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 )

O O O O B-GENEPROD O B-EXP_ASSAY I-EXP_ASSAY O B-ORGANISM O B-ORGANISM O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O O O O O O O B-ORGANISM O O O O O O O B-ORGANISM O O O O O O O O B-GENEPROD O O O O O O O

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 + / + )

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 )

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 + / + )

O O O O O O O O O O O O O O O O O O O O O O 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 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 )

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 )

O O O O B-TISSUE O B-SMALL_MOLECULE O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-SMALL_MOLECULE O O O O O B-ORGANISM I-ORGANISM 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-SMALL_MOLECULE O O O O O O O O O O O O O O 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-GENEPROD O O O O O B-GENEPROD O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-TISSUE O B-SMALL_MOLECULE O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O

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 B-TISSUE O B-SMALL_MOLECULE O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-ORGANISM I-ORGANISM O O O O O O O O O O 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-GENEPROD O O O O O B-GENEPROD O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-TISSUE O B-SMALL_MOLECULE O O B-EXP_ASSAY O O O O O O O O O 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-SMALL_MOLECULE O O O B-SMALL_MOLECULE 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-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-ORGANISM O O O O B-ORGANISM O O O B-ORGANISM O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O 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-SMALL_MOLECULE O O O B-SMALL_MOLECULE 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 .

O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-GENEPROD O O O O O O O O O O O O O O O O O O O O

(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 B-EXP_ASSAY O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O 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-CELL_TYPE I-CELL_TYPE O B-GENEPROD O O O O O O B-ORGANISM O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 B-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O O O O O O O O O O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-GENEPROD O O B-ORGANISM O O B-GENEPROD 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 B-EXP_ASSAY O O B-GENEPROD O O O O O O O O O O O O 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-CELL_TYPE I-CELL_TYPE O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O B-ORGANISM O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 .

O O O B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O O O O O O B-SUBCELLULAR O B-SUBCELLULAR O B-SUBCELLULAR O B-SUBCELLULAR O B-SUBCELLULAR O O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O B-SUBCELLULAR O B-SUBCELLULAR O B-SUBCELLULAR O O O O O O O O O B-SUBCELLULAR O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O B-SUBCELLULAR O B-SUBCELLULAR I-SUBCELLULAR O B-SUBCELLULAR O O O O B-GENEPROD I-GENEPROD O B-GENEPROD O O O O O B-SUBCELLULAR O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O B-SUBCELLULAR O O

(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 .

O O O B-EXP_ASSAY I-EXP_ASSAY O B-SUBCELLULAR O O O B-GENEPROD O O O O B-SUBCELLULAR O O O B-GENEPROD O O O O B-SUBCELLULAR O O O B-GENEPROD O O O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O

(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 ) .

O O O B-EXP_ASSAY O O O O O O B-SUBCELLULAR O O O O O O O O B-ORGANISM O O O 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-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 ) .

O O O B-ORGANISM O B-EXP_ASSAY O O O B-ORGANISM O 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-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-CELL_LINE O O O O O O O O O O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(E) 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 .

O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-ORGANISM B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 B-EXP_ASSAY O O O O O O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 .

O O O O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O B-CELL_TYPE I-CELL_TYPE O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O 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-SUBCELLULAR I-SUBCELLULAR O O O B-SUBCELLULAR I-SUBCELLULAR O O O 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) 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 .

O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD B-ORGANISM O O O O O O O O O O O O O B-GENEPROD O B-EXP_ASSAY O O O O O B-GENEPROD O B-EXP_ASSAY O O B-TISSUE O B-TISSUE I-TISSUE O

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 .

B-EXP_ASSAY O O O B-GENEPROD O B-GENEPROD O O O O O O O

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 .

O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O O O O 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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-TISSUE O O O O B-GENEPROD O O O B-GENEPROD O

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 .

B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O 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-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 .

B-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O 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 .

B-EXP_ASSAY I-EXP_ASSAY O O B-CELL_TYPE I-CELL_TYPE O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O O B-TISSUE O O O O O O O O B-TISSUE O O O O O O O O O O O O O O O O O O B-TISSUE I-TISSUE O O O O O O O O O O O O O O B-EXP_ASSAY O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O B-CELL_TYPE I-CELL_TYPE 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 B-EXP_ASSAY O O O O 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-EXP_ASSAY O 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 .

B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O O O O O O O O O B-TISSUE I-TISSUE O B-TISSUE O O B-GENEPROD O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O B-GENEPROD O O O O B-TISSUE O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY 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 .

B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O O O O O B-ORGANISM I-ORGANISM O O O O O O O B-GENEPROD O B-GENEPROD O O O O O B-TISSUE O B-TISSUE 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 B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O 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-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-GENEPROD O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY 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 B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD B-ORGANISM I-ORGANISM B-EXP_ASSAY O B-GENEPROD O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE B-EXP_ASSAY O O O O O O O B-GENEPROD O B-EXP_ASSAY O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-GENEPROD O O O 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 .

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O B-GENEPROD O 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 .

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD 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 .

B-EXP_ASSAY O O B-GENEPROD O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O B-EXP_ASSAY 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 .

B-EXP_ASSAY O O B-GENEPROD O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O B-EXP_ASSAY 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 .

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O O B-CELL_LINE I-CELL_LINE I-CELL_LINE 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 .

B-EXP_ASSAY O O B-GENEPROD O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O B-EXP_ASSAY 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 .

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O B-GENEPROD O O O O O O B-ORGANISM B-GENEPROD O B-CELL_TYPE O O B-CELL_TYPE O O O O O O O O O O O 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 .

B-EXP_ASSAY O O B-GENEPROD O O O B-ORGANISM B-GENEPROD O B-CELL_TYPE O O B-CELL_TYPE O O O O O O O O O O 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 .

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O B-EXP_ASSAY 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 .

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 .

B-EXP_ASSAY O O B-GENEPROD O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O O O O B-EXP_ASSAY O O O O

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 .

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 .

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O O O O O O O O O O O O O O B-EXP_ASSAY O O O O

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 .

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 .

B-EXP_ASSAY O O B-GENEPROD O O O O O O O O O O B-EXP_ASSAY 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 .

B-EXP_ASSAY O O B-GENEPROD O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O B-EXP_ASSAY 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 B-SMALL_MOLECULE O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O B-ORGANISM I-ORGANISM O B-SMALL_MOLECULE 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 ) .

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O O O O B-GENEPROD O O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O O O O B-GENEPROD O O O O O O O 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-SMALL_MOLECULE O B-SMALL_MOLECULE 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 .

B-EXP_ASSAY O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O B-SUBCELLULAR O O O B-SMALL_MOLECULE 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 ) .

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O B-GENEPROD O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE 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 ) .

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O O O O O O O B-GENEPROD O O O O B-GENEPROD O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE 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 ) .

B-EXP_ASSAY O O B-GENEPROD O O O O O O B-GENEPROD O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE 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 .

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O O O O O O B-SMALL_MOLECULE 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 ) .

B-EXP_ASSAY O O B-GENEPROD O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE 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 .

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O O O O B-GENEPROD O O B-GENEPROD O O O B-ORGANISM B-GENEPROD O B-GENEPROD O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O O O O O O O B-SMALL_MOLECULE 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 .

B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE 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 .

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O O O B-GENEPROD O B-CELL_LINE O O B-GENEPROD O B-GENEPROD O B-CELL_LINE 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 .

B-EXP_ASSAY I-EXP_ASSAY O O O O O B-GENEPROD O B-GENEPROD O B-CELL_LINE O O O O O O O O O O O O O O O O O O O O O O O O O O

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 .

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O B-GENEPROD O O B-CELL_LINE O O O O O O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE 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 .

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O O O O B-GENEPROD O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE 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 .

B-EXP_ASSAY O O B-GENEPROD O O O O O B-GENEPROD O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE 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 .

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O O O O O O B-GENEPROD O O O O B-GENEPROD O O B-ORGANISM O O O O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE 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 .

B-EXP_ASSAY O O B-GENEPROD O O O O O O O B-GENEPROD O B-ORGANISM O O O O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE 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 .

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O O O O B-GENEPROD O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE 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 .

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE 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 .

B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O B-GENEPROD O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE 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 ) .

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O O O O B-GENEPROD O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O B-GENEPROD O O O O O O O O B-GENEPROD O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE 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 ) .

B-EXP_ASSAY O O B-GENEPROD O O O O O O O O B-GENEPROD O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O B-GENEPROD O O O O O O O O B-GENEPROD O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE 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-GENEPROD O O O O O O O B-SMALL_MOLECULE O O O B-EXP_ASSAY 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 .

B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O B-EXP_ASSAY O

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 .

B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O B-SMALL_MOLECULE O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O B-SMALL_MOLECULE O O O O O O O O

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 .

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 ) .

B-EXP_ASSAY O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O B-GENEPROD O O B-EXP_ASSAY O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O 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 .

O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O B-GENEPROD O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O

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 .

B-EXP_ASSAY O B-GENEPROD O O O B-GENEPROD O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O B-SUBCELLULAR O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O

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 .

O B-EXP_ASSAY O O O O O B-GENEPROD O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O B-SUBCELLULAR O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O

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 B-SMALL_MOLECULE O O O O O B-GENEPROD O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O B-SMALL_MOLECULE O O O 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-EXP_ASSAY O B-EXP_ASSAY O O O O O O O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O B-SMALL_MOLECULE O 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 .

B-EXP_ASSAY I-EXP_ASSAY O O O O O B-GENEPROD O B-GENEPROD O B-SMALL_MOLECULE O B-GENEPROD O O B-EXP_ASSAY O B-SMALL_MOLECULE I-SMALL_MOLECULE O O B-GENEPROD O B-GENEPROD O B-SMALL_MOLECULE O B-GENEPROD O O O O O O B-SMALL_MOLECULE O O O O O B-EXP_ASSAY O B-EXP_ASSAY 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 .

B-EXP_ASSAY I-EXP_ASSAY O O O O O B-SMALL_MOLECULE O B-GENEPROD O O B-EXP_ASSAY O B-SMALL_MOLECULE O O O B-GENEPROD O B-GENEPROD O B-SMALL_MOLECULE O B-GENEPROD O O O O B-EXP_ASSAY 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 .

B-EXP_ASSAY I-EXP_ASSAY O O O O O B-GENEPROD O B-GENEPROD O B-SMALL_MOLECULE O B-GENEPROD O O B-EXP_ASSAY O B-SMALL_MOLECULE O O O B-GENEPROD O B-GENEPROD O B-SMALL_MOLECULE O B-GENEPROD O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE 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 .

B-EXP_ASSAY I-EXP_ASSAY O O O O O B-GENEPROD O B-GENEPROD O O B-EXP_ASSAY O B-SMALL_MOLECULE O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O B-SMALL_MOLECULE O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O B-EXP_ASSAY O B-SMALL_MOLECULE 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 .

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 .

B-EXP_ASSAY O O B-GENEPROD O O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 .

B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O O B-EXP_ASSAY 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 .

O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O

(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 .

O O O O O O O B-GENEPROD O O B-SMALL_MOLECULE O O O O O O O O O O O B-EXP_ASSAY O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O

(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 .

O O O O O B-GENEPROD O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O 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-EXP_ASSAY O O O O O O O O O O

(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-GENEPROD O O B-SMALL_MOLECULE O O O O O O O O B-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O O O O O 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 .

O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O B-SMALL_MOLECULE O O O O O O O O B-EXP_ASSAY O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O 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) 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 .

O O O O O B-GENEPROD O O B-SUBCELLULAR O B-GENEPROD O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-SUBCELLULAR I-SUBCELLULAR O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-SUBCELLULAR O O O B-SUBCELLULAR O B-GENEPROD O O O O O O O

(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 ) .

O O O O O O O B-GENEPROD O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O B-EXP_ASSAY O O O O B-EXP_ASSAY 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-EXP_ASSAY O O O O O O O O O

(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-GENEPROD O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O B-SMALL_MOLECULE O O O O O O B-EXP_ASSAY O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O 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-GENEPROD O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O 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-EXP_ASSAY O O O B-GENEPROD O O O B-GENEPROD O O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD 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 .

O O O O O B-GENEPROD O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O 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-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O O

(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-GENEPROD O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O B-EXP_ASSAY O O O O B-EXP_ASSAY I-EXP_ASSAY O 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-GENEPROD O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O O B-EXP_ASSAY O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY 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 B-EXP_ASSAY I-EXP_ASSAY O O O O O B-SUBCELLULAR O B-SUBCELLULAR O O O O O O O O O O O B-SUBCELLULAR O O B-SUBCELLULAR O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O B-SMALL_MOLECULE 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-SMALL_MOLECULE O O O O O O O O O O O B-GENEPROD B-EXP_ASSAY O B-EXP_ASSAY O B-ORGANISM O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O B-EXP_ASSAY O O O B-EXP_ASSAY O B-GENEPROD O O O O O O O O B-GENEPROD O O O O O O O B-EXP_ASSAY O B-SMALL_MOLECULE 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 ) .

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O 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-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O 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-GENEPROD 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).


RT - PCR validations of regulated IR - transcripts through splicing upon BDNF stimulation ( D ) 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 .

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O 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-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O

RT-PCR validations of regulated IR-transcripts through splicing upon BDNF stimulation (D) 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.


( A ) Heatmap of the IR - transcript expression values in control , bicuculline - stimulated and BDNF - stimulated conditions . IR - transcripts regulated in at least one condition are displayed ( fold change of intron - retaining transcript expression ≥ 20 % and | z - score | ≥ 1 . 5 ) .

O O O O O O O O O B-EXP_ASSAY O O O O B-SMALL_MOLECULE O O O B-GENEPROD O O O O O O O O O O O O O O O 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) Heatmap of the IR-transcript expression values in control, bicuculline-stimulated and BDNF-stimulated conditions. IR-transcripts regulated in at least one condition are displayed (fold change of intron-retaining transcript expression ≥ 20% and |z-score| ≥ 1.5).


RT - PCR validations of IR - transcripts commonly regulated ( C ) Expression of the IR - transcripts and the spliced isoforms were analyzed by semi - quantitative PCR ( left panels ) . Means and SDs of PIR values are shown beneath each panel . In addition , fold changes ( FC ) in the expression of the IR - transcripts ( red and orange ) and spliced isoforms ( dark grey ) were assessed with real - time qPCR .

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O 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-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O

RT-PCR validations of IR-transcripts commonly regulated (C) Expression of the IR-transcripts and the spliced isoforms were analyzed by semi-quantitative PCR (left panels). Means and SDs of PIR values are shown beneath each panel. In addition, fold changes (FC) in the expression of the IR-transcripts (red and orange) and spliced isoforms (dark grey) were assessed with real-time qPCR.


RT - PCR validations of IR - transcripts , specifically regulated upon bicuculline stimulation ( D ) , specifically regulated upon BDNF stimulation ( E ) , and bi - directionally regulated ( F ) . Expression of the IR - transcripts and the spliced isoforms were analyzed by semi - quantitative PCR ( left panels ) . Means and SDs of PIR values are shown beneath each panel . In addition , fold changes ( FC ) in the expression of the IR - transcripts ( red and orange ) and spliced isoforms ( dark grey ) were assessed with real - time qPCR .

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O B-GENEPROD O O O 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-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O 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-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O

RT-PCR validations of IR-transcripts , specifically regulated upon bicuculline stimulation (D), specifically regulated upon BDNF stimulation (E), and bi-directionally regulated (F). Expression of the IR-transcripts and the spliced isoforms were analyzed by semi-quantitative PCR (left panels). Means and SDs of PIR values are shown beneath each panel. In addition, fold changes (FC) in the expression of the IR-transcripts (red and orange) and spliced isoforms (dark grey) were assessed with real-time qPCR.


A A representative photograph showing the co - cultured DRG and DH neurons . The DRG neurons were expressed with Spy - pHluorin for imaging the synaptic transmission . Scale bar , 10 μm . B Images of a presynaptic bouton marked in ( A ) showing the Spy - pHluorin fluorescence at 20 s before ( - 20 s , pre - stimulus ) , 20 s , 40 s , and 120 s after electrical stimulation ( post - stimulus ) in 0Ca2 + ( upper panel ) or 2Ca2 + ( lower panel ) solution . C Averaged fluorescence changes ( ΔF / F0 ) of Spy - pHluorin in 0Ca2 + ( left ) or 2Ca2 + solution ( right ) in response to the same electrical stimulation ( 20 Hz , 20 s ) ( n = 45 puncta from 6 cells for 0Ca2 + and 57 puncta from 6 cells for 2Ca2 + ) . The shadow in the traces represents the error bars ( s . e . m ) of each point .

O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY B-TISSUE O B-TISSUE B-CELL_TYPE O O B-TISSUE B-CELL_TYPE O O O B-GENEPROD O B-GENEPROD O O O B-SUBCELLULAR O O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O 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-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O 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-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O

A A representative photograph showing the co-cultured DRG and DH neurons. The DRG neurons were expressed with Spy-pHluorin for imaging the synaptic transmission. Scale bar, 10 μm. B Images of a presynaptic bouton marked in (A) showing the Spy-pHluorin fluorescence at 20 s before (-20 s, pre-stimulus), 20 s, 40 s, and 120 s after electrical stimulation (post-stimulus) in 0Ca2+ (upper panel) or 2Ca2+ (lower panel) solution. C Averaged fluorescence changes (ΔF/F0) of Spy-pHluorin in 0Ca2+ (left) or 2Ca2+ solution (right) in response to the same electrical stimulation (20 Hz, 20 s) (n = 45 puncta from 6 cells for 0Ca2+ and 57 puncta from 6 cells for 2Ca2+). The shadow in the traces represents the error bars (s.e.m) of each point.


D - F The same as in ( A - C ) , but the experiments were performed in cultured hippocampal neurons ( n = 70 puncta from 3 cells for 0Ca2 + and 72 puncta from 3 cells for 2Ca2 + ) .

O O O O O O O O O O O O O O O O O O O O B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O O

D-F The same as in (A-C), but the experiments were performed in cultured hippocampal neurons (n = 70 puncta from 3 cells for 0Ca2+ and 72 puncta from 3 cells for 2Ca2+).


B Representative western blots ( upper ) and analysis ( lower ) of the expression levels of Syb2 in control ( Ctrl ) and Syb2 - cKO DRG neurons ( n = 3 per group ) . Control DRG neurons is from homozygous floxed Syb2 - null mice infected with AAV5 - CAG - EGFP virus .

O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O B-GENEPROD O O O O O O B-GENEPROD O O B-TISSUE B-CELL_TYPE O O O O O O O O O B-TISSUE B-CELL_TYPE O O O O B-GENEPROD O O B-ORGANISM O O B-ORGANISM O O O B-GENEPROD O O

B Representative western blots (upper) and analysis (lower) of the expression levels of Syb2 in control (Ctrl) and Syb2-cKO DRG neurons (n = 3 per group). Control DRG neurons is from homozygous floxed Syb2-null mice infected with AAV5-CAG-EGFP virus.


C Evoked EPSCs and statistics from DH neurons co - cultured with control ( Ctrl ) or Syb2 - cKO DRG cells ( cKO ) in 0Ca2 + solution ( n = 13 cells for Ctrl and 11 for cKO ) .

O O B-EXP_ASSAY O O O B-TISSUE B-CELL_TYPE B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O B-GENEPROD O O B-TISSUE O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O

C Evoked EPSCs and statistics from DH neurons co-cultured with control (Ctrl) or Syb2-cKO DRG cells (cKO) in 0Ca2+ solution (n = 13 cells for Ctrl and 11 for cKO).


D Evoked EPSCs and statistics from DH neurons co - cultured with DRG neurons before ( black ) and after ( red ) applying 1 μM ω - conotoxin - GVIA ( GVIA ) in 0Ca2 + bath ( n = 9 cells ) .

O O B-EXP_ASSAY O O O B-TISSUE B-CELL_TYPE B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-TISSUE B-CELL_TYPE O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O O O O O O

D Evoked EPSCs and statistics from DH neurons co-cultured with DRG neurons before (black) and after (red) applying 1 μM ω-conotoxin-GVIA (GVIA) in 0Ca2+ bath (n = 9 cells).


F Left panels , evoked EPSCs from DH neurons co - cultured with scrambled shRNA control ( Ctrl ) or Cav2 . 2 - KD ( sh - 1 / sh - 2 ) DRG neurons in 0Ca2 + solution . Right panel , quantification of evoked EPSCs ( n = 17 cells for Ctrl , 12 for sh - 1 , and 19 for sh - 2 ) . EPSCs were evoked by local electrical stimulation ( Estim , at arrows ) .

O O O O O B-EXP_ASSAY O B-TISSUE B-CELL_TYPE O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O B-TISSUE B-CELL_TYPE O O B-SMALL_MOLECULE O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O

F Left panels, evoked EPSCs from DH neurons co-cultured with scrambled shRNA control (Ctrl) or Cav2.2-KD (sh-1/sh-2) DRG neurons in 0Ca2+ solution. Right panel, quantification of evoked EPSCs (n = 17 cells for Ctrl, 12 for sh-1, and 19 for sh-2). EPSCs were evoked by local electrical stimulation (Estim, at arrows).


C Western blots of the lysates of HEK293T cells , resolved on SDS - PAGE and stained with a pan - arrestin antibody . mt = mock transfected . Bpa : Benzoyl - Phenylalanine . Arrestins were equipped with a 3xHA tag at the C - terminus , which increases their size of about 3 kDa compared to the endogenous proteins ( βarr1 47 kDa , βarr2 46 kDa ) . βarr1 and βarr2 were transfected at 1 / 3 DNA compared to the corresponding TAG - mutants .

O B-EXP_ASSAY I-EXP_ASSAY O O O O B-CELL_LINE O O O O B-SMALL_MOLECULE O B-EXP_ASSAY O B-EXP_ASSAY O O O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O 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-GENEPROD O O O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O

C Western blots of the lysates of HEK293T cells, resolved on SDS-PAGE and stained with a pan-arrestin antibody. mt = mock transfected. Bpa: Benzoyl-Phenylalanine. Arrestins were equipped with a 3xHA tag at the C-terminus, which increases their size of about 3 kDa compared to the endogenous proteins (βarr1 47 kDa, βarr2 46 kDa). βarr1 and βarr2 were transfected at 1/3 DNA compared to the corresponding TAG-mutants.


Each panel represents immunoblots of whole cell lysates stained with an anti - HA antibody detecting arrestin . In the left panels GPCRs were combined with βarr1 variants , in the right panels with βarr2 variants . Residues replaced by Bpa are indicated above each lane . Covalent arrestin - receptor complexes are expected between 100 - 200 kDa , considering the size of the receptors ( V2R 41 . 5 kDa , PTH1R 63 . 8 kDa , CRF1R , 46 . 3 kDa ) and their glycosylation . A PTH1R , agonist PTH ( 1 - 34 ) B CRF1R , agonist Urocortin 1 ( Ucn1 ) C V2R , agonist arginine - vasopressin ( AVP ) D No receptor .

O O O B-EXP_ASSAY O O O O B-EXP_ASSAY O O O O B-GENEPROD O O O O O O O O O O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O O B-SMALL_MOLECULE O O 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-GENEPROD O O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O O O O O O O B-GENEPROD O O B-SMALL_MOLECULE O O O O O O B-GENEPROD O O B-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-GENEPROD O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O

Each panel represents immunoblots of whole cell lysates stained with an anti-HA antibody detecting arrestin. In the left panels GPCRs were combined with βarr1 variants, in the right panels with βarr2 variants. Residues replaced by Bpa are indicated above each lane. Covalent arrestin-receptor complexes are expected between 100-200 kDa, considering the size of the receptors (V2R 41.5 kDa, PTH1R 63.8 kDa, CRF1R, 46.3 kDa) and their glycosylation. A PTH1R, agonist PTH(1-34) B CRF1R, agonist Urocortin 1 (Ucn1) C V2R, agonist arginine-vasopressin (AVP) D No receptor.


Aliquots from agonist - treated samples shown in Figure 2 were deglycosylated with PNGaseF , run on SDS - PAGE and immunoblotted with antibodies detecting arrestin ( upper panels , α - HA ) and the GPCRs ( as indicated , CRF1R is equipped with a N - terminal FLAG , V2R carries a C - terminal 1D4 epitope ) .

O O O O O O O O O O O O O B-GENEPROD O O O B-SMALL_MOLECULE O B-EXP_ASSAY O B-EXP_ASSAY O O O O O O O O O O B-GENEPROD O O O O O O O O B-GENEPROD O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O

Aliquots from agonist-treated samples shown in Figure 2 were deglycosylated with PNGaseF, run on SDS-PAGE and immunoblotted with antibodies detecting arrestin (upper panels, α-HA) and the GPCRs (as indicated, CRF1R is equipped with a N-terminal FLAG, V2R carries a C-terminal 1D4 epitope).


A SDS - PAGE and immunoblots of immunoprecipitated samples . Sample 1 was immunoprecipitated without previous UV treatment . Sample 2 is the product of irradiation of sample 1 . Sample 3 was treated with UV light before immunoprecipitation . All samples were run in parallel on the same gel .

O B-SMALL_MOLECULE O B-EXP_ASSAY O B-EXP_ASSAY O B-EXP_ASSAY O O O O O B-EXP_ASSAY 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-EXP_ASSAY O O O O O O O O O O O O

A SDS-PAGE and immunoblots of immunoprecipitated samples. Sample 1 was immunoprecipitated without previous UV treatment. Sample 2 is the product of irradiation of sample 1. Sample 3 was treated with UV light before immunoprecipitation. All samples were run in parallel on the same gel.


B Size exclusion chromatography ( SEC ) of Sample 3 .

B Size exclusion chromatography (SEC) of Sample 3.


C Western blot of the fractions eluted by SEC .

O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-EXP_ASSAY O

C Western blot of the fractions eluted by SEC.


MALDI - TOF analysis of the SEC fractions VR2 and VR3 . The spectrum of sample VR2 shows a [ M + H ] + peak featuring twice the mass ( ~ 108 000 Da ) of the [ M + H ] + peak of the βarr1 monomer ( sample VR3 , ~ 50 400 Da ) .

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O 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-GENEPROD O O O O O O O O O O O

MALDI-TOF analysis of the SEC fractions VR2 and VR3. The spectrum of sample VR2 shows a [M+H]+ peak featuring twice the mass (~108 000 Da) of the [M+H]+ peak of the βarr1 monomer (sample VR3, ~50 400 Da).


C Western blot of whole cell lysates from HEK293T cells expressing βarr1 mutated as indicated in the top lines . All samples were run in parallel on the same gel .

O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-CELL_LINE O O B-GENEPROD O O O O O O O O O O O O O O O O O O O

C Western blot of whole cell lysates from HEK293T cells expressing βarr1 mutated as indicated in the top lines. All samples were run in parallel on the same gel.


( E ) Western blot of whole cells lysates stained with an α - HA antibody . Residues exchanged with BrEtY are indicated in the upper row , mutations at PTH1R are indicated on the left side . The red arrows indicate the two most prominent signals . Red squares indicate signals that vanish upon removal of specific Cys residues in PTH1R .

O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-EXP_ASSAY O O O O B-GENEPROD O O O O O B-SMALL_MOLECULE O O O O O O O O O B-GENEPROD O O O 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-SMALL_MOLECULE O O B-GENEPROD O

(E) Western blot of whole cells lysates stained with an α-HA antibody. Residues exchanged with BrEtY are indicated in the upper row, mutations at PTH1R are indicated on the left side. The red arrows indicate the two most prominent signals. Red squares indicate signals that vanish upon removal of specific Cys residues in PTH1R.


A . Western blot detection of PEX13 , ATG7 , SMURF1 , and FANCC in HeLa / GFP - LC3cells transfected with the indicated siRNA . Asterisk denotes non - specific band .

O O B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O B-GENEPROD O B-CELL_LINE O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O

A. Western blot detection of PEX13, ATG7, SMURF1, and FANCC in HeLa/GFP-LC3cells transfected with the indicated siRNA. Asterisk denotes non-specific band.


B . Representative images of GFP - LC3 colocalization with mCherry - capsid at 10 h after Sindbis virus ( strain AO30 ) infection of HeLa / GFP - LC3 cells treated with the indicated siRNA . siPEX13 oligo # 2 is shown ; similar results were observed with three other siPEX13oligos . Arrowheads denote representative colocalized GFP - LC3 / mCherry - capsid puncta . Scale bars , 10 μm .

O O O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O O O O B-ORGANISM I-ORGANISM O O O O O O B-CELL_LINE O B-GENEPROD O B-GENEPROD O O O 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-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O

B. Representative images of GFP-LC3 colocalization with mCherry-capsid at 10 h after Sindbis virus (strain AO30) infection of HeLa/GFP-LC3 cells treated with the indicated siRNA. siPEX13 oligo #2 is shown; similar results were observed with three other siPEX13oligos. Arrowheads denote representative colocalized GFP-LC3/mCherry-capsid puncta. Scale bars, 10 μm.