Figure 2Expression 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 .

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Figure 2Expression 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.


Figure 3Flow 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 .

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Figure 3Flow 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.


Figure 4Flow 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 . 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 . Pdx1 and Sox17 expression levels in E9 . 5 VPR and DPR cells .

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

Figure 4Flow 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.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.Pdx1 and Sox17 expression levels in E9.5 VPR and DPR cells.


Figure 5Sagittal 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 ) .

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Figure 5Sagittal 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).


Figure 1qRT - 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 . 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 . 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 . 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 .

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Figure 1qRT-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.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.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.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.


Figure 2qRT - 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 . 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 . 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 . 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 .

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Figure 2qRT-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.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.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.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.


Figure 3Frequencies 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 ) . Immunostaining for EEA1 ( red ) ( F ) and LAMP2 ( red ) ( G ) in THP - 1 cells stimulated with FITC - ecGAMP ( 5 μg / ml , green ) for 30 min , nucleus in blue ( DAPI ) . Data are representative of 3 independent experiments . Scale bar , 10 μm . qRT - PCR detection of IFNB1 and IL6 mRNA in THP - 1 cells ( H , I ) and mBMDMs ( J , K ) stimulated with ecGAMP ( 5 μg / ml ) and icGAMP ( 0 . 1 μg / ml ) , respectively , for 4 h in presence of DMSO or bafilomycin A1 ( BafA ) ( 1 μM ) .

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

Figure 3Frequencies 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).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).


Figure 4qRT - 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 ) . 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 ) . 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 . 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 . 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 .

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Figure 4qRT-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).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).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.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.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.


Figure 5qRT - 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 . 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 . 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 . 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 ) . 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 ) .

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Figure 5qRT-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.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.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.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).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).


Figure 6Purified 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 . 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 . 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 B-PANEL_START O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6Purified 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.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.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.


Figure 7Immunostaining 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 . 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 . 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 . Western blot detection of the presence of cGAS and STING in the immunoprecipitates of streptavidin beads . THP - 1 cells were stimulated with biotin - ecGAMP ( 5 μg / ml ) or biotin - icGAMP ( 1 μg / ml ) for indicated time and cell lysates were harvested for IP with streptavidin beads . Data ( A - B , D - G ) are representative of 3 independent experiments .

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

Figure 7Immunostaining 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.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.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.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.


Figure 8qRT - 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 . 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 .

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Figure 8qRT-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.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.


Figure 1 ( A ) WT and atg1Δ cells grown in YEPD medium were transferred to SD - N or SD - N + 50 mM MES - KOH ( pH 6 . 2 ) medium for 120 hours , and dead cells stained by phloxine B were observed by fluorescent microscopy . Scale bar , 25 µm . ( B - C ) WT and atg1Δ cells grown nitrogen starved as ( A ) for the indicated times . Cell viability ( B ) and medium pH ( C ) were examined by phloxine B staining and pH meter , respectively . These data represent the average of three independent experiments and bars indicate standard deviations .

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Figure 1(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.


Figure 2 ( 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 . ( 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2(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.(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.


Figure 3 ( 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 ) . ( 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3(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).(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


Figure 4 ( 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4(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.


Figure 5 ( 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5(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.


Figure 1 ( 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 )

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Figure 1(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)


Figure 2Efficiencies 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 )

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Figure 2Efficiencies 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)


Figure 3RT - 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 .

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Figure 3RT-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.


Figure 5 ( 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 . 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 .

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

Figure 5(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.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.


Figure 2A 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 + ) .

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Figure 2A 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+).


Figure 5B 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 ) . 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 ) .

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Figure 5B 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).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).


Figure 1C 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 .

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Figure 1C 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.


Figure 2Each 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 .

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Figure 2Each 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.


Figure 3Aliquots 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 ) .

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Figure 3Aliquots 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).


Figure 4A 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 . 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 ) .

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Figure 4A 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.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).


Figure 5C 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 .

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Figure 5C 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.


Figure 1A . 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 . C . Quantification of colocalized GFP - LC3 and mCherry - capsid puncta normalized to the number of mCherry - capsid puncta per cell ( % colocalization ) in the experiment shown in ( B ) . Bars are mean ± SEM of triplicate samples ( ~ 100 cells analyzed per sample ) . Similar results were observed in more than three independent experiments . * * * p < 0 . 001 ; one - way ANOVA with adjustment for multiple comparisons . See Fig EV1A and B for quantification of mCherry - capsid puncta per cell and GFP - LC3 puncta per cell from the same experiment . D . Western blot detection of Pex13 in MEFs of indicated genotype . Asterisks denote non - specific bands . E . Quantification of colocalized GFP - LC3 and mCherry - capsid puncta normalized to the number of mCherry - capsid puncta per cell ( % colocalization ) at 8 h after Sindbis virus ( AO28 strain ) infection in MEFs of the indicated genotype . Bars are mean ± SEM of triplicate samples ( ~ 100 cells analyzed per sample ) . Similar results were observed in more than three independent experiments . * p < 0 . 05 ; two - tailed unpaired t - test . See Fig EV1G and H for quantification of mCherry - capsid puncta per cell and GFP - LC3 puncta per cell from the same experiment . F . Western blot detection of p62 and LC3 in MEFs of indicated genotype treated with 100 nM Baf A1 or DMSO vehicle and cultured in normal medium ( starvation " - " ) or EBSS ( starvation " + " ) for 3 h . Similar results were observed in three independent experiments . G . Quantification of GFP - LC3 puncta in MEFs of indicated genotype treated with 10 nM Baf A1 or DMSO vehicle and cultured in normal medium or HBSS ( starvation " + " ) for 3 h . Bars are mean ± SEM of triplicate samples ( ~ 100 cells analyzed per sample ) . * p < 0 . 05 ; two - tailed unpaired t - test . NC , nonsilencing control .

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Figure 1A. 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.C. Quantification of colocalized GFP-LC3 and mCherry-capsid puncta normalized to the number of mCherry-capsid puncta per cell (% colocalization) in the experiment shown in (B). Bars are mean ± SEM of triplicate samples (~100 cells analyzed per sample). Similar results were observed in more than three independent experiments. ***p<0.001; one-way ANOVA with adjustment for multiple comparisons. See Fig EV1A and B for quantification of mCherry-capsid puncta per cell and GFP-LC3 puncta per cell from the same experiment.D. Western blot detection of Pex13 in MEFs of indicated genotype. Asterisks denote non-specific bands.E. Quantification of colocalized GFP-LC3 and mCherry-capsid puncta normalized to the number of mCherry-capsid puncta per cell (% colocalization) at 8 h after Sindbis virus (AO28 strain) infection in MEFs of the indicated genotype. Bars are mean ± SEM of triplicate samples (~100 cells analyzed per sample). Similar results were observed in more than three independent experiments. *p<0.05; two-tailed unpaired t-test. See Fig EV1G and H for quantification of mCherry-capsid puncta per cell and GFP-LC3 puncta per cell from the same experiment.F. Western blot detection of p62 and LC3 in MEFs of indicated genotype treated with 100 nM Baf A1 or DMSO vehicle and cultured in normal medium (starvation "-") or EBSS (starvation "+") for 3 h. Similar results were observed in three independent experiments.G. Quantification of GFP-LC3 puncta in MEFs of indicated genotype treated with 10 nM Baf A1 or DMSO vehicle and cultured in normal medium or HBSS (starvation "+") for 3 h. Bars are mean ± SEM of triplicate samples (~100 cells analyzed per sample). *p<0.05; two-tailed unpaired t-test. NC, nonsilencing control.


Figure 2A . Representative images of Parkin - mediated clearance of mitochondrial outer membrane protein TOMM20 in HeLa / Parkin cells treated with indicated siRNA 16 h after treatment with 10 μM CCCP or DMSO vehicle control . siPEX13 oligo # 2 is shown ; similar results were observed with three other siPEX13 oligos . Scale bars , 20 μm . B . Quantification of TOMM20 clearance in the experiment shown in ( A ) . Results represent mean ± SEM of triplicate samples ( ~ 100 cells analyzed per sample ) . Similar results were observed in more than three independent experiments . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ; one - way ANOVA with adjustment for multiple comparisons . C . Representative images of Parkin - mediated clearance of mitochondrial double - stranded DNA ( mtDNA ) in HeLa / HA - Parkin cells treated with indicated siRNA 8 h after treatment with 2 . 5 μM oligomycin and 250 nM antimycin A ( OA ) or DMSO vehicle control . siPEX13 oligo # 1 is shown ; similar results were observed with three other siPEX13 oligos . Nuclear DNA staining was masked using DAPI . Scale bars , 20 μm . D . Quantification of mtDNA clearance in the experiment shown in ( C ) . Results represent box plots of ~ 300 cells analyzed per sample . Whiskers represent 5 % - 95 % range , and each outlier is represented by a dot . Similar results were observed in three independent experiments . * * * * p < 0 . 0001 ; Kruskal - Wallis H - test . E . Representative images of TOMM20 fragmentation or compaction around perinuclear region in MEFs of the indicated genotype 24 h after treatment with 30 μM CCCP or DMSO vehicle control . White lines indicate cell borders . Scale bars , 20 μm . F . Quantification of percentage of cells in experiment shown in ( E ) with accumulation of fragmented mitochondria after CCCP treatment . Results represent mean ± SEM of triplicate samples ( ~ 100 cells analyzed per sample ) . Similar results were observed in three independent experiments . * * p < 0 . 01 ; two - tailed unpaired t - test . G . Electron microscopic analysis of E18 . 5 mouse embryonic liver , heart , and skeletal muscle . Shown are representative images from one mouse . Similar results were observed in three mice per genotype . Arrowheads indicate damaged mitochondria . Scale bars , 1 μm .

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Figure 2A. Representative images of Parkin-mediated clearance of mitochondrial outer membrane protein TOMM20 in HeLa/Parkin cells treated with indicated siRNA 16 h after treatment with 10 μM CCCP or DMSO vehicle control. siPEX13 oligo #2 is shown; similar results were observed with three other siPEX13 oligos. Scale bars, 20 μm.B. Quantification of TOMM20 clearance in the experiment shown in (A). Results represent mean ± SEM of triplicate samples (~100 cells analyzed per sample). Similar results were observed in more than three independent experiments. *p<0.05, **p<0.01, ***p<0.001; one-way ANOVA with adjustment for multiple comparisons.C. Representative images of Parkin-mediated clearance of mitochondrial double-stranded DNA (mtDNA) in HeLa/HA-Parkin cells treated with indicated siRNA 8 h after treatment with 2.5 μM oligomycin and 250 nM antimycin A (OA) or DMSO vehicle control. siPEX13 oligo #1 is shown; similar results were observed with three other siPEX13 oligos. Nuclear DNA staining was masked using DAPI. Scale bars, 20 μm.D. Quantification of mtDNA clearance in the experiment shown in (C). Results represent box plots of ~300 cells analyzed per sample. Whiskers represent 5%-95% range, and each outlier is represented by a dot. Similar results were observed in three independent experiments. ****p<0.0001; Kruskal-Wallis H-test.E. Representative images of TOMM20 fragmentation or compaction around perinuclear region in MEFs of the indicated genotype 24 h after treatment with 30 μM CCCP or DMSO vehicle control. White lines indicate cell borders. Scale bars, 20 μm.F. Quantification of percentage of cells in experiment shown in (E) with accumulation of fragmented mitochondria after CCCP treatment. Results represent mean ± SEM of triplicate samples (~100 cells analyzed per sample). Similar results were observed in three independent experiments. **p<0.01; two-tailed unpaired t-test.G. Electron microscopic analysis of E18.5 mouse embryonic liver, heart, and skeletal muscle. Shown are representative images from one mouse. Similar results were observed in three mice per genotype. Arrowheads indicate damaged mitochondria. Scale bars, 1 μm.


Figure 3A . Western blot detection of PEX13 in HeLa / Parkin cells transfected with indicated siRNA and siRNA - resistant PEX13 plasmid . To achieve similar PEX13 protein expression levels , 0 . 75 μg PEX13 I326T and PEX13 W313G plasmids were transfected compared to 0 . 25 μg WT PEX13 . Total plasmid level was adjusted using empty vector . Asterisk denotes non - specific band . B . Representative images of Parkin - mediated clearance of TOMM20 in HeLa / Parkin cells treated with PEX13 siRNA and transfected with indicated PEX13 siRNA - resistant plasmid and then treated with CCCP ( 10 μM , 16 h ) . Scale bars , 20 μm . See Fig EV3B for representative images of mitochondrial morphology ( TOMM20 staining ) in control cells treated with DMSO . C . Quantification of experiment shown in ( B ) . Results represent mean ± SEM of triplicate samples ( ~ 100 cells analyzed per sample ) . Similar results were observed in three independent experiments . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ; two - tailed unpaired t - test . D . Western blot detection of PEX13 expression in wild - type ( WT ) and PEX13 W313G ( W313G ) mutant primary human fibroblasts . E . Representative images of Parkin - mediated clearance of TOMM20 in wild - type ( WT ) and PEX13 W313G ( W313G ) mutant primary human fibroblasts treated with Oligomycin ( 2 . 5 μM ) + Antimycin A ( 250 nM ) for 24 h . Scale bars , 20 μm . W313G cells varied in morphology ; outlined cell in upper right panel shows a representative cell with larger size and abnormal mitochondrial morphology ( see Fig EV3C for higher resolution imaging ) and outlined cell in lower right panel shows a representative cell with accumulation of fragmented mitochondria that would be scored as positive in ( F ) . F . Quantification of experiment shown in ( E ) . Results represent mean ± SEM of 5 groups of 10 images of random fields of cells ( > 350 cells analyzed per sample ) . Similar results were observed in two independent experiments . * * * p < 0 . 0001 ; two - tailed unpaired t - test .

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

Figure 3A. Western blot detection of PEX13 in HeLa/Parkin cells transfected with indicated siRNA and siRNA-resistant PEX13 plasmid. To achieve similar PEX13 protein expression levels, 0.75 μg PEX13 I326T and PEX13 W313G plasmids were transfected compared to 0.25 μg WT PEX13. Total plasmid level was adjusted using empty vector. Asterisk denotes non-specific band.B. Representative images of Parkin-mediated clearance of TOMM20 in HeLa/Parkin cells treated with PEX13 siRNA and transfected with indicated PEX13 siRNA-resistant plasmid and then treated with CCCP (10 μM, 16 h). Scale bars, 20 μm. See Fig EV3B for representative images of mitochondrial morphology (TOMM20 staining) in control cells treated with DMSO.C. Quantification of experiment shown in (B). Results represent mean ± SEM of triplicate samples (~100 cells analyzed per sample). Similar results were observed in three independent experiments. *p<0.05, **p<0.01,***p<0.001; two-tailed unpaired t-test.D. Western blot detection of PEX13 expression in wild-type (WT) and PEX13 W313G (W313G) mutant primary human fibroblasts.E. Representative images of Parkin-mediated clearance of TOMM20 in wild-type (WT) and PEX13 W313G (W313G) mutant primary human fibroblasts treated with Oligomycin (2.5 μM) + Antimycin A (250 nM) for 24 h. Scale bars, 20 μm. W313G cells varied in morphology; outlined cell in upper right panel shows a representative cell with larger size and abnormal mitochondrial morphology (see Fig EV3C for higher resolution imaging) and outlined cell in lower right panel shows a representative cell with accumulation of fragmented mitochondria that would be scored as positive in (F).F. Quantification of experiment shown in (E). Results represent mean ± SEM of 5 groups of 10 images of random fields of cells (>350 cells analyzed per sample). Similar results were observed in two independent experiments. ***p<0.0001; two-tailed unpaired t-test.


Figure 4A - C Representative images of PEX13 and PMP70 ( A ) , PEX13 and TOMM20 ( B ) , or PEX13 - Flag and WIPI2 ( C ) colocalization in HeLa / Parkin cells transfected with PEX13 after 4 h DMSO or CCCP treatment . Scale bars , 20 μm ( A - B ) and 5 μm ( C ) . D Western blot detection of endogenous PEX13 in HeLa / Parkin cells treated with 10 μM CCCP for the indicated time .

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Figure 4A-C Representative images of PEX13 and PMP70 (A), PEX13 and TOMM20 (B), or PEX13-Flag and WIPI2 (C) colocalization in HeLa/Parkin cells transfected with PEX13 after 4 h DMSO or CCCP treatment. Scale bars, 20 μm (A-B) and 5 μm (C).D Western blot detection of endogenous PEX13 in HeLa/Parkin cells treated with 10 μM CCCP for the indicated time.


Figure 5A . Quantitative real - time PCR detection of mRNA levels for PEX3 , PEX13 , PEX14 , and PEX19 in HeLa cells transfected with the indicated siRNA . * * * p < 0 . 001 ; two - tailed unpaired t - test . B . Quantification of Parkin - mediated TOMM20 clearance in HeLa / Parkin cells transfected with the indicated siRNA 16 h after treatment with 10 µM CCCP . Results represent mean ± SEM of triplicate samples ( ~ 100 cells analyzed per sample ) . Similar results were observed in three independent experiments . * * p < 0 . 01 , * * * p < 0 . 001 ; one - way ANOVA with adjustment for multiple comparisons . C . Quantification of mtDNA clearance in HeLa / HA - Parkin cells transfected with the indicated siRNA 8 h after treatment with 2 . 5 μM oligomycin and 250 nM antimycin A ( OA ) . Results represent box plots of ~ 300 cells analyzed per sample . Whiskers represent 5 % - 95 % range , and each outlier is represented by a dot . Similar results were observed in three independent experiments . * * * * p < 0 . 0001 , NS = not significant ; Kruskal - Wallis H - test . D . Quantification of GFP - LC3 puncta in HeLa / GFP - LC3 cells treated with 10 nM Baf A1 or DMSO vehicle and cultured in normal medium or 3 h HBSS starvation media . Results represent mean ± SEM in triplicate samples ( ~ 100 cells analyzed per sample ) . Similar results were observed in three independent experiments . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , NS = not significant ; one - way ANOVA with adjustment for multiple comparisons . Statistical analyses refer to the differences between Pex or ATG7 siRNAs vs . NC siRNA within each treatment group .

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Figure 5A. Quantitative real-time PCR detection of mRNA levels for PEX3, PEX13, PEX14, and PEX19 in HeLa cells transfected with the indicated siRNA. ***p<0.001; two-tailed unpaired t-test.B. Quantification of Parkin-mediated TOMM20 clearance in HeLa/Parkin cells transfected with the indicated siRNA 16 h after treatment with 10 µM CCCP. Results represent mean ± SEM of triplicate samples (~100 cells analyzed per sample). Similar results were observed in three independent experiments. **p<0.01, ***p<0.001; one-way ANOVA with adjustment for multiple comparisons.C. Quantification of mtDNA clearance in HeLa/HA-Parkin cells transfected with the indicated siRNA 8 h after treatment with 2.5 μM oligomycin and 250 nM antimycin A (OA). Results represent box plots of ~300 cells analyzed per sample. Whiskers represent 5%-95% range, and each outlier is represented by a dot. Similar results were observed in three independent experiments. ****p<0.0001, NS= not significant; Kruskal-Wallis H-test.D. Quantification of GFP-LC3 puncta in HeLa/GFP-LC3 cells treated with 10 nM Baf A1 or DMSO vehicle and cultured in normal medium or 3 h HBSS starvation media. Results represent mean ± SEM in triplicate samples (~100 cells analyzed per sample). Similar results were observed in three independent experiments. *p<0.05, **p<0.01, ***p<0.001, NS= not significant; one-way ANOVA with adjustment for multiple comparisons. Statistical analyses refer to the differences between Pex or ATG7 siRNAs vs. NC siRNA within each treatment group.


Figure 1 ( A ) Hematoxylin and Eosin ( H & E ) staining of WT and K14ΔNLef1 HF from back skin at 11 . 5 weeks ( early anagen ) . White arrowheads : ectopic SG and epidermal cysts . Data information Scale bars : 50 μm . ( B ) Tail epidermal whole - mounts from WT and K14ΔNLef1 mice labelled with Fabp5 , Krt15 and counterstained with Dapi . Data information : Scale bars : 50 μm . ( C ) Average signal intensity of ΔNLef1 binding sites detected by a Lef1 antibody ( red ) as compared to input alone ( grey ) and negative control ( Neg . Ctl . , black ) . DNA motif analysis revealed Lef1 classical consensus sequence . Representative plots of ChIP - Seq reads aligned to the Vgll4 , Runx1 and Gata6 loci . ( D ) Heat - map ( Pearson correlation ) of differentially expressed genes ( DEG ) between different micro - dissected regions ( IFE , HF and SG ) from WT mice ( left panel ) ΔNLef1 Chip - seq data show ΔNLef1 direct transcriptional targets in black ( right panel ) . ( E ) Gene Set Enrichment Analysis comparing gene location of ΔNLef1 peaks and SG gene expression signature . ( F ) RT - qPCR analysis of Gata6 in WT and K14ΔNLef1 primary keratinocytes transfected with siRNA targeting ΔNLef1 or a scrambled sequence ( scr . ) . Data are means ± SEM of three independent wells . Data information Statistical analyses were performed with an ordinary one - way ANOVA : ( ns ) not significant ; ( * ) p - value < 0 . 05 ; ( * * * ) p - value < 0 . 0005 . ( G ) RT - qPCR analysis of Gata6 in human SebE6E7 sebocytes transfected for 48h with a mock plasmid or a Lef1 - or ΔN34Lef1 - expressing plasmid . ∆ N34Lef1 is the human ortholog of murine ∆ N32Lef1 , which is expressed in K14 ∆ NLef1 transgenic mice Data are means ± SEM of three independent experiments . Data information : Statistical analyses were performed with an ordinary one - way ANOVA : ( ns ) not significant ; ( * ) p - value < 0 . 05 ; ( * * * ) p - value < 0 . 0005 . ( H ) Dorsal skin sections of WT and K14ΔNLef1 mice stained for Krt14 and Gata6 . Data information Scale bars : 50 μm . ( I ) Heat - maps of DEG from WT - or K14ΔNLef1 - unfractionated ( all ) or sorted ( Itga6 + Cd34 - , basal ; Itga6 + Cd34 + , HFSC ) keratinocytes in comparison with IFE , SG , and HF gene signatures ranked from high to low expression . ( J ) Heat - map depicting similarities , as Pearson ' s correlation coefficient , between DEG from keratinocytes from WT or K14ΔNLef1 mice and IFE vs SG vs HF transcriptome analysis ( left panel ) . Additional correlation with ΔNLef1 Chip - seq data is depicted in the right heat - map .

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

Figure 1(A) Hematoxylin and Eosin (H&E) staining of WT and K14ΔNLef1 HF from back skin at 11.5 weeks (early anagen). White arrowheads: ectopic SG and epidermal cysts. Data information Scale bars: 50 μm.(B) Tail epidermal whole-mounts from WT and K14ΔNLef1 mice labelled with Fabp5, Krt15 and counterstained with Dapi. Data information: Scale bars: 50 μm.(C) Average signal intensity of ΔNLef1 binding sites detected by a Lef1 antibody (red) as compared to input alone (grey) and negative control (Neg. Ctl., black). DNA motif analysis revealed Lef1 classical consensus sequence. Representative plots of ChIP-Seq reads aligned to the Vgll4, Runx1 and Gata6 loci.(D) Heat-map (Pearson correlation) of differentially expressed genes (DEG) between different micro-dissected regions (IFE, HF and SG) from WT mice (left panel) ΔNLef1 Chip-seq data show ΔNLef1 direct transcriptional targets in black (right panel).(E) Gene Set Enrichment Analysis comparing gene location of ΔNLef1 peaks and SG gene expression signature.(F) RT-qPCR analysis of Gata6 in WT and K14ΔNLef1 primary keratinocytes transfected with siRNA targeting ΔNLef1 or a scrambled sequence (scr.). Data are means ± SEM of three independent wells. Data information Statistical analyses were performed with an ordinary one-way ANOVA: (ns) not significant; (*) p-value < 0.05; (***) p-value < 0.0005.(G) RT-qPCR analysis of Gata6 in human SebE6E7 sebocytes transfected for 48h with a mock plasmid or a Lef1- or ΔN34Lef1-expressing plasmid. ∆N34Lef1 is the human ortholog of murine ∆N32Lef1, which is expressed in K14∆NLef1 transgenic mice Data are means ± SEM of three independent experiments. Data information: Statistical analyses were performed with an ordinary one-way ANOVA: (ns) not significant; (*) p-value < 0.05; (***) p-value < 0.0005.(H) Dorsal skin sections of WT and K14ΔNLef1 mice stained for Krt14 and Gata6. Data information Scale bars: 50 μm.(I) Heat-maps of DEG from WT- or K14ΔNLef1-unfractionated (all) or sorted (Itga6+Cd34-, basal; Itga6+Cd34+, HFSC) keratinocytes in comparison with IFE, SG, and HF gene signatures ranked from high to low expression.(J) Heat-map depicting similarities, as Pearson's correlation coefficient, between DEG from keratinocytes from WT or K14ΔNLef1 mice and IFE vs SG vs HF transcriptome analysis (left panel). Additional correlation with ΔNLef1 Chip-seq data is depicted in the right heat-map.


Figure 2 ( A ) Sections of WT embryonic skin at different HF stages stained for Gata6 , Lef1 , Sox9 and counterstained with Dapi . Black asterisks indicate overexposed areas of nonspecific Lef1 staining in the suprabasal epidermis . Quantification of the percentage of cells labelled for both Gata6 and Lef1 , or Gata6 and Sox9 in stages 4 - 5 HF are shown ( upper right panel ) . Data are means ± SD and were obtained from 9 HF from 3 mice . ( B ) Sections of E18 . 5 WT and Lef1 - / - mouse skin stained for Lef1 and Gata6 . Deletion of Lef1 does not impair Gata6 expression . Data information : Scale bars : 50 μm . ( C ) Sections of WT and K14ΔNβcateninER ( K14ΔNβ - catER D2 and D4 strains ) adult dorsal skin stained with antibodies against Krt14 and Gata6 . Topical treatment with 1 or 6 doses of 4OHT activates β - catenin leading to anagen induction and ectopic hair follicles but not Gata6 expression . Data information : Scale bars : 50 μm . ( D ) Sections of E18 . 5 WT and K14Cre / βCat Flox ( ex3 ) / + mouse skin stained for Sox9 and Gata6 . Activation of Wnt / β - catenin signaling during epidermis development does not induce Gata6 expression but results in ectopic expression of the HFSC marker Sox9 . Data information : Scale bar : 25 μm .

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Figure 2(A) Sections of WT embryonic skin at different HF stages stained for Gata6, Lef1, Sox9 and counterstained with Dapi. Black asterisks indicate overexposed areas of nonspecific Lef1 staining in the suprabasal epidermis. Quantification of the percentage of cells labelled for both Gata6 and Lef1, or Gata6 and Sox9 in stages 4-5 HF are shown (upper right panel). Data are means ± SD and were obtained from 9 HF from 3 mice. (B) Sections of E18.5 WT and Lef1-/- mouse skin stained for Lef1 and Gata6. Deletion of Lef1 does not impair Gata6 expression. Data information: Scale bars: 50 μm.(C) Sections of WT and K14ΔNβcateninER (K14ΔNβ-catER D2 and D4 strains) adult dorsal skin stained with antibodies against Krt14 and Gata6. Topical treatment with 1 or 6 doses of 4OHT activates β-catenin leading to anagen induction and ectopic hair follicles but not Gata6 expression. Data information: Scale bars: 50 μm.(D) Sections of E18.5 WT and K14Cre/βCat Flox(ex3)/+ mouse skin stained for Sox9 and Gata6. Activation of Wnt/β-catenin signaling during epidermis development does not induce Gata6 expression but results in ectopic expression of the HFSC marker Sox9. Data information: Scale bar: 25 μm.