is a single - cell , and each row represents one gene . Cluster - specific TFs are listed on the right . The genes in bold are known to have roles in pancreatic development . L , GFPlow ; H , GFPhigh .

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Hierarchical clustering of 815 genes correlating with the first two PCs identified three groups of cell types and four major distinct clusters (a-d) of genes (p < 1 ×10-9, cell cycle-related genes are excluded). Each column is a single-cell, and each row represents one gene. Cluster-specific TFs are listed on the right. The genes in bold are known to have roles in pancreatic development. L, GFPlow; H, GFPhigh.


Expression levels of group - specific genes in individual cells . The gene expression values were projected onto the PCA ( left ) and box ( right ) plots . Each point represents a cell . Points were colored based on the hierarchical clustering in Fig 1E . The y - axes of the box plots represent the expression levels ( RPKM ) . Central line in the box represents median ; box limits represent the first and third quartile ; whiskers represent 1 . 5 × interquartile range . Unpaired Wilcoxon rank - sum test . * p - value < 1 x 10 - 3 ; * * p - value < 1 x 10 - 6 ; * * * p - value < 1 x 10 - 9 .

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

Expression levels of group-specific genes in individual cells. The gene expression values were projected onto the PCA (left) and box (right) plots. Each point represents a cell. Points were colored based on the hierarchical clustering in Fig 1E. The y-axes of the box plots represent the expression levels (RPKM). Central line in the box represents median; box limits represent the first and third quartile; whiskers represent 1.5 × interquartile range. Unpaired Wilcoxon rank-sum test. *p-value < 1 x 10-3; **p-value <1 x 10-6; ***p-value <1 x 10-9.


Flow cytometric analysis of BrdU incorporation rates in GFPlow and GFPhigh cells from the DPR and VPR . Statistical analysis of BrdU + in different cell populations . The Data show the mean ± SEM ; n = number of independent biological replicates , unpaired t - test . * p - value < 0 . 05 ; * * * p - value < 0 . 001 .

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

Flow cytometric analysis of BrdU incorporation rates in GFPlow and GFPhigh cells from the DPR and VPR. Statistical analysis of BrdU+ in different cell populations. The Data show the mean ± SEM; n = number of independent biological replicates, unpaired t-test. *p-value < 0.05; ***p-value < 0.001.


Flow cytometric analysis of endocrine progenitors and β - cells in the ventral pancreases ( V . P . ) and dorsal pancreases ( D . P . ) . ( A , D and G ) Dissected ventral and dorsal pancreatic tissues . Scale bar : 500 µm . ( B , E and H ) FACS gating for sorting Ngn3 - GFP + or Ins1 - RFP + cells from the V . P . and D . P . ( C and F ) statistical analysis of percentages of Ngn3 - GFPhigh cells among Ngn3 - GFP + cells in the V . P . and D . P . ( I ) Statistical analysis of percentages of Ins1 - RFP + cells among total pancreatic cells in the V . P . and D . P .

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

Flow cytometric analysis of endocrine progenitors and β-cells in the ventral pancreases (V. P.) and dorsal pancreases (D. P.). (A, D and G) Dissected ventral and dorsal pancreatic tissues. Scale bar: 500 µm. (B, E and H) FACS gating for sorting Ngn3-GFP+ or Ins1-RFP+ cells from the V. P. and D. P. (C and F) statistical analysis of percentages of Ngn3-GFPhigh cells among Ngn3-GFP+ cells in the V. P. and D. P. (I) Statistical analysis of percentages of Ins1-RFP+ cells among total pancreatic cells in the V. P. and D. P.


Differentially expressed genes among cells from VPR , DPR , the V . P . and D . P . at various developmental stages . The numbers indicate significantly highly expressed genes in the ventral ( blue ) or dorsal ( orange ) regions . The dotted lines represent the two - fold change threshold .

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

Differentially expressed genes among cells from VPR, DPR, the V. P. and D. P. at various developmental stages. The numbers indicate significantly highly expressed genes in the ventral (blue) or dorsal (orange) regions. The dotted lines represent the two-fold change threshold.


Flow cytometric analyses of Liv2 + cells in the VPR , DPR and the liver buds ( LB ) . Liv2 + cells from the VPR - L ( brown arrow ) , Liv2 - cells from the VPR - L ( yellow arrow ) and Liv2 + cells from the LB ( black arrow ) were sorted for single - cell RNA - seq .

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

Flow cytometric analyses of Liv2+ cells in the VPR, DPR and the liver buds (LB). Liv2+ cells from the VPR-L (brown arrow), Liv2- cells from the VPR-L (yellow arrow) and Liv2+ cells from the LB (black arrow) were sorted for single-cell RNA-seq.


Immunofluorescence of PDX1 and Liv2 on sagittal section of E10 . 5 mouse embryo . The white and yellow arrows denote PDX1low / Liv2 + cells in in the liver and EHBD regions , respectively . Scale bar : 50 µm .

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

Immunofluorescence of PDX1 and Liv2 on sagittal section of E10.5 mouse embryo. The white and yellow arrows denote PDX1low/Liv2+ cells in in the liver and EHBD regions, respectively. Scale bar: 50 µm.


Hierarchical clustering of 694 genes correlated with the first two PCs identified three major distinct cell lineages ( p < 1 × 10 - 6 ) . Each column represents a single - cell and each row represents one gene . Cells were re - ordered according to the value of PC1 or PC2 . Genes in clusters ' a , b ' and ' c , d ' were re - ordered by correlations with PC2 and PC1 , respectively . The color of the cell type and cell source are indicated in ( C ) . Lineage - specific TFs are listed on the right .

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

Hierarchical clustering of 694 genes correlated with the first two PCs identified three major distinct cell lineages (p < 1 × 10-6). Each column represents a single-cell and each row represents one gene. Cells were re-ordered according to the value of PC1 or PC2. Genes in clusters 'a, b' and 'c, d' were re-ordered by correlations with PC2 and PC1, respectively. The color of the cell type and cell source are indicated in (C). Lineage-specific TFs are listed on the right.


The gene expression values of lineage marker genes were projected onto the PCA ( E ) and box ( F ) plots . Each point represents a cell . The y - axes of the box plots represent the expression levels ( RPKM ) . Central line in the box represents median ; box limits represent the first and third quartile ; whiskers represent 1 . 5 × interquartile range . Unpaired Wilcoxon rank - sum test . * * p - value < 1 x 10 - 5 ; * * * p - value < 1 x 10 - 10 .

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

The gene expression values of lineage marker genes were projected onto the PCA (E) and box (F) plots. Each point represents a cell. The y-axes of the box plots represent the expression levels (RPKM). Central line in the box represents median; box limits represent the first and third quartile; whiskers represent 1.5 × interquartile range. Unpaired Wilcoxon rank-sum test. **p-value <1 x 10-5; ***p-value <1 x 10-10.


Pdx1 and Sox17 expression levels in E9 . 5 VPR and DPR cells .

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

Pdx1 and Sox17 expression levels in E9.5 VPR and DPR cells.


Sagittal section of E12 . 5 Pdx1 - Cre ; Rosa26 - LacZ mouse embryo counterstained for ALB ( brown ) . X - gal staining ( blue ) showing cells derived from Pdx1 - expressing cells . The arrowheads indicate cells co - stained with X - gal and ALB ( See Fig EV5D for more images of different sections ) . Scale bar : 50 µm ( left ) or 20 µm ( right ) .

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

Sagittal section of E12.5 Pdx1-Cre; Rosa26-LacZ mouse embryo counterstained for ALB (brown). X-gal staining (blue) showing cells derived from Pdx1-expressing cells. The arrowheads indicate cells co-stained with X-gal and ALB (See Fig EV5D for more images of different sections). Scale bar: 50 µm (left) or 20 µm (right).


qRT - PCR detection of the fold induction of IFNB1 mRNA relative to unstimulated condition in different cell types . Cells were stimulated with ecGAMP ( 5 μg / ml ) for 4 h .

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

qRT-PCR detection of the fold induction of IFNB1 mRNA relative to unstimulated condition in different cell types. Cells were stimulated with ecGAMP (5 μg/ml) for 4 h.


qRT - PCR detection of Ifnb1 mRNA abundance in mBMDMs treated with different eCDNs ( 5 μg / ml ) for 4 h .

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

qRT-PCR detection of Ifnb1 mRNA abundance in mBMDMs treated with different eCDNs (5 μg/ml) for 4 h.


ELISA detection of IFNβ release by mBMDMs treated for 4 h or 24 h with extracellular 2 ' 3 ' - cGAMP at indicated concentrations .

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

ELISA detection of IFNβ release by mBMDMs treated for 4 h or 24 h with extracellular 2'3'-cGAMP at indicated concentrations.


ELISA detection of IFNβ release by mBMDMs treated for 4 h or 24 h with extracellular c - di - AMP at indicated concentrations .

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

ELISA detection of IFNβ release by mBMDMs treated for 4 h or 24 h with extracellular c-di-AMP at indicated concentrations.


qRT - PCR detection of IFNB1 mRNA in THP - 1 cells stimulated with indicated eCDNs ( 5 μg / ml ) for 4 h .

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

qRT-PCR detection of IFNB1 mRNA in THP-1 cells stimulated with indicated eCDNs (5 μg/ml) for 4 h.


ELISA detection of IFNβ in supernatants of THP - 1 cells stimulated with indicated eCDNs at indicated concentrations for 4 h .

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

ELISA detection of IFNβ in supernatants of THP-1 cells stimulated with indicated eCDNs at indicated concentrations for 4 h.


qRT - PCR detection of the fold induction of IFNB1 mRNA relative to unstimulated condition in human CD14 + monocytes derived from PBMC stimulated with indicated eCDNs ( 5 μg / ml ) for 4 h and 8 h . Each symbol represents one individual donor . Data are means + SD averaged from 10 healthy donors .

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

qRT-PCR detection of the fold induction of IFNB1 mRNA relative to unstimulated condition in human CD14+ monocytes derived from PBMC stimulated with indicated eCDNs (5 μg/ml) for 4 h and 8 h. Each symbol represents one individual donor. Data are means+SD averaged from 10 healthy donors.


ELISA detection of IFNβ in supernatants of human CD14 + monocytes derived from PBMC stimulated with indicated eCDNs ( 5 μg / ml ) for 4 h . Each symbol represents result from one individual donor . Data are means + SD averaged from 10 healthy donors .

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

ELISA detection of IFNβ in supernatants of human CD14+ monocytes derived from PBMC stimulated with indicated eCDNs (5 μg/ml) for 4 h. Each symbol represents result from one individual donor. Data are means+SD averaged from 10 healthy donors.


qRT - PCR detection of IFNB1 mRNA abundance in THP - 1 cells treated with ecGAMP and icGAMP at indicated concentrations for 4 h .

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

qRT-PCR detection of IFNB1 mRNA abundance in THP-1 cells treated with ecGAMP and icGAMP at indicated concentrations for 4 h.


qRT - PCR detection of IFNB1 mRNA abundance in THP - 1 cells treated with ec - di - AMP and ic - di - AMP ( B ) at indicated concentrations for 4 h .

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

qRT-PCR detection of IFNB1 mRNA abundance in THP-1 cells treated with ec-di-AMP and ic-di-AMP (B) at indicated concentrations for 4 h.


ELISA detection of IFNβ release from THP - 1 cells stimulated with ecGAMP and icGAMP ( C ) at indicated concentrations for 4 h .

ELISA detection of IFNβ release from THP-1 cells stimulated with ecGAMP and icGAMP (C) at indicated concentrations for 4 h.


ELISA detection of IFNβ release from THP - 1 cells stimulated with ec - di - AMP and ic - di - AMP ( D ) at indicated concentrations for 4 h .

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

ELISA detection of IFNβ release from THP-1 cells stimulated with ec-di-AMP and ic-di-AMP (D) at indicated concentrations for 4 h.


qRT - PCR detection of Ifnb1 mRNA abundance in mBMDMs treated with ecGAMP and icGAMP ( E ) at indicated concentrations for 4 h .

qRT-PCR detection of Ifnb1 mRNA abundance in mBMDMs treated with ecGAMP and icGAMP (E) at indicated concentrations for 4 h.


qRT - PCR detection of Ifnb1 mRNA abundance in mBMDMs treated with ec - di - AMP and ic - di - AMP ( F ) at indicated concentrations for 4 h .

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

qRT-PCR detection of Ifnb1 mRNA abundance in mBMDMs treated with ec-di-AMP and ic-di-AMP (F) at indicated concentrations for 4 h.


ELISA detection of IFNβ release from mBMDMs stimulated with ecGAMP and icGAMP at indicated concentrations for 4 h .

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

ELISA detection of IFNβ release from mBMDMs stimulated with ecGAMP and icGAMP at indicated concentrations for 4 h.


ELISA detection of IFNβ release from mBMDMs stimulated with ec - di - AMP and ic - di - AMP ( H ) at indicated concentrations for 4 h .

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

ELISA detection of IFNβ release from mBMDMs stimulated with ec-di-AMP and ic-di-AMP (H) at indicated concentrations for 4 h.


Frequencies of FITC + THP - 1 cells stimulated with FITC - ecGAMP for 1 h in presence of DMSO or indicated inhibitors including Dynasore ( 10 μM ) , chlorpromazine ( CPZ , 10 μM ) , dimethylamiloride ( DMA , 100 μM ) , polyinosinic acid ( Poly I , 50 μg / ml ) or mannans from Sacharomyces cerevesiae ( Mannans , 1 mg / ml ) .

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

Frequencies of FITC+ THP-1 cells stimulated with FITC-ecGAMP for 1 h in presence of DMSO or indicated inhibitors including Dynasore (10 μM), chlorpromazine (CPZ, 10 μM), dimethylamiloride (DMA, 100 μM), polyinosinic acid (Poly I, 50 μg/ml) or mannans from Sacharomyces cerevesiae (Mannans, 1 mg/ml).


qRT - PCR detection of IFNB1 ( B ) and IL6 ( C ) mRNA in THP - 1 cells stimulated with ecGAMP ( 5 μg / ml ) or intracellular 2 ′ 3 ′ - cGAMP ( icGAMP ) ( 0 . 1 μg / ml ) for 4 h in presence of DMSO or dynasore ( 10 μM ) . qRT - PCR detection of Ifnb1 ( D ) and Il6 ( E ) mRNA in mBMDMs stimulated with ecGAMP ( 5 μg / ml ) or icGAMP ( 0 . 1 μg / ml ) for 4 h in presence of DMSO or dynasore ( 10 μM ) .

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

qRT-PCR detection of IFNB1 (B) and IL6 (C) mRNA in THP-1 cells stimulated with ecGAMP (5 μg/ml) or intracellular 2′3′-cGAMP (icGAMP) (0.1 μg/ml) for 4 h in presence of DMSO or dynasore (10 μM). qRT-PCR detection of Ifnb1 (D) and Il6 (E) mRNA in mBMDMs stimulated with ecGAMP (5 μg/ml) or icGAMP (0.1 μg/ml) for 4 h in presence of DMSO or dynasore (10 μM).


Immunostaining for EEA1 ( red ) ( F ) and LAMP2 ( red ) ( G ) in THP - 1 cells stimulated with FITC - ecGAMP ( 5 μg / ml , green ) for 30 min , nucleus in blue ( DAPI ) . Data are representative of 3 independent experiments . Scale bar , 10 μm .

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

Immunostaining for EEA1 (red) (F) and LAMP2 (red) (G) in THP-1 cells stimulated with FITC-ecGAMP (5 μg/ml, green) for 30 min, nucleus in blue (DAPI). Data are representative of 3 independent experiments. Scale bar, 10 μm.


qRT - PCR detection of IFNB1 and IL6 mRNA in THP - 1 cells ( H , I ) and mBMDMs ( J , K ) stimulated with ecGAMP ( 5 μg / ml ) and icGAMP ( 0 . 1 μg / ml ) , respectively , for 4 h in presence of DMSO or bafilomycin A1 ( BafA ) ( 1 μM ) .

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

qRT-PCR detection of IFNB1 and IL6 mRNA in THP-1 cells (H, I) and mBMDMs (J, K) stimulated with ecGAMP (5 μg/ml) and icGAMP (0.1 μg/ml), respectively, for 4 h in presence of DMSO or bafilomycin A1 (BafA) (1 μM).


qRT - PCR detection of IFNB1 mRNA in scrambled ( Scramble ) or STING shRNA stably transfected ( STING KD ) THP - 1 cells stimulated with ec - di - AMP ( 5 μg / ml ) and ecGAMP ( 5 μg / ml ) or transfected with ISD or poly ( I : C ) .

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

qRT-PCR detection of IFNB1 mRNA in scrambled (Scramble) or STING shRNA stably transfected (STING KD) THP-1 cells stimulated with ec-di-AMP (5 μg/ml) and ecGAMP (5 μg/ml) or transfected with ISD or poly(I:C).


ELISA detection of IFNβ protein in supernatants of Scramble or STING KD THP - 1 cells stimulated with indicated eCDNs ( 5 μg / ml ) or transfected with ISD or poly ( I : C ) .

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

ELISA detection of IFNβ protein in supernatants of Scramble or STING KD THP-1 cells stimulated with indicated eCDNs (5 μg/ml) or transfected with ISD or poly(I:C).


qRT - PCR detection of Ifnb1 mRNA in WT and Sting - / - ( Sting KO ) mBMDMs stimulated with indicated eCDNs ( 5 μg / ml ) or transfected with ISD .

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

qRT-PCR detection of Ifnb1 mRNA in WT and Sting-/- (Sting KO) mBMDMs stimulated with indicated eCDNs (5 μg/ml) or transfected with ISD.


ELISA detection of IFNβ protein secretion in supernatants of mBMDMs stimulated with indicated eCDNs ( 5 μg / ml ) or transfected with ISD or poly ( I : C ) .

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

ELISA detection of IFNβ protein secretion in supernatants of mBMDMs stimulated with indicated eCDNs (5 μg/ml) or transfected with ISD or poly (I:C).


qRT - PCR detection of Ifnb1 mRNA in WT , Sting KO or Sting KO complemented with mouse STING ( Sting KO + mSTING ) RAW264 . 7 cells stimulated with indicated eCDNs ( 5 μg / ml ) or transfected with ISD .

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

qRT-PCR detection of Ifnb1 mRNA in WT, Sting KO or Sting KO complemented with mouse STING (Sting KO+mSTING) RAW264.7 cells stimulated with indicated eCDNs (5 μg/ml) or transfected with ISD.


Western blot detection of indicated proteins in lysates of WT and Sting - / - ( KO ) mBMDMs stimulated with ecGAMP ( 5 μg / ml ) or transfected with ISD . Data are representative of 3 independent experiments .

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

Western blot detection of indicated proteins in lysates of WT and Sting-/- (KO) mBMDMs stimulated with ecGAMP (5 μg/ml) or transfected with ISD. Data are representative of 3 independent experiments.


qRT - PCR detection of IFNB1 mRNA levels in STING stable HEK293T cells ( HA - STING - HEK293T ) stimulated with ecGAMP or icGAMP at 5 μg / ml for indicated times .

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

qRT-PCR detection of IFNB1 mRNA levels in STING stable HEK293T cells (HA-STING-HEK293T) stimulated with ecGAMP or icGAMP at 5 μg/ml for indicated times.


Western blot detection of indicated proteins in HA - STING - HEK293T cells stimulated with increasing amounts of ecGAMP and icGAMP at 5 μg / ml for 24 h . Data are representative of 3 independent experiments .

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

Western blot detection of indicated proteins in HA-STING-HEK293T cells stimulated with increasing amounts of ecGAMP and icGAMP at 5 μg/ml for 24 h. Data are representative of 3 independent experiments.


qRT - PCR detection of the induction of IFNB1 mRNA in HEK293T cells stably transfected with pcDNA3 . 1 - HA ( HA ) , HA - STING and HA - STING + HA - cGAS stimulated with ecGAMP ( 5 μg / ml ) , icGAMP ( 0 . 1 μg / ml ) or transfected with ISD .

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

qRT-PCR detection of the induction of IFNB1 mRNA in HEK293T cells stably transfected with pcDNA3.1-HA (HA), HA-STING and HA-STING+HA-cGAS stimulated with ecGAMP (5 μg/ml), icGAMP (0.1 μg/ml) or transfected with ISD.


qRT - PCR detection of IFNB1 mRNA in WT and CGAS KO THP - 1 cells stimulated with indicated eCDNs ( 5 μg / ml ) or transfected with ISD or poly ( I : C ) for 4 h .

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

qRT-PCR detection of IFNB1 mRNA in WT and CGAS KO THP-1 cells stimulated with indicated eCDNs (5 μg/ml) or transfected with ISD or poly (I:C) for 4 h.


ELISA detection of IFNβ in supernatants of WT and CGAS KO THP - 1 cells stimulated with indicated eCDNs ( 5 μg / ml ) or transfected with ISD or poly ( I : C ) for 4 h .

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

ELISA detection of IFNβ in supernatants of WT and CGAS KO THP-1 cells stimulated with indicated eCDNs (5 μg/ml) or transfected with ISD or poly(I:C) for 4 h.


qRT - PCR detection of Ifnb1 mRNA in mBMDMs from WT and Cgas - / - ( Cgas KO ) mice stimulated with indicated eCDNs ( 5 μg / ml ) or transfected with ISD or poly ( I : C ) for 4 h .

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

qRT-PCR detection of Ifnb1 mRNA in mBMDMs from WT and Cgas-/- (Cgas KO) mice stimulated with indicated eCDNs (5 μg/ml) or transfected with ISD or poly(I:C) for 4 h.


ELISA detection of Ifnb1 in supernatants of mBMDMs from WT and Cgas KO mice stimulated with indicated eCDNs ( 5 μg / ml ) or transfected with ISD or poly ( I : C ) for 4 h .

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

ELISA detection of Ifnb1 in supernatants of mBMDMs from WT and Cgas KO mice stimulated with indicated eCDNs (5 μg/ml) or transfected with ISD or poly(I:C) for 4 h.


qRT - PCR detection of IFNB1 mRNA in WT and CGAS KO THP - 1 cells stimulated with ecGAMP ( 5 μg / ml ) or icGMAP ( 0 . 1 μg / ml ) or transfected with ISD .

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

qRT-PCR detection of IFNB1 mRNA in WT and CGAS KO THP-1 cells stimulated with ecGAMP (5 μg/ml) or icGMAP (0.1 μg/ml) or transfected with ISD.


qRT - PCR detection of Ifnb1 mRNA in mBMDMs from WT and Cgas KO stimulated with ecGAMP ( 5 μg / ml ) or icGMAP ( 0 . 1 μg / ml ) or transfected with ISD .

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

qRT-PCR detection of Ifnb1 mRNA in mBMDMs from WT and Cgas KO stimulated with ecGAMP (5 μg/ml) or icGMAP (0.1 μg/ml) or transfected with ISD.


Western blot detection of indicated proteins in lysates of mBMDMs from WT and Cgas KO mice stimulated with ecGAMP ( 5 μg / ml ) , icGAMP ( 0 . 1 μg / ml ) or transfected with ISD . Data are representative of 3 independent experiments .

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

Western blot detection of indicated proteins in lysates of mBMDMs from WT and Cgas KO mice stimulated with ecGAMP (5 μg/ml), icGAMP (0.1 μg/ml) or transfected with ISD. Data are representative of 3 independent experiments.


qRT - PCR detection of IFNB1 mRNA of WT , CGAS KO , CGAS KO complemented with cGAS ( CGAS KO + cGAS ) or CGAS KO stably transfected with the empty vector ( CGAS KO + Vector ) THP - 1 cells stimulated with ecGAMP ( 5 μg / ml ) .

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

qRT-PCR detection of IFNB1 mRNA of WT, CGAS KO, CGAS KO complemented with cGAS (CGAS KO+cGAS) or CGAS KO stably transfected with the empty vector (CGAS KO+Vector) THP-1 cells stimulated with ecGAMP (5 μg/ml).


ELISA detection of IFNβ in supernatants ( I ) of WT , CGAS KO , CGAS KO complemented with cGAS ( CGAS KO + cGAS ) or CGAS KO stably transfected with the empty vector ( CGAS KO + Vector ) THP - 1 cells stimulated with ecGAMP ( 5 μg / ml ) .

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

ELISA detection of IFNβ in supernatants (I) of WT, CGAS KO, CGAS KO complemented with cGAS (CGAS KO+cGAS) or CGAS KO stably transfected with the empty vector (CGAS KO+Vector) THP-1 cells stimulated with ecGAMP (5 μg/ml).


Purified h - cGAS protein was precipitated with Ctrl beads or cGAMP beads and then immunoblotted .

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

Purified h-cGAS protein was precipitated with Ctrl beads or cGAMP beads and then immunoblotted.


THP - 1 cells were stimulated with biotin - cGAMP for indicated times and then whole cell lysates ( WCL ) were precipitated with streptavidin beads followed by immunoblotting .

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THP-1 cells were stimulated with biotin-cGAMP for indicated times and then whole cell lysates (WCL) were precipitated with streptavidin beads followed by immunoblotting.


Immunofluorescent staining of cGAS ( red ) in THP - 1 cells treated with FITC - ecGAMP ( 5 μg / ml ) ( green ) or FITC - icGAMP ( 0 . 1 μg / ml ) ( green ) for 4 h , nucleus in blue ( DAPI ) . Scale bar , 10 μm .

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Immunofluorescent staining of cGAS (red) in THP-1 cells treated with FITC-ecGAMP (5 μg/ml) (green) or FITC-icGAMP (0.1 μg/ml) (green) for 4 h, nucleus in blue (DAPI). Scale bar, 10 μm.


Purified h - cGAS protein was precipitated with beads coupled with cGAMP , c - di - GMP or c - di - AMP followed by immunoblotting .

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

Purified h-cGAS protein was precipitated with beads coupled with cGAMP, c-di-GMP or c-di-AMP followed by immunoblotting.


Binding curves of surface - immobilized 2 ' 3 ' - cGAMP with His - cGAS at indicated concentrations . Vertical lines mark the start of association and dissociation phases of the binding events . The dashed lines are global fits to a Langmuir reaction model ; global fitting parameters are listed in the table below the plot . ( n = 4 independent experiment ) .

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

Binding curves of surface-immobilized 2'3'-cGAMP with His-cGAS at indicated concentrations. Vertical lines mark the start of association and dissociation phases of the binding events. The dashed lines are global fits to a Langmuir reaction model; global fitting parameters are listed in the table below the plot. (n=4 independent experiment).


The small - angle X - ray scattering analysis of the full - length cGAS with cGAMP . The EOM fit of the measured SAXS data . The goodness - of - the fit χ2 = 1 . 1 .

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The small-angle X-ray scattering analysis of the full-length cGAS with cGAMP. The EOM fit of the measured SAXS data. The goodness-of-the fit χ2 = 1.1.


Immunostaining of STING ( anti - STING , red ) in THP - 1 cells stimulated with FITC - ecGAMP ( 5 μg / ml , green ) for 2 h , nucleus in blue ( DAPI ) . Data are representative of 3 independent experiments . Scale bar , 10 μm .

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Immunostaining of STING (anti-STING, red) in THP-1 cells stimulated with FITC-ecGAMP (5 μg/ml, green) for 2 h, nucleus in blue (DAPI). Data are representative of 3 independent experiments. Scale bar, 10 μm.


Cellular localization of ecGAMP in WT and CGAS KO THP - 1 cells stimulated with FITC - ecGAMP ( 5 μg / ml , green ) for 2 h , nucleus in blue ( DAPI ) . Data are presentative of 5 independent experiments . Scale bar , 10 μm .

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Cellular localization of ecGAMP in WT and CGAS KO THP-1 cells stimulated with FITC-ecGAMP (5 μg/ml, green) for 2 h, nucleus in blue (DAPI). Data are presentative of 5 independent experiments. Scale bar, 10 μm.


Frequency of perinuclear accumulation of FITC - ecGAMP in WT and CGAS KO THP - 1 cells stimulated with FITC - ecGAMP ( 5 μg / ml ) for 2 h . Data are means + SD averaged from 5 independent experiments and approximately 100 cells were imaged and counted in each experiment . Each symbol represents the percentage of THP - 1 cells with perinuclear cGAMP aggregates in every independent experiment . Mann - Whitney U test was used for statistical analysis . * * p < 0 . 01 .

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 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

Frequency of perinuclear accumulation of FITC-ecGAMP in WT and CGAS KO THP-1 cells stimulated with FITC-ecGAMP (5 μg/ml) for 2 h. Data are means+SD averaged from 5 independent experiments and approximately 100 cells were imaged and counted in each experiment. Each symbol represents the percentage of THP-1 cells with perinuclear cGAMP aggregates in every independent experiment. Mann-Whitney U test was used for statistical analysis. **p<0.01.


Western blot detection of the presence of GST - cGAS and His - STING in the immunoprecipitates of cGAMP agarose . Purified GST - cGAS and His - STING were incubated separately or together with cGAMP agarose . The input and immunoprecipitates were immunoblotted .

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

Western blot detection of the presence of GST-cGAS and His-STING in the immunoprecipitates of cGAMP agarose. Purified GST-cGAS and His-STING were incubated separately or together with cGAMP agarose. The input and immunoprecipitates were immunoblotted.


Western blot detection of the presence of His - STING in the immunoprecipitates of Glutathione Sepharose . Purified GST - cGAS and His - STING were incubated together in IP lysis buffer overnight at 4 ° C .

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

Western blot detection of the presence of His-STING in the immunoprecipitates of Glutathione Sepharose. Purified GST-cGAS and His-STING were incubated together in IP lysis buffer overnight at 4°C.


Western blot detection of the presence of GST - cGAS and His - STING in the immunoprecipitates of Glutathione Sepharose . Purified GST - cGAS and His - STING were incubated together in the absence or presence of increasing 2 ′ 3 ′ - cGAMP .

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

Western blot detection of the presence of GST-cGAS and His-STING in the immunoprecipitates of Glutathione Sepharose. Purified GST-cGAS and His-STING were incubated together in the absence or presence of increasing 2′3′-cGAMP.


Western blot detection of the presence of cGAS and STING in the immunoprecipitates of streptavidin beads . THP - 1 cells were stimulated with biotin - ecGAMP ( 5 μg / ml ) or biotin - icGAMP ( 1 μg / ml ) for indicated time and cell lysates were harvested for IP with streptavidin beads . Data ( A - B , D - G ) are representative of 3 independent experiments .

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

Western blot detection of the presence of cGAS and STING in the immunoprecipitates of streptavidin beads. THP-1 cells were stimulated with biotin-ecGAMP (5 μg/ml) or biotin-icGAMP (1 μg/ml) for indicated time and cell lysates were harvested for IP with streptavidin beads. Data (A-B, D-G) are representative of 3 independent experiments.


qRT - PCR detection of Ifnb1 mRNA ( A ) of mBMDMs infected with HSV - 1 at indicated MOI together with stimulation with ecGAMP at indicated concentrations for 4 h . Data are means + SD averaged from 3 independent experiments performed with technical triplicates . Each symbol represents the mean of technical triplicates . Two - way ANOVA followed by Bonferroni ' s post hoc test was used for statistical analysis . * , p < 0 . 05 ; * * p < 0 . 01 ; * * * , 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 O O O O O O O O O O O O O O

qRT-PCR detection of Ifnb1 mRNA (A) of mBMDMs infected with HSV-1 at indicated MOI together with stimulation with ecGAMP at indicated concentrations for 4 h. Data are means+SD averaged from 3 independent experiments performed with technical triplicates. Each symbol represents the mean of technical triplicates. Two-way ANOVA followed by Bonferroni's post hoc test was used for statistical analysis. *, p<0.05; **p<0.01; ***, p<0.001.


ELISA detection of IFNβ in supernatants ( B ) of mBMDMs infected with HSV - 1 at indicated MOI together with stimulation with ecGAMP at indicated concentrations for 4 h . Data are means + SD averaged from 3 independent experiments performed with technical triplicates . Each symbol represents the mean of technical triplicates . Two - way ANOVA followed by Bonferroni ' s post hoc test was used for statistical analysis . * , p < 0 . 05 ; * * p < 0 . 01 ; * * * , p < 0 . 001 .

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

ELISA detection of IFNβ in supernatants (B) of mBMDMs infected with HSV-1 at indicated MOI together with stimulation with ecGAMP at indicated concentrations for 4 h. Data are means+SD averaged from 3 independent experiments performed with technical triplicates. Each symbol represents the mean of technical triplicates. Two-way ANOVA followed by Bonferroni's post hoc test was used for statistical analysis. *, p<0.05; **p<0.01; ***, p<0.001.


Western blot detection of indicated proteins in lysates of mBMDMs infected with HSV - 1 at indicated MOI together with stimulation with ecGAMP at indicated concentrations for 4 h . Data are representative of 3 independent experiments .

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

Western blot detection of indicated proteins in lysates of mBMDMs infected with HSV-1 at indicated MOI together with stimulation with ecGAMP at indicated concentrations for 4 h. Data are representative of 3 independent experiments.


( A ) WT and atg1Δ cells grown in YEPD medium were transferred to SD - N or SD - N + 50 mM MES - KOH ( pH 6 . 2 ) medium for 120 hours , and dead cells stained by phloxine B were observed by fluorescent microscopy . Scale bar , 25 µm .

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(A) WT and atg1Δ cells grown in YEPD medium were transferred to SD-N or SD-N +50 mM MES-KOH (pH 6.2) medium for 120 hours, and dead cells stained by phloxine B were observed by fluorescent microscopy. Scale bar, 25 µm.


( B - C ) WT and atg1Δ cells grown nitrogen starved as ( A ) for the indicated times . Cell viability ( B ) and medium pH ( C ) were examined by phloxine B staining and pH meter , respectively . These data represent the average of three independent experiments and bars indicate standard deviations .

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(B-C) WT and atg1Δ cells grown nitrogen starved as (A) for the indicated times. Cell viability (B) and medium pH (C) were examined by phloxine B staining and pH meter, respectively. These data represent the average of three independent experiments and bars indicate standard deviations.


( A ) WT and atg1Δ cells grown in YEPD medium were transferred to SD - N + 50 mM MES - KOH ( pH 6 . 2 ) . After 120 hours , cultures were diluted 5 . 0 × 105 fold and plated onto YEPD agar . The plates were incubated at 30 ° C for four days . Insets indicate the plate overlaid with TTC agar to examine the respiratory competency of formed colonies .

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(A) WT and atg1Δ cells grown in YEPD medium were transferred to SD-N +50 mM MES-KOH (pH 6.2). After 120 hours, cultures were diluted 5.0×105 fold and plated onto YEPD agar. The plates were incubated at 30°C for four days. Insets indicate the plate overlaid with TTC agar to examine the respiratory competency of formed colonies.


( B ) WT , and atg1Δ cells were nitrogen - starved as ( A ) . Cell viability and respiratory competency was determined by phloxine B staining and TTC overlay technique as ( A ) , respectively . The black and gray areas indicate the percentage of viable cells that are respiratory competent or respiratory deficient , respectively .

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

(B) WT, and atg1Δ cells were nitrogen-starved as (A). Cell viability and respiratory competency was determined by phloxine B staining and TTC overlay technique as (A), respectively. The black and gray areas indicate the percentage of viable cells that are respiratory competent or respiratory deficient, respectively.


( C ) WT , atg1Δ , atg2Δ , atg7Δ , atg11Δ , atg15Δ , and atg32Δ cells were nitrogen - starved as ( A ) . Cell viability and respiratory competency was determined by phloxine B staining and TTC overlay technique as ( A ) , respectively . The black and gray areas indicate the percentage of viable cells that are respiratory competent or respiratory deficient , respectively . These data represent the average of three independent experiments and bars indicate standard deviations .

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(C) WT, atg1Δ, atg2Δ, atg7Δ, atg11Δ, atg15Δ, and atg32Δ cells were nitrogen-starved as (A). Cell viability and respiratory competency was determined by phloxine B staining and TTC overlay technique as (A), respectively. The black and gray areas indicate the percentage of viable cells that are respiratory competent or respiratory deficient, respectively. These data represent the average of three independent experiments and bars indicate standard deviations.


( A ) WT and atg1Δ cells expressing mitochondria targeted mCherry cultured in SD - N + 50 mM MES - KOH ( pH 6 . 2 ) medium for 120 hours were observed by fluorescent microscopy . Mitochondrial DNA was stained with SYBR green I , and mitochondria were visualized by mitochondria targeted mCherry . Scale bar , 2 µm .

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(A) WT and atg1Δ cells expressing mitochondria targeted mCherry cultured in SD-N +50 mM MES-KOH (pH 6.2) medium for 120 hours were observed by fluorescent microscopy. Mitochondrial DNA was stained with SYBR green I, and mitochondria were visualized by mitochondria targeted mCherry. Scale bar, 2 µm.


( B - C ) WT and atg1Δ cells were transferred to SD - N + 50 mM MES - KOH ( pH 6 . 2 ) medium for the indicated time . ROS accumulation was detected by DHE staining ( B ) . Each photo contains about 200 cells . Scale bar , 20 µm . ( C ) shows quantification of ROS accumulated cells ( n > 200 cells ) .

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(B-C) WT and atg1Δ cells were transferred to SD-N +50 mM MES-KOH (pH 6.2) medium for the indicated time. ROS accumulation was detected by DHE staining (B). Each photo contains about 200 cells. Scale bar, 20 µm. (C) shows quantification of ROS accumulated cells (n>200 cells).


( D ) WT and atg1Δ cells were transferred to SD - N + 50 mM MES - KOH ( pH 6 . 2 ) with 10 mM NAC for the indicated time . Cell viability was determined by phloxine B staining . Cells from these cultures were plated on YEPD agar and overlaid with TTC agar to examine respiratory competency . The black and gray areas indicate the percentage of viable cells that are respiratory competent or respiratory deficient , respectively . These data represent the average of three independent experiments and bars indicate standard

O O O O O B-CONTROLLED_VAR O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) WT and atg1Δ cells were transferred to SD-N +50 mM MES-KOH (pH 6.2) with 10 mM NAC for the indicated time. Cell viability was determined by phloxine B staining. Cells from these cultures were plated on YEPD agar and overlaid with TTC agar to examine respiratory competency. The black and gray areas indicate the percentage of viable cells that are respiratory competent or respiratory deficient, respectively. These data represent the average of three independent experiments and bars indicate standard deviations


( A ) WT and atg1Δ cells were transferred to SD - N + 50 mM MES - KOH ( pH 6 . 2 ) medium for the indicated time . Lysates were prepared using the alkaline - trichloroacetic acid method and subjected to immunoprecipitation with anti - Cox2 , anti - Cox4 , anti - Tim17 and anti - Pgk1 . Pgk1 was used as loading control .

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

(A) WT and atg1Δ cells were transferred to SD-N +50 mM MES-KOH (pH 6.2) medium for the indicated time. Lysates were prepared using the alkaline-trichloroacetic acid method and subjected to immunoprecipitation with anti-Cox2, anti-Cox4, anti-Tim17 and anti-Pgk1. Pgk1 was used as loading control.


( B ) WT and atg1Δ cells expressing Cta1 - 3 × FLAG or Ctt1 - 3 × FLAG were nitrogen - starved as in ( A ) for the indicated time . Cell lysates were prepared as ( A ) and subjected to immune precipitation with anti - FLAG and anti - Pgk1 . Pgk1 was used as loading control .

(B) WT and atg1Δ cells expressing Cta1-3×FLAG or Ctt1-3×FLAG were nitrogen-starved as in (A) for the indicated time. Cell lysates were prepared as (A) and subjected to immune precipitation with anti-FLAG and anti-Pgk1. Pgk1 was used as loading control.


( A ) WT and atg1Δ cells were transferred to non - buffered SD - N medium for the indicated time . Cell viability was determined by phloxine B staining . Nitrogen - starved cells were plated onto YEPD agar and overlaid with TTC agar to examine the respiratory competency of formed colonies . The black and gray areas indicate the percentage of viable cells that are respiratory competent or respiratory deficient , respectively .

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

(A) WT and atg1Δ cells were transferred to non-buffered SD-N medium for the indicated time. Cell viability was determined by phloxine B staining. Nitrogen-starved cells were plated onto YEPD agar and overlaid with TTC agar to examine the respiratory competency of formed colonies. The black and gray areas indicate the percentage of viable cells that are respiratory competent or respiratory deficient, respectively.


( B ) WT , atg1Δ , rho0 , and rho0 atg1Δ cells grown in YEPD medium were transferred to SD - N with or without 10 mM NAC for the indicated time . In the presence of NAC , medium pH was adjusted by using KOH . Cell viability and medium pH were examined by phloxine B staining and pH meter , respectively . These data represent the average of three independent experiments and bars indicate standard deviations .

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

(B) WT, atg1Δ, rho0, and rho0 atg1Δ cells grown in YEPD medium were transferred to SD-N with or without 10 mM NAC for the indicated time. In the presence of NAC, medium pH was adjusted by using KOH. Cell viability and medium pH were examined by phloxine B staining and pH meter, respectively. These data represent the average of three independent experiments and bars indicate standard deviations.


( A ) Quality of the cell fractionation assays was controlled by western blot by assessing the distribution of nuclear and cytosolic markers in the whole cell extract ( WCE ) , the cytosol and the nucleus . Protein lysates were isolated from mouse primary neocortical cells ( 14 days in culture ) treated for 3hrs with the transcription inhibitor triptolide ( 1µM ) . ( four independent cultures )

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Quality of the cell fractionation assays was controlled by western blot by assessing the distribution of nuclear and cytosolic markers in the whole cell extract (WCE), the cytosol and the nucleus. Protein lysates were isolated from mouse primary neocortical cells (14 days in culture) treated for 3hrs with the transcription inhibitor triptolide (1µM). (four independent cultures)


Efficiencies of bicuculline ( A ) stimulation were assessed by controlling the induction of ERK phosphorylation by immunostaining . Mouse primary neocortical cells ( 14 days in culture ) were stimulated with bicuculline ( 20µM ) to elevate the neuronal network excitation Cells were stained with anti - phospho - ERK ( green , bottom ) and anti - MAP2 ( white , top ) antibodies and counterstained with Hoechst ( blue , top ) . ( four independent cultures )

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

Efficiencies of bicuculline (A) stimulation were assessed by controlling the induction of ERK phosphorylation by immunostaining. Mouse primary neocortical cells (14 days in culture) were stimulated with bicuculline (20µM) to elevate the neuronal network excitation Cells were stained with anti-phospho-ERK (green, bottom) and anti-MAP2 (white, top) antibodies and counterstained with Hoechst (blue, top). (four independent cultures)


RT - PCR validations of regulated IR - transcripts through splicing upon bicuculline stimulation ( C ) Expression of the IR - isoforms and the spliced isoforms were analyzed by semi - quantitative PCR ( left panels ) . Means and SDs ( standard deviations ) of PIR values are shown beneath each panel . In addition , fold changes in the expression of the IR - transcripts ( red and orange ) and spliced ( dark grey ) isoforms were assessed with real - time qPCR using three different primer sets , as represented in the top scheme . Note that the Gria3 spliced transcript ( C ) does not correspond to the canonical mRNAs and presumably arise from a first step of recursive splicing and thereby likely require splicing completion to generate fully mature Gria3 transcripts ( Sibley et al , 2015 ) .

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

RT-PCR validations of regulated IR-transcripts through splicing upon bicuculline stimulation (C) Expression of the IR-isoforms and the spliced isoforms were analyzed by semi-quantitative PCR (left panels). Means and SDs (standard deviations) of PIR values are shown beneath each panel. In addition, fold changes in the expression of the IR-transcripts (red and orange) and spliced (dark grey) isoforms were assessed with real-time qPCR using three different primer sets, as represented in the top scheme. Note that the Gria3 spliced transcript (C) does not correspond to the canonical mRNAs and presumably arise from a first step of recursive splicing and thereby likely require splicing completion to generate fully mature Gria3 transcripts (Sibley et al, 2015).