C ) Human colon organoids cultured in full medium ( FM ) condition ( upper panel ) or FM treated with IFN - γ ( lower panel ) . Left : bright field images of 7 - day cultured human colon organoids , scale bar : 100 µm . Right : Immunofluorescence for KRT20 ( green ; note increased expression upon IFN - γ treatment ) . Scale bar : 25 µm .

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

C) Human colon organoids cultured in full medium (FM) condition (upper panel) or FM treated with IFN-γ (lower panel). Left: bright field images of 7-day cultured human colon organoids, scale bar: 100 µm. Right: Immunofluorescence for KRT20 (green; note increased expression upon IFN-γ treatment). Scale bar: 25 µm.


D ) Human colon organoids cultured in basic medium ( upper panel ) or additionally treated with IFN - γ ( lower panel ) . Left : bright field images of organoids that were cultured for 4 days in basic medium and in addition for 3 days with IFN - γ ( lower panel ) scale bar : 100 µm . Middle : Immunofluorescence labelling for KRT20 ( green ) Right : Immunofluorescence labelling for E - cadherin ( green ) to visualize epithelial cell boundaries ; insets with higher magnification indicate elongation of epithelial cells that are grown in basic medium and treated with IFN - γ . Scale bar : 25 µm .

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

D) Human colon organoids cultured in basic medium (upper panel) or additionally treated with IFN-γ (lower panel). Left: bright field images of organoids that were cultured for 4 days in basic medium and in addition for 3 days with IFN-γ (lower panel) scale bar: 100 µm. Middle: Immunofluorescence labelling for KRT20 (green) Right: Immunofluorescence labelling for E-cadherin (green) to visualize epithelial cell boundaries; insets with higher magnification indicate elongation of epithelial cells that are grown in basic medium and treated with IFN-γ. Scale bar: 25 µm.


Comparison of E ) KRT20 , F ) MUC2 , G ) CHGA , H ) LGR5 mRNA expression in organoids grown in FM or in basic medium and either untreated or treated with IFN - γ ( n = 3 ) . Data are presented as mean + / - SD , * : p ≤ 0 . 05 , * * : p ≤ 0 . 001 , * * * : p ≤ 0 . 0001 , as determined by one - way ANOVA , followed by Tukey ' s multiple comparisons

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

Comparison of E) KRT20, F) MUC2, G) CHGA, H) LGR5 mRNA expression in organoids grown in FM or in basic medium and either untreated or treated with IFN-γ (n=3). Data are presented as mean +/- SD ,*: p≤0.05, **: p≤0.001, ***: p≤0.0001, as determined by one-way ANOVA, followed by Tukey's multiple comparisons test


ACE2 mRNA expression in organoids grown in FM or in basic medium and either untreated or treated with IFN - γ ( n = 3 ) . Data are presented as mean + / - SD , * : p ≤ 0 . 05 , * * : p ≤ 0 . 001 , * * * : p ≤ 0 . 0001 , as determined by one - way ANOVA , followed by Tukey ' s multiple comparisons

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

ACE2 mRNA expression in organoids grown in FM or in basic medium and either untreated or treated with IFN-γ (n=3). Data are presented as mean +/- SD ,*: p≤0.05, **: p≤0.001, ***: p≤0.0001, as determined by one-way ANOVA, followed by Tukey's multiple comparisons test


J ) Immunofluorescence staining for ACE2 ( red ) and KRT20 ( green ) of organoids grown in full medium ( upper ) or basic medium ( lower ) and additionally treated with IFN - γ ( right ) , indicating expression of ACE2 in differentiated enterocytes . White arrows point to ACE2 . Scale bar : 10

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

J) Immunofluorescence staining for ACE2 (red) and KRT20 (green) of organoids grown in full medium (upper) or basic medium (lower) and additionally treated with IFN-γ (right), indicating expression of ACE2 in differentiated enterocytes. White arrows point to ACE2. Scale bar: 10 µm


A ) Immunofluorescence for N - protein ( green ) 48 h after SARS - CoV - 2 infection of human colon organoids cultured in full medium ( upper ) , full medium + IFN - γ ( upper right ) , basic medium ( lower left ) and basic medium + IFN - γ ( lower right ) . Yellow arrows point to N - protein positive cells . Scale bar : 25 µm .

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

A) Immunofluorescence for N-protein (green) 48 h after SARS-CoV-2 infection of human colon organoids cultured in full medium (upper), full medium + IFN-γ (upper right), basic medium (lower left) and basic medium + IFN-γ (lower right). Yellow arrows point to N-protein positive cells. Scale bar: 25 µm.


B ) qPCR data displaying the relative virus load of SARS - CoV - 2 measured by viral genome quantity in full medium vs . basic medium organoids 48 h after infection and normalized to GAPDH ( n = 6 ) . C ) qPCR data displaying the relative virus load of SARS - CoV - 2 measured by viral genome quantity in full medium vs . full medium + IFN - γ treated organoids 48 h after infection and normalized to GAPDH ( n = 9 ) . D ) qPCR data displaying the relative virus load of SARS - CoV - 2 measured by viral genome quantity in basic medium vs . basic medium + IFN - γ treated organoids 48 h after infection and normalized to GAPDH ( n = 4 ) . E ) qPCR data displaying the relative virus load of SARS - CoV - 2 measured by viral genome quantity in full medium + IFN - γ vs . basic medium + IFN - γ treated organoids 48 h after infection and normalized to GAPDH ( n = 4 ) . ( B - E ) Data are presented as mean + / - SD , * : p ≤ 0 . 05 , * * : p ≤ 0 . 001 , * * * : p ≤ 0 . 0001 , as determined by Student ' s t - test

O O B-EXP_ASSAY O O O O B-EXP_ASSAY I-EXP_ASSAY O B-ORGANISM I-ORGANISM I-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 B-GENEPROD O O O O O O O O B-EXP_ASSAY O O O O B-EXP_ASSAY I-EXP_ASSAY O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O O 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 B-GENEPROD O O O O O O O O B-EXP_ASSAY O O O O B-EXP_ASSAY I-EXP_ASSAY O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O O 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 B-GENEPROD O O O O O O O O B-EXP_ASSAY O O O O B-EXP_ASSAY I-EXP_ASSAY O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-GENEPROD I-GENEPROD I-GENEPROD 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 O 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 data displaying the relative virus load of SARS-CoV-2 measured by viral genome quantity in full medium vs. basic medium organoids 48 h after infection and normalized to GAPDH (n=6). C) qPCR data displaying the relative virus load of SARS-CoV-2 measured by viral genome quantity in full medium vs. full medium + IFN-γ treated organoids 48 h after infection and normalized to GAPDH (n=9). D) qPCR data displaying the relative virus load of SARS-CoV-2 measured by viral genome quantity in basic medium vs. basic medium + IFN-γ treated organoids 48 h after infection and normalized to GAPDH (n=4). E) qPCR data displaying the relative virus load of SARS-CoV-2 measured by viral genome quantity in full medium + IFN-γ vs. basic medium+ IFN-γ treated organoids 48 h after infection and normalized to GAPDH (n=4). (B-E) Data are presented as mean +/- SD ,*: p≤0.05, **: p≤0.001, ***: p≤0.0001, as determined by Student's t-test


F ) qPCR data comparing the relative virus load of SARS - CoV - 2 measured by viral genome quantity normalized to GAPDH at 24 and 48 h after infection in full medium ( left ) and basic medium ( right ) untreated or treated with IFN - γ , indicating increase in virus load in differentiated and IFN - γ treated conditions ( n = 8 ) . Data are presented as mean + / - SD , * : p ≤ 0 . 05 , * * : p ≤ 0 . 001 , * * * : p ≤ 0 . 0001 , as determined by one - way ANOVA , followed by Tukey ' s multiple comparisons

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

F) qPCR data comparing the relative virus load of SARS-CoV-2 measured by viral genome quantity normalized to GAPDH at 24 and 48 h after infection in full medium (left) and basic medium (right) untreated or treated with IFN-γ, indicating increase in virus load in differentiated and IFN-γ treated conditions (n=8). Data are presented as mean +/- SD ,*: p≤0.05, **: p≤0.001, ***: p≤0.0001, as determined by one-way ANOVA, followed by Tukey's multiple comparisons test


G ) Electron microscopy imaging of organoids grown in basic medium , pretreated with IFN - γ for 3 days and infected for 48 h with SARS - CoV - 2 . Upper : Overview showing large virus - loaded vesicles in an epithelial cell . Scale bar : 500nm . Lower : Disintegrated organoid cell containing high virus load , scale bar : 2 . 5µm , higher magnification to visualize virus particles , scale bar : 100nm .

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

G) Electron microscopy imaging of organoids grown in basic medium, pretreated with IFN-γ for 3 days and infected for 48 h with SARS-CoV-2. Upper: Overview showing large virus-loaded vesicles in an epithelial cell. Scale bar: 500nm. Lower: Disintegrated organoid cell containing high virus load, scale bar: 2.5µm, higher magnification to visualize virus particles, scale bar: 100nm.


H ) Immunofluorescence for cleaved - caspase3 ( red ) and E - cadherin ( green ) 48 h after SARS - CoV - 2 infection of human colon organoids cultured in full medium ( upper ) , full medium + IFN - γ ( upper right ) , basic medium ( lower left ) and basic medium + IFN - γ ( lower right ) . Scale bar : 50 µm . ( below ) Quantification of apoptotic cells , indicating increased cell death in the differentiated and IFN - γ treated condition after SARS - CoV - 2 infection ( n = 7 ) . Data are presented as mean + / - SD , * : p ≤ 0 . 05 , * * : p ≤ 0 . 001 , * * * : p ≤ 0 . 0001 , as determined by one - way ANOVA , followed by Tukey ' s multiple comparisons

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

H) Immunofluorescence for cleaved-caspase3 (red) and E-cadherin (green) 48 h after SARS-CoV-2 infection of human colon organoids cultured in full medium (upper), full medium + IFN-γ (upper right), basic medium (lower left) and basic medium + IFN-γ (lower right). Scale bar: 50 µm. (below) Quantification of apoptotic cells, indicating increased cell death in the differentiated and IFN-γ treated condition after SARS-CoV-2 infection (n=7). Data are presented as mean +/- SD ,*: p≤0.05, **: p≤0.001, ***: p≤0.0001, as determined by one-way ANOVA, followed by Tukey's multiple comparisons test


C ) Analysis of discordance and concordance of transcriptomic responses ( DISCO ) between untreated vs . SARS - CoV - 2 infected and untreated vs IFN - γ treated organoids . The 50 most regulated genes are highlighted ; grey : discordant and black : concordant .

O O O O O O O O O O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM 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

C) Analysis of discordance and concordance of transcriptomic responses (DISCO) between untreated vs. SARS-CoV-2 infected and untreated vs IFN-γ treated organoids. The 50 most regulated genes are highlighted; grey: discordant and black: concordant.


D ) Relative expression of selected enterocyte - specific genes upon SARS - CoV - 2 infection , normalized to GAPDH . Differences in gene expression were assessed using the linear model ' lmFit ' and ' makeContrasts ' in limma .

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

D) Relative expression of selected enterocyte-specific genes upon SARS-CoV-2 infection, normalized to GAPDH. Differences in gene expression were assessed using the linear model 'lmFit' and 'makeContrasts' in limma.


E ) Immunofluorescence for KRT20 ( green ) in human colon organoids cultured in basic medium and either non - infected ( left ) or 48 h after SARS - CoV - 2 infection ( right ) , indicating a strong increase in KRT20 expression after viral infection . Scale bar : 50 µm . Quantification of fluorescence intensity below ( n = 8 ) . Data are presented as mean + / - SD , * * * * : p ≤ 0 . 0001 , as determined by Student ' s t -

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

E) Immunofluorescence for KRT20 (green) in human colon organoids cultured in basic medium and either non-infected (left) or 48 h after SARS-CoV-2 infection (right), indicating a strong increase in KRT20 expression after viral infection. Scale bar: 50 µm. Quantification of fluorescence intensity below (n=8). Data are presented as mean +/- SD ,****: p≤0.0001, as determined by Student's t-test


F ) qPCR data displaying the increase of INF - γ target genes ( CXCL11 , IFIT3 ) ( upper ) and enterocyte marker genes ( KRT20 , ACE2 ) ( lower ) after SARS - CoV - 2 infection , validating the results from the microarray analysis ( n = 3 ) . Data are presented as mean + / - SD , * * * * : p ≤ 0 . 0001 , as determined by Student ' s t -

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

F) qPCR data displaying the increase of INF-γ target genes (CXCL11, IFIT3) (upper) and enterocyte marker genes (KRT20, ACE2) (lower) after SARS-CoV-2 infection, validating the results from the microarray analysis (n=3). Data are presented as mean +/- SD ,****: p≤0.0001, as determined by Student's t-test


Montages of representative DAPI - stained nuclei are shown for gene knockdowns that led to reduced nuclear size . Cell - to - cell variability in the amount of ELYS knockdown likely accounts for apparent nuclear size variability . Ten nuclei per column , organized by maximum nuclear size z - score . Scale bar , 25 µm . Median nuclear size z - scores are plotted for gene knockdowns that led to reduced nuclear size . Data are based on three different siRNA oligo sequences for each gene and two biological replicates . Error bars represent the SEM for biological replicates .

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

Montages of representative DAPI-stained nuclei are shown for gene knockdowns that led to reduced nuclear size. Cell-to-cell variability in the amount of ELYS knockdown likely accounts for apparent nuclear size variability. Ten nuclei per column, organized by maximum nuclear size z-score. Scale bar, 25 µm. Median nuclear size z-scores are plotted for gene knockdowns that led to reduced nuclear size. Data are based on three different siRNA oligo sequences for each gene and two biological replicates. Error bars represent the SEM for biological replicates.


MCF - 10AT1k . cl2 cells were transfected with control or ELYS siRNA . ( A ) Representative images of cells stained for lamin B2 and B1 are shown . ( B ) Representative images of cells stained for lamin A / C and B1 are shown . ( C ) For each experiment , nuclear lamin B1 , B2 , and A / C staining intensities were quantified for 44 - 159 nuclei per condition ( 96 nuclei on average ) and normalized to the negative control . Three biological replicates , data from one representative experiment shown . ( D ) Representative images of cells stained for FG - Nups ( mAb414 ) and lamin B1 are shown . ( E ) Representative images of cells stained for Nup133 and lamin B1 are shown . ( F ) For each experiment , nuclear mAb414 and Nup133 staining intensities were quantified for 31 - 83 nuclei per condition ( 60 nuclei on average ) and normalized to the negative control . To measure NPC densities , confocal NE surface images were acquired for mAb414 and Nup133 stained nuclei . NPC numbers were counted per unit area for 18 - 50 nuclei per condition ( 30 nuclei on average ) and normalized to the negative control . Two biological replicates , data from one representative experiment shown .

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

MCF-10AT1k.cl2 cells were transfected with control or ELYS siRNA. (A) Representative images of cells stained for lamin B2 and B1 are shown. (B) Representative images of cells stained for lamin A/C and B1 are shown. (C) For each experiment, nuclear lamin B1, B2, and A/C staining intensities were quantified for 44-159 nuclei per condition (96 nuclei on average) and normalized to the negative control. Three biological replicates, data from one representative experiment shown. (D) Representative images of cells stained for FG-Nups (mAb414) and lamin B1 are shown. (E) Representative images of cells stained for Nup133 and lamin B1 are shown. (F) For each experiment, nuclear mAb414 and Nup133 staining intensities were quantified for 31-83 nuclei per condition (60 nuclei on average) and normalized to the negative control. To measure NPC densities, confocal NE surface images were acquired for mAb414 and Nup133 stained nuclei. NPC numbers were counted per unit area for 18-50 nuclei per condition (30 nuclei on average) and normalized to the negative control. Two biological replicates, data from one representative experiment shown.


MCF - 10AT1k . cl2 cells were transfected with control or ELYS siRNA . Representative images of cells stained for Ran and lamin B1 are shown . ( H ) Representative images of cells stained for NTF2 and lamin A / C are shown . ( I ) Nuclear Ran and NTF2 staining intensities were quantified and normalized to the negative control . To measure the N / C staining intensity ratio of a given cell , the average fluorescence intensity of a nuclear region was divided by the average fluorescence intensity of a cytoplasmic region . For each experiment , 54 - 216 nuclei were quantified per condition ( 112 nuclei on average ) . Two biological replicates , data from one representative experiment shown .

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

MCF-10AT1k.cl2 cells were transfected with control or ELYS siRNA. Representative images of cells stained for Ran and lamin B1 are shown. (H) Representative images of cells stained for NTF2 and lamin A/C are shown. (I) Nuclear Ran and NTF2 staining intensities were quantified and normalized to the negative control. To measure the N/C staining intensity ratio of a given cell, the average fluorescence intensity of a nuclear region was divided by the average fluorescence intensity of a cytoplasmic region. For each experiment, 54-216 nuclei were quantified per condition (112 nuclei on average). Two biological replicates, data from one representative experiment shown.


MCF - 10AT1k . cl2 cells were co - transfected with control or ELYS siRNA and plasmids expressing eGFP alone or siRNA - resistant eGFP - ELYS . Cell lysates were analyzed by western blot and probed for ELYS and tubulin . ELYS band intensity was normalized to tubulin and then normalized to the eGFP / negative siRNA control . ELYS concentrations are indicated in the row labeled [ ELYS ] ( average ± SD ) . Quantification was performed on two biological replicates . Cells were stained with an antibody against lamin B1 . Representative images are shown . ( B ) For each experiment , nuclear cross - sectional areas were quantified for 68 - 298 nuclei per condition ( 162 nuclei on average ) and averaged . Three biological replicates , data from one representative experiment shown . ( C ) For each experiment , nuclear lamin B2 staining intensities were quantified for 29 - 81 nuclei per condition ( 60 nuclei on average ) and normalized to the eGFP / negative siRNA control . Two biological replicates , data from one representative experiment shown .

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

MCF-10AT1k.cl2 cells were co-transfected with control or ELYS siRNA and plasmids expressing eGFP alone or siRNA-resistant eGFP-ELYS. Cell lysates were analyzed by western blot and probed for ELYS and tubulin. ELYS band intensity was normalized to tubulin and then normalized to the eGFP/negative siRNA control. ELYS concentrations are indicated in the row labeled [ELYS] (average ± SD). Quantification was performed on two biological replicates. Cells were stained with an antibody against lamin B1. Representative images are shown. (B) For each experiment, nuclear cross-sectional areas were quantified for 68-298 nuclei per condition (162 nuclei on average) and averaged. Three biological replicates, data from one representative experiment shown. (C) For each experiment, nuclear lamin B2 staining intensities were quantified for 29-81 nuclei per condition (60 nuclei on average) and normalized to the eGFP/negative siRNA control. Two biological replicates, data from one representative experiment shown.


Cells with and without cytoplasmic lamin B2 aggregates were counted and the percentage of cells with aggregates was calculated . For each experiment , 58 - 299 cells were examined per condition . Three biological replicates , data from one representative experiment shown .

O O O O B-SUBCELLULAR B-GENEPROD I-GENEPROD 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

Cells with and without cytoplasmic lamin B2 aggregates were counted and the percentage of cells with aggregates was calculated. For each experiment, 58-299 cells were examined per condition. Three biological replicates, data from one representative experiment shown.


Cells were stained with antibodies against Ran and NTF2 . For each experiment , 62 - 130 nuclei were quantified per condition ( 92 nuclei on average ) . Two biological replicates , data from one representative experiment shown . ( E ) Nuclear Ran and NTF2 staining intensities were quantified and normalized to the eGFP control . ( F ) To measure the N / C staining intensity ratio of a given cell , the average fluorescence intensity of a nuclear region was divided by the average fluorescence intensity of a cytoplasmic region .

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

Cells were stained with antibodies against Ran and NTF2. For each experiment, 62-130 nuclei were quantified per condition (92 nuclei on average). Two biological replicates, data from one representative experiment shown. (E) Nuclear Ran and NTF2 staining intensities were quantified and normalized to the eGFP control. (F) To measure the N/C staining intensity ratio of a given cell, the average fluorescence intensity of a nuclear region was divided by the average fluorescence intensity of a cytoplasmic region.


Transfections were performed as in ( A ) and cells were stained with an antibody against FG - Nups ( mAb414 ) and Nup133 . Representative images for mAb414 staining are shown . Confocal imaging was performed through nuclear cross - sections as well as on the surface of nuclei . ( H ) To measure NPC densities , confocal NE surface images were acquired for mAb414 and Nup133 stained nuclei . NPC numbers were counted per unit area for 25 - 116 nuclei per condition ( 53 nuclei on average ) and normalized to the negative control . Two biological replicates , data from one representative experiment shown .

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

Transfections were performed as in (A) and cells were stained with an antibody against FG-Nups (mAb414) and Nup133. Representative images for mAb414 staining are shown. Confocal imaging was performed through nuclear cross-sections as well as on the surface of nuclei. (H) To measure NPC densities, confocal NE surface images were acquired for mAb414 and Nup133 stained nuclei. NPC numbers were counted per unit area for 25-116 nuclei per condition (53 nuclei on average) and normalized to the negative control. Two biological replicates, data from one representative experiment shown.


MCF - 10AT1k . cl2 cells were co - transfected with control or ELYS siRNA and plasmids expressing mCherry alone or mCherry - importin α . Cells were stained with an antibody against lamin B2 . Representative images are shown . Scale bar , 10 µm . ( B ) For each experiment , nuclear cross - sectional areas were quantified for 30 - 172 nuclei per condition ( 84 nuclei on average ) and averaged . Two biological replicates , data from one representative experiment shown .

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

MCF-10AT1k.cl2 cells were co-transfected with control or ELYS siRNA and plasmids expressing mCherry alone or mCherry-importin α. Cells were stained with an antibody against lamin B2. Representative images are shown. Scale bar, 10 µm. (B) For each experiment, nuclear cross-sectional areas were quantified for 30-172 nuclei per condition (84 nuclei on average) and averaged. Two biological replicates, data from one representative experiment shown.


Cells with and without cytoplasmic lamin B2 aggregates were counted and the percentage of cells with aggregates was calculated . For each experiment , 74 - 218 cells were examined per condition . Two biological replicates , data from one representative experiment shown .

Cells with and without cytoplasmic lamin B2 aggregates were counted and the percentage of cells with aggregates was calculated. For each experiment, 74-218 cells were examined per condition. Two biological replicates, data from one representative experiment shown.


For each experiment , nuclear lamin B2 staining intensities were quantified for 34 - 187 nuclei per condition ( 85 nuclei on average ) and normalized to the mCherry / negative siRNA control . To measure bulk import capacity for importin α / β cargos , cells were co - transfected as in ( A ) along with a plasmid expressing GFP - 3x SV40 NLS . For each experiment , nuclear GFP - NLS intensities were quantified for 22 - 188 nuclei per condition ( 73 nuclei on average ) and normalized to the mCherry / negative siRNA control . Two biological replicates , data from one representative experiment shown .

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

For each experiment, nuclear lamin B2 staining intensities were quantified for 34-187 nuclei per condition (85 nuclei on average) and normalized to the mCherry/negative siRNA control. To measure bulk import capacity for importin α/β cargos, cells were co-transfected as in (A) along with a plasmid expressing GFP-3x SV40 NLS. For each experiment, nuclear GFP-NLS intensities were quantified for 22-188 nuclei per condition (73 nuclei on average) and normalized to the mCherry/negative siRNA control. Two biological replicates, data from one representative experiment shown.


MCF - 10AT1k . cl2 cells were transfected with plasmids expressing mCherry alone or mCherry - RanQ69L and stained with an antibody against lamin B2 . Representative images are shown .

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

MCF-10AT1k.cl2 cells were transfected with plasmids expressing mCherry alone or mCherry-RanQ69L and stained with an antibody against lamin B2. Representative images are shown.


MCF - 10AT1k . cl2 cells were treated with 20 µM importazole or an equivalent volume of DMSO as a control for 24 hours and stained with an antibody against lamin B1 . Representative images are shown .

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

MCF-10AT1k.cl2 cells were treated with 20 µM importazole or an equivalent volume of DMSO as a control for 24 hours and stained with an antibody against lamin B1. Representative images are shown.


For each experiment , nuclear cross - sectional areas were quantified for 32 - 271 nuclei per condition ( 123 nuclei on average ) and averaged . Three biological replicates , data from one representative experiment shown .

O O O O B-SUBCELLULAR O O O O O O O O O O B-SUBCELLULAR O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O

For each experiment, nuclear cross-sectional areas were quantified for 32-271 nuclei per condition (123 nuclei on average) and averaged. Three biological replicates, data from one representative experiment shown.


To measure bulk import capacity for importin α / β cargos , experiments were performed as in ( A - B ) except that cells were also transfected with a plasmid expressing GFP - 3x SV40 NLS . For each experiment , nuclear GFP - NLS intensities were quantified for 11 - 75 nuclei per condition ( 35 nuclei on average ) and normalized to the appropriate control . Two biological replicates , data from one representative experiment shown .

O O O O O O B-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 B-GENEPROD O O B-ORGANISM O O O O O O B-SUBCELLULAR B-GENEPROD O O B-EXP_ASSAY O O O O O O B-SUBCELLULAR O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O

To measure bulk import capacity for importin α/β cargos, experiments were performed as in (A-B) except that cells were also transfected with a plasmid expressing GFP-3x SV40 NLS. For each experiment, nuclear GFP-NLS intensities were quantified for 11-75 nuclei per condition (35 nuclei on average) and normalized to the appropriate control. Two biological replicates, data from one representative experiment shown.


Cells with and without cytoplasmic lamin B aggregates were counted and the percentage of cells with aggregates was calculated . For each experiment , 31 - 130 cells were examined per condition . Three biological replicates , data from one representative experiment shown .

Cells with and without cytoplasmic lamin B aggregates were counted and the percentage of cells with aggregates was calculated. For each experiment, 31-130 cells were examined per condition. Three biological replicates, data from one representative experiment shown.


Cell lysates from control and XPO1 siRNA transfected MCF - 10AT1k . cl2 cells were analyzed by western blot and probed for XPO1 and tubulin . One representative western blot is shown . XPO1 band intensity was normalized to tubulin . Quantification from two biological replicates is shown .

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

Cell lysates from control and XPO1 siRNA transfected MCF-10AT1k.cl2 cells were analyzed by western blot and probed for XPO1 and tubulin. One representative western blot is shown. XPO1 band intensity was normalized to tubulin. Quantification from two biological replicates is shown.


MCF - 10AT1k . cl2 cells were transfected with control or XPO1 siRNA . Lamin B1 and lamin A / C nuclear staining intensities were quantified based on experiments using two different XPO1 siRNA sequences and two biological replicates . For each experiment , 108 - 865 nuclei were quantified per condition ( 416 nuclei on average ) . Average and SD values based on averages from these four experiments are plotted , normalized to the negative siRNA control .

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

MCF-10AT1k.cl2 cells were transfected with control or XPO1 siRNA. Lamin B1 and lamin A/C nuclear staining intensities were quantified based on experiments using two different XPO1 siRNA sequences and two biological replicates. For each experiment, 108-865 nuclei were quantified per condition (416 nuclei on average). Average and SD values based on averages from these four experiments are plotted, normalized to the negative siRNA control.


MCF - 10AT1k . cl2 cells were transfected with the indicated siRNAs and nuclear cross - sectional areas were quantified . For each experiment , 203 - 604 nuclei were quantified per condition ( 403 nuclei on average ) . Average and SD values based on averages from four biological replicates are plotted , normalized to the negative siRNA control .

B-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O B-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

MCF-10AT1k.cl2 cells were transfected with the indicated siRNAs and nuclear cross-sectional areas were quantified. For each experiment, 203-604 nuclei were quantified per condition (403 nuclei on average). Average and SD values based on averages from four biological replicates are plotted, normalized to the negative siRNA control.


MCF - 10AT1k . cl2 cells were transfected with control siRNA or with siRNA against XPO1 . Cells were stained with DAPI and quantification of DNA staining intensity was used to estimate the fraction of cells in various stages of the cell cycle by high - throughput imaging as previously described [ 123 ] ( see Methods ) . The stacked bars represent the means of the fractions for each cell cycle phase calculated over 3 biological replicates .

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

MCF-10AT1k.cl2 cells were transfected with control siRNA or with siRNA against XPO1. Cells were stained with DAPI and quantification of DNA staining intensity was used to estimate the fraction of cells in various stages of the cell cycle by high-throughput imaging as previously described [123] (see Methods). The stacked bars represent the means of the fractions for each cell cycle phase calculated over 3 biological replicates.


MCF - 10AT1k . cl2 cells were transfected with control or XPO1 siRNA . For the bottom row of images , cells transfected with control siRNA were treated with 20 ng / ml leptomycin B for 24 hours . Cells were fixed and stained with 2 µg / ml FITC . Representative images are shown . Scale bar , 20 µm . For each experiment , nuclear cross - sectional areas and nuclear FITC staining intensities were quantified for 105 - 620 nuclei per condition ( 267 nuclei on average ) and normalized to the negative control . Two biological replicates , data from one representative experiment shown .

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

MCF-10AT1k.cl2 cells were transfected with control or XPO1 siRNA. For the bottom row of images, cells transfected with control siRNA were treated with 20 ng/ml leptomycin B for 24 hours. Cells were fixed and stained with 2 µg/ml FITC. Representative images are shown. Scale bar, 20 µm. For each experiment, nuclear cross-sectional areas and nuclear FITC staining intensities were quantified for 105-620 nuclei per condition (267 nuclei on average) and normalized to the negative control. Two biological replicates, data from one representative experiment shown.


( B ) COUP - TFII expression ( green ) is increased in human kidneys from patients with thrombotic microangiopathy ( TMA ) and diabetic nephropathy ( DN ) ( n = 2 ) . Quantification by confocal micrographs in 400x hpf . * * * * p < 0 . 0001 by one - way ANOVA ; mean ± SD . The majority of these cells were localized in the interstitial region . More than 50 % COUP - TFII ( + ) cells ( green ) are co - localized with expanded αSMA - positive ( red ) areas of fibrosis . Pearson correlation coefficient ( PCC ) = 0 . 56 ± 0 . 1 for patient # 1 and 0 . 65 ± 0 . 13 for patient # 2 .

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

(B) COUP-TFII expression (green) is increased in human kidneys from patients with thrombotic microangiopathy (TMA) and diabetic nephropathy (DN) (n=2). Quantification by confocal micrographs in 400x hpf. ****p<0.0001 by one-way ANOVA; mean ± SD. The majority of these cells were localized in the interstitial region. More than 50% COUP-TFII (+) cells (green) are co-localized with expanded αSMA-positive (red) areas of fibrosis. Pearson correlation coefficient (PCC) = 0.56 ± 0.1 for patient#1 and 0.65 ± 0.13 for patient#2.


( C ) COUP - TFII expression ( green ) is increased in lungs from patients with IPF ( n = 2 ) by immunofluorescence . Quantification by confocal micrographs in 400x hpf . * * p < 0 . 01 ; * * * * p < 0 . 0001 by one - way ANOVA , mean ± SD .

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

(C) COUP-TFII expression (green) is increased in lungs from patients with IPF (n=2) by immunofluorescence. Quantification by confocal micrographs in 400x hpf. **p<0.01; ****p<0.0001 by one-way ANOVA, mean ± SD.


( E ) COUP - TFII expression increased significantly in organoids treated with TGFβ1 ( 10ng / ml ) , cobalt chloride ( 100 μM ) , IL - 1β ( 10ng / ml ) , but not PDGF ( 50ng / ml ) . Quantification by confocal micrographs in 400x hpf . * p < 0 . 05 ; * * p < 0 . 01 ; * * * * p < 0 . 0001 by one - way ANOVA , mean ± SD . The higher magnification of the insert from IL - 1β φησwed co - localization of COUP - TFII ( green ) and αSMA ( red ) staining .

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

(E) COUP-TFII expression increased significantly in organoids treated with TGFβ1 (10ng/ml), cobalt chloride (100 μM), IL-1β (10ng/ml), but not PDGF (50ng/ml). Quantification by confocal micrographs in 400x hpf. *p<0.05; **p<0.01; ****p<0.0001 by one-way ANOVA, mean ± SD. The higher magnification of the insert from IL-1β φησwed co-localization of COUP-TFII (green) and αSMA (red) staining.


( C ) Majority COUP - TFII + cells ( green ) were also positive for Foxd1 ( tdTomato - labeled as red ) in the non - injured mice kidney ( n = 2 ) . Quantification by confocal micrographs in 400x hpf .

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

(C) Majority COUP-TFII+ cells (green) were also positive for Foxd1 (tdTomato-labeled as red) in the non-injured mice kidney (n=2). Quantification by confocal micrographs in 400x hpf.


( A ) COUP - TFII expression is significantly increased and co - localized with αSMA + cells at day 10 of UUO and day 14 of UIRI in mice ( n = 3 animals per group ) . Quantification by confocal micrographs in 200x hpf . * p < 0 . 05 * * p < 0 . 01 by paired t test ; mean ± SD .

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

(A) COUP-TFII expression is significantly increased and co-localized with αSMA+ cells at day 10 of UUO and day 14 of UIRI in mice (n=3 animals per group). Quantification by confocal micrographs in 200x hpf. *p<0.05 **p<0.01 by paired t test; mean ± SD.


( B ) In a time course of UUO , COUP - TFII expression increased as early as day 2 and preceded the increased expression of αSMA ( n = 2 animals per time point ) . Quantification by confocal micrographs in 200x hpf . * p < 0 . 05 ; * * * p < 0 . 001 by two - way ANOVA test ; mean ± SD .

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

(B) In a time course of UUO, COUP-TFII expression increased as early as day 2 and preceded the increased expression of αSMA (n=2 animals per time point). Quantification by confocal micrographs in 200x hpf. *p<0.05; ***p<0.001 by two-way ANOVA test; mean ± SD.


( E & F ) Gli1 - tdTomato + cells expanded primarily in the outer medullary region . In contrast , COUP - TFII + cells are distributed both in cortex and medulla . A subset of COUP - TFII + cells ( green ) overlap with genetically labeled Gli1 + pericyte / perivascular cells ( red ) in UUO injury model ( n = 3 animals per group ) . CTL : contralateral kidney . Quantification by confocal micrographs in 200x hpf .

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

(E&F) Gli1-tdTomato+ cells expanded primarily in the outer medullary region. In contrast, COUP-TFII+ cells are distributed both in cortex and medulla. A subset of COUP-TFII+ cells (green) overlap with genetically labeled Gli1+ pericyte/perivascular cells (red) in UUO injury model (n=3 animals per group). CTL: contralateral kidney. Quantification by confocal micrographs in 200x hpf.


( D ) COUP - TFII + cells decrease significantly in the UUO kidney in TAM - treated homozygous ( F / F ; Cre / + ) mice ( KO group , n = 6 ) compared to wild - type littermates ( WT group , n = 4 ) . Expression of αSMA ( red ) and collagen1 ( yellow ) are also markedly reduced . Masson Trichrome staining shows less kidney fibrosis in KO compared to WT group . Quantification by confocal micrographs in 200x hpf . * * p < 0 . 01 by unpaired t test , mean ± SD .

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

(D) COUP-TFII+ cells decrease significantly in the UUO kidney in TAM-treated homozygous (F/F;Cre/+) mice (KO group, n=6) compared to wild-type littermates (WT group, n=4). Expression of αSMA (red) and collagen1 (yellow) are also markedly reduced. Masson Trichrome staining shows less kidney fibrosis in KO compared to WT group. Quantification by confocal micrographs in 200x hpf. **p<0.01 by unpaired t test, mean ± SD.


( D ) COUP - TFII - OE cells , in the absence of TGFβ1 stimulation , displayed an elongated fibroblast shape with increased αSMA expression , similar to WT cells treated with TGFβ1 . Quantification by confocal micrographs in 400x hpf , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 by one - way ANOVA , mean ± SD .

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

(D) COUP-TFII-OE cells, in the absence of TGFβ1 stimulation, displayed an elongated fibroblast shape with increased αSMA expression, similar to WT cells treated with TGFβ1. Quantification by confocal micrographs in 400x hpf, *p<0.05, **p<0.01, ***p<0.001 by one-way ANOVA, mean ± SD.


( E ) Overexpression of COUP - TFII alone without TGFβ1 induces collagen1 production ( n = 3 ) . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 by one - way ANOVA , mean ± SD .

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

(E) Overexpression of COUP-TFII alone without TGFβ1 induces collagen1 production (n=3). *p<0.05, **p<0.01, ***p<0.001 by one-way ANOVA, mean ± SD.


( D ) WT , COUP - TFII overexpression ( OE ) and COUP - TFII knockout ( KO ) cells were seeded in Seahorse XF - 24 cell culture microplates . The cells were rendered quiescent in 0 . 5 % FBS DMEM overnight and then treated with or without 10 ng / ml TGFβ1 for 24 hours . All the cells were incubated in the glycolysis stress test medium without glucose and pyruvate , followed by sequential treatments with glucose ( 10mM ) , oligomycin ( 5μg / ml ) and 2 - DG ( 50mM ) . Real - time extracellular acidification rate ( ECAR ) was recorded as the baseline ( before glucose ) , the rate of glycolysis ( after glucose ) , glycolytic capacity ( after oligomycin ) , and glycolytic reserve ( after 2 - DG ) ( n = 10 ) .

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

(D) WT, COUP-TFII overexpression (OE) and COUP-TFII knockout (KO) cells were seeded in Seahorse XF-24 cell culture microplates. The cells were rendered quiescent in 0.5% FBS DMEM overnight and then treated with or without 10 ng/ml TGFβ1 for 24 hours. All the cells were incubated in the glycolysis stress test medium without glucose and pyruvate, followed by sequential treatments with glucose (10mM), oligomycin (5μg/ml) and 2-DG (50mM). Real-time extracellular acidification rate (ECAR) was recorded as the baseline (before glucose), the rate of glycolysis (after glucose), glycolytic capacity (after oligomycin), and glycolytic reserve (after 2-DG) (n = 10).


( E ) COUP - TFII OE significantly increased , while COUP - TFII KO decreased , in the baseline , rate of glycolysis , glycolytic capacity , and glycolytic reserve when compared to WT cells under TGFβ1 stimulation . Without TGFβ1 treatment , there is no difference on the baseline and glycolysis among WT , OE , and KO cells . COUP - TFII KO significantly decreased glycolytic capacity and reserve compared to WT , even without TGFβ1 stimulation ( n = 10 ) , * p < 0 . 05 ; * * p < 0 . 01 ; * * * * p < 0 . 0001 by one - way ANOVA , mean ± SD .

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

(E) COUP-TFII OE significantly increased, while COUP-TFII KO decreased, in the baseline, rate of glycolysis, glycolytic capacity, and glycolytic reserve when compared to WT cells under TGFβ1 stimulation. Without TGFβ1 treatment, there is no difference on the baseline and glycolysis among WT, OE, and KO cells. COUP-TFII KO significantly decreased glycolytic capacity and reserve compared to WT, even without TGFβ1 stimulation (n=10), *p<0.05; **p<0.01; ****p<0.0001 by one-way ANOVA, mean ± SD.


( A ) Volcano plot of all detected proteins from proteomics of TGFβ  - treated naïve ( WT ) and COUP - TFII overexpressing ( COUP - TFII - OE ) cells . PDK4 expression is significantly decreased in OE cells ( n = 2 ) .

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

(A) Volcano plot of all detected proteins from proteomics of TGFβ-treated naïve (WT) and COUP-TFII overexpressing (COUP-TFII-OE) cells. PDK4 expression is significantly decreased in OE cells (n=2).


( E ) PGC1α OE significantly increased expression of Cpt1 and PDK4 , both are involved in key steps of FAO . However , overexpression of PGC1α did not abrogate the TGFβ1 up - regulated genes in glycolysis and αSMA ( n = 6 ) . * p < 0 . 05 ; * * p < 0 . 01 ; * * * * p < 0 . 0001 by one - way ANOVA , mean ± SD .

O O O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O 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 O O O O O O O O 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) PGC1α OE significantly increased expression of Cpt1 and PDK4, both are involved in key steps of FAO. However, overexpression of PGC1α did not abrogate the TGFβ1 up-regulated genes in glycolysis and αSMA (n=6). *p<0.05; **p<0.01; ****p<0.0001 by one-way ANOVA, mean ± SD.


( B ) Non - supervised clustering heatmap plot indicates substantial clustering of transcripts from each tissue but not of transcripts from patients compared to controls . PD samples indicated in red .

O O O 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-DISEASE O O O O O

(B) Non-supervised clustering heatmap plot indicates substantial clustering of transcripts from each tissue but not of transcripts from patients compared to controls. PD samples indicated in red.


( D ) qPCR validation of TH levels in SN samples of PD and controls , normalized to beta - actin mRNA . T - test p * = 0 . 025 , Data presented as mean ± SD . n = 18 for CT and 24 for PD .

O O O B-EXP_ASSAY O O B-GENEPROD B-EXP_ASSAY O B-TISSUE O O B-DISEASE 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 B-DISEASE O

(D) qPCR validation of TH levels in SN samples of PD and controls, normalized to beta-actin mRNA. T-test p*=0.025, Data presented as mean ± SD. n=18 for CT and 24 for PD.


( D ) Heatmap of circRNA abundance , indicating significant tissue - based SN clustering , PD samples indicated in red .

O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O B-TISSUE O O B-DISEASE O O O O O

(D) Heatmap of circRNA abundance, indicating significant tissue-based SN clustering, PD samples indicated in red.


( A ) Total circRNAs detected in the SN , MTG and AMG of both PD and control donors , p * = 0 . 02 p * = 0 . 026 and p * = 0 . 022 for comparison between PD and CT in SN , MTG and AMG . T - test for total SN vs MTG p * = 2 . 27E - 8 and SN vs AMG p * = 5 . 86E - 10 . n = 8 for Amygdala control , 15 for Amygdala PD , 8 for MTG control and 13 for MTG PD , 10 for SN control and 15 for SN PD . The box is drawn from Q1 to Q3 with a horizontal line drawn in the middle to denote the median and x marks the average . Whiskers mark minimum or maximum values . P value was calculated by T - test .

O O O O B-SMALL_MOLECULE O O O B-TISSUE O B-TISSUE O B-TISSUE O O B-DISEASE 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 B-DISEASE O O O B-TISSUE O B-TISSUE O B-TISSUE O O O O O O B-TISSUE O B-TISSUE O O O O O O O O O B-TISSUE O B-TISSUE O O O O O O O O O O O O O B-TISSUE O O O O B-TISSUE B-DISEASE O O O B-TISSUE O O O O B-TISSUE B-DISEASE O O O B-TISSUE O O O O B-TISSUE B-DISEASE O O O O O O O O O O O O O O O O O O O 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) Total circRNAs detected in the SN, MTG and AMG of both PD and control donors, p*=0.02 p*=0.026 and p*=0.022 for comparison between PD and CT in SN, MTG and AMG. T-test for total SN vs MTG p*=2.27E-8 and SN vs AMG p*=5.86E-10. n=8 for Amygdala control, 15 for Amygdala PD, 8 for MTG control and 13 for MTG PD, 10 for SN control and 15 for SN PD. The box is drawn from Q1 to Q3 with a horizontal line drawn in the middle to denote the median and x marks the average. Whiskers mark minimum or maximum values. P value was calculated by T-test.


( B ) Global editing levels in the 3 brain regions is based on the Alu editing index , representing the weighted average editing level across all expressed Alu sequences , Wilcoxon , corrected p * = 0 . 038 , 0 . 055 and 0 . 42 for Amygdala , MTG and SN . Wilcoxon test between the Alu editing index of control and PD samples .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-TISSUE O O O O O O B-EXP_ASSAY I-EXP_ASSAY 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 B-TISSUE O B-TISSUE O B-TISSUE O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-DISEASE O O

(B) Global editing levels in the 3 brain regions is based on the Alu editing index, representing the weighted average editing level across all expressed Alu sequences, Wilcoxon, corrected p*=0.038, 0.055 and 0.42 for Amygdala, MTG and SN. Wilcoxon test between the Alu editing index of control and PD samples.


( C ) Global RNA editing levels based on the Alu editing index , studied only in Alu elements within circRNA exons and their flanking introns , in the 3 brain regions , for circRNAs corrected p * = 0 . 025 and 0 . 040 for the AMG and MTG and p = 0 . 3 for SN . For flanking introns p * = 0 . 02 and 0 . 040 for AMG and MTG and p = 0 . 34 for SN , Wilcoxon test . n = 8 for Amygdala control , 15 for Amygdala PD , 8 for MTG control and 13 for MTG PD , 10 for SN control and 15 for SN PD . The box is drawn from Q1 to Q3 with a horizontal line drawn in the middle to denote the median and x marks the average . Whiskers mark minimum or maximum values .

O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-SMALL_MOLECULE O O O O O O O O O B-TISSUE O O O B-SMALL_MOLECULE O O O O O O O O O O O O O B-TISSUE O B-TISSUE O O O O O O O B-TISSUE O O O O O O O O O O O O O O O B-TISSUE O B-TISSUE O O O O O O O B-TISSUE O O O O O O O O B-TISSUE O O O O B-TISSUE B-DISEASE O O O B-TISSUE O O O O B-TISSUE B-DISEASE O O O B-TISSUE O O O O B-TISSUE B-DISEASE O O O O O O O O O O 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) Global RNA editing levels based on the Alu editing index, studied only in Alu elements within circRNA exons and their flanking introns, in the 3 brain regions, for circRNAs corrected p*= 0.025 and 0.040 for the AMG and MTG and p=0.3 for SN. For flanking introns p*= 0.02 and 0.040 for AMG and MTG and p=0.34 for SN, Wilcoxon test. n=8 for Amygdala control, 15 for Amygdala PD, 8 for MTG control and 13 for MTG PD, 10 for SN control and 15 for SN PD. The box is drawn from Q1 to Q3 with a horizontal line drawn in the middle to denote the median and x marks the average. Whiskers mark minimum or maximum values.


( D ) Negative correlation between total RNA editing and circRNA abundance , correlation = - 0 . 37 , Correlation test p value = 0 . 0015 .

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

(D) Negative correlation between total RNA editing and circRNA abundance, correlation = -0.37, Correlation test p value = 0.0015.


( E ) Adar1 expression levels in all tissues . The box is drawn from Q1 to Q3 with a horizontal line drawn in the middle to denote the median and x marks the average . Whiskers mark minimum or maximum values .

O O O B-GENEPROD 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

(E) Adar1 expression levels in all tissues. The box is drawn from Q1 to Q3 with a horizontal line drawn in the middle to denote the median and x marks the average. Whiskers mark minimum or maximum values.


( F , G ) Correlations between circRNAs detected in each sample and the age of the control ( green , correlation = 0 . 68 , p - value = 0 . 032 ) and PD donors ( blue , correlation = - 0 . 14 p value = 0 . 62 ) .

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 B-DISEASE B-ORGANISM O O O O O O O O O O O O O O O O O

(F,G) Correlations between circRNAs detected in each sample and the age of the control (green, correlation = 0.68, p-value = 0.032) and PD donors (blue, correlation = -0.14 p value = 0.62).


( H ) Volcano plot of DE circRNAs in PD vs control tissues , red dots indicate statistically significant DE circRNAs according to FDR correction of Wald test ( DEseq2 analysis ) . CircSLC8A1 is marked is red .

O O O O O O O B-SMALL_MOLECULE O B-DISEASE 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 B-GENEPROD O O O O O

(H) Volcano plot of DE circRNAs in PD vs control tissues, red dots indicate statistically significant DE circRNAs according to FDR correction of Wald test (DEseq2 analysis). CircSLC8A1 is marked is red.


( B , C ) CircSLC8A1 and linear SLC8A1 levels in the SN of control and PD brains , T - test p = 0 . 025 , p = 0 . 118 respectively .

O O O O O B-GENEPROD O O B-GENEPROD B-EXP_ASSAY O O B-TISSUE O O O B-DISEASE B-TISSUE O O O O O O O O O O O O O O O O O

(B, C) CircSLC8A1 and linear SLC8A1 levels in the SN of control and PD brains, T-test p=0.025, p= 0.118 respectively.


( D ) qPCR validations of circSLC8A1 and SLC8A1 mRNA changes in the SN , T - test p * = 0 . 025 . n = 18 for CT and 24 for PD , x defines mean values .

O O O B-EXP_ASSAY O O B-GENEPROD O B-GENEPROD O O O O B-TISSUE O O O O O O O O O O O O O O O O O O O B-DISEASE O O O O O O

(D) qPCR validations of circSLC8A1 and SLC8A1 mRNA changes in the SN, T-test p*=0.025. n=18 for CT and 24 for PD, x defines mean values.


( E ) no difference in circSLC8A1 levels in females and males , T - test p = 0 . 583 for control and p = 0 . 262 for PD . n = 15 females and 16 males . . The box is drawn from Q1 to Q3 with a horizontal line drawn in the middle to denote the median and x marks the average .

O O O O O O B-GENEPROD 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-DISEASE O O O O O O O O O O O O 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) no difference in circSLC8A1 levels in females and males, T-test p=0.583 for control and p=0.262 for PD. n=15 females and 16 males. . The box is drawn from Q1 to Q3 with a horizontal line drawn in the middle to denote the median and x marks the average.


( F , G ) CircSLC8A1 and linear SLC8A1 mRNA levels are correlated in control , R2 = 0 . 47 T - test p = 0 . 0017 but not in PD samples , R2 = 0 . 016 , p > 0 . 05 . n = 18 for CT and 23 for PD .

O O O O O B-GENEPROD O O B-GENEPROD 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 B-DISEASE O O O O O O O O O O O O O O O O O O O O O O B-DISEASE O

(F, G) CircSLC8A1 and linear SLC8A1 mRNA levels are correlated in control, R2=0.47 T-test p=0.0017 but not in PD samples, R2= 0.016, p>0.05. n=18 for CT and 23 for PD.


( H ) Increased CircSLC8A1 RNA levels in PQ - exposed SH - SY cells ( normalized to Tubb3 and RPL19 ) while other circRNAs remain

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

(H) Increased CircSLC8A1 RNA levels in PQ-exposed SH-SY cells (normalized to Tubb3 and RPL19) while other circRNAs remain unchanged


( I , J ) Immunoblot decline ( western blot and quantification ) of the SLC8A1 protein in PQ - exposed SH - SY cells , with Tubulin as a loading control , T - test p * = 0 . 015 . n = 3 biological replicas for each condition . Data presented as mean ± SD .

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

(I,J) Immunoblot decline (western blot and quantification) of the SLC8A1 protein in PQ-exposed SH-SY cells, with Tubulin as a loading control, T-test p*=0.015. n=3 biological replicas for each condition. Data presented as mean ± SD.


( A ) qPCR measurements of circSLC8A1 and SLC8A1 mRNA after SH - SY treatment of statins or the PF LRRK2 inhibitor , T - test p * * = 0 . 007 and p * * = 0 . 01 for PF - 06447475 and p * = 0 . 047 and p = * 0 . 0136 for Statins for cirSLC8A1 and SLC8A1 mRNA

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

(A) qPCR measurements of circSLC8A1 and SLC8A1 mRNA after SH-SY treatment of statins or the PF LRRK2 inhibitor, T-test p**=0.007 and p**=0.01 for PF-06447475 and p*=0.047 and p=*0.0136 for Statins for cirSLC8A1 and SLC8A1 mRNA respectively


( B , C ) Protein gel and quantification of SLC8A1 and Tubulin as a loading control in Statin and PF treated neuronal cell cultures , T - test p * = 0 . 027 for Statins and p * = 0 . 011 for PF - 06447475 , n = 3 biological replicas for each condition . Data presented as mean ± SD .

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

(B,C) Protein gel and quantification of SLC8A1 and Tubulin as a loading control in Statin and PF treated neuronal cell cultures, T-test p*=0.027 for Statins and p*=0.011 for PF-06447475, n=3 biological replicas for each condition. Data presented as mean ± SD.


( D ) SLC8A1 immunostaining of statin - treated SH - SY cells .

(D) SLC8A1 immunostaining of statin-treated SH-SY cells.


( B ) qPCR quantification from nuclear / cytoplasmic fractionations of SH - SY cells , T - test p = 0 . 005 for circSLC8A1 , p = 0 . 009 for NEAT1 and p = 0 . 009 for Rpl19 . n = 3 biological replicas for each condition .

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

(B) qPCR quantification from nuclear/cytoplasmic fractionations of SH-SY cells, T-test p=0.005 for circSLC8A1, p=0.009 for NEAT1 and p=0.009 for Rpl19. n=3 biological replicas for each condition.


( C ) qPCR quantification of Ago2 - bound RNA , immunoprecipitated ( IP ) / Input values normalized to negative control for a known Ago2 target , circCDR1as and circSLC8A1 , T - test p = 0 . 029 for circSLC8A1 and p = 0 . 001 for circCDR1as , n = 3 biological replicas for each condition .

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

(C) qPCR quantification of Ago2-bound RNA, immunoprecipitated (IP)/Input values normalized to negative control for a known Ago2 target, circCDR1as and circSLC8A1, T-test p=0.029 for circSLC8A1 and p=0.001 for circCDR1as, n=3 biological replicas for each condition.


( G ) relative expression of known miR - 128 targets from SN RNA - seq , p * * = 0 . 0004 for BMI1 , p * = 0 . 011 for SIRT1 and p * = 0 . 005 for AXIN1 .

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

(G) relative expression of known miR-128 targets from SN RNA-seq, p**=0.0004 for BMI1, p*=0.011 for SIRT1 and p*=0.005 for AXIN1.


( H ) relative expression of known miR - 128 targets in SH - SY cells treated with Paraquat , T - test p * = 0 . 03 , p * = 0 . 02 and p * = 0 . 002 for AXIN1 , BM1 and SIRT1 respectively , Data presented as mean ± SD with 4 - 6 biological replicas for each condition .

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

(H) relative expression of known miR-128 targets in SH-SY cells treated with Paraquat, T-test p*=0.03, p*=0.02 and p*=0.002 for AXIN1, BM1 and SIRT1 respectively, Data presented as mean ± SD with 4-6 biological replicas for each condition.


A In silico analysis of Sirt7 mRNA expression in different tissues of the BXD genetic reference population ( www . genenetwork . org ) . Adr : Adrenal Gland mRNA , Bla : Bladder mRNA , Bone : Bone Tibia mRNA , CNS : CNS mRNA , Cec : Cecum mRNA , Epi : Epididymis mRNA , Eso : Esophagus mRNA , Eye : Eye mRNA , Fat : Peritoneal Fat mRNA , GI : GI Track mRNA , Hea : Heart mRNA , Kid : Kidney mRNA , Liv : Liver mRNA , Lung : Lung mRNA , Mus : Gastrocnemius Muscle mRNA , Ova : Ovary mRNA , Pro : Prostate mRNA , Sal : Salivary Gland mRNA , Skin : Skin Back mRNA , Sn : Sciatic Nerve mRNA , Spl : Spleen mRNA , Tes : Testis mRNA , Thy : Thymus mRNA , Ton : Tongue mRNA , Tra : Trachea mRNA , and Ute : Uterus mRNA .

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A In silico analysis of Sirt7 mRNA expression in different tissues of the BXD genetic reference population (www.genenetwork.org). Adr: Adrenal Gland mRNA, Bla: Bladder mRNA, Bone: Bone Tibia mRNA, CNS: CNS mRNA, Cec: Cecum mRNA, Epi: Epididymis mRNA, Eso: Esophagus mRNA, Eye: Eye mRNA, Fat: Peritoneal Fat mRNA, GI: GI Track mRNA, Hea: Heart mRNA, Kid: Kidney mRNA, Liv: Liver mRNA, Lung: Lung mRNA, Mus: Gastrocnemius Muscle mRNA, Ova: Ovary mRNA, Pro: Prostate mRNA, Sal: Salivary Gland mRNA, Skin: Skin Back mRNA, Sn: Sciatic Nerve mRNA, Spl: Spleen mRNA, Tes: Testis mRNA, Thy: Thymus mRNA, Ton: Tongue mRNA, Tra: Trachea mRNA, and Ute: Uterus mRNA.


B Correlation plot for kidney mRNA expression of Sirt7 and Slc12a7 that codifies KCC4 in the mouse BxD genetic reference population ( www . genenetwork . org ) .

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B Correlation plot for kidney mRNA expression of Sirt7 and Slc12a7 that codifies KCC4 in the mouse BxD genetic reference population (www.genenetwork.org).


C Oocytes injected with cRNA encoding KCC4 were incubated with NAD + 200 μM and NAM 10 mM for 4 h . Lysates were immunoprecipitated with an anti - KCC4 antibody and then analyzed by SDS - PAGE / immunoblotting using an anti - acetylated - lysine ( AcK ) or anti - KCC4 antibody . Non - injected oocytes ( UI ) were used as a control .

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C Oocytes injected with cRNA encoding KCC4 were incubated with NAD+ 200 μM and NAM 10 mM for 4 h. Lysates were immunoprecipitated with an anti-KCC4 antibody and then analyzed by SDS-PAGE/immunoblotting using an anti-acetylated-lysine (AcK) or anti-KCC4 antibody. Non-injected oocytes (UI) were used as a control.


D Oocytes expressing KCC4 were incubated with NAM for the indicated times , and KCC4 protein levels ( upper panel ) , and activity ( lower panel ) were analyzed by SDS - PAGE / immunoblotting or Cl - - dependent 86Rb + uptake under hypotonic conditions .

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D Oocytes expressing KCC4 were incubated with NAM for the indicated times, and KCC4 protein levels (upper panel), and activity (lower panel) were analyzed by SDS-PAGE/immunoblotting or Cl--dependent 86Rb+ uptake under hypotonic conditions.


E Oocytes injected with H20 or KCC4 were incubated for the indicated times , and KCC4 protein levels were analyzed by SDS - PAGE / immunoblotting .

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E Oocytes injected with H20 or KCC4 were incubated for the indicated times, and KCC4 protein levels were analyzed by SDS-PAGE/immunoblotting.


F Oocytes were injected with either WT , K114R or K114Q KCC4 and incubated with vehicle ( V ) , NAD + or NAM for 48 h , and KCC4 protein levels ( upper pannel ) , and activity ( lower pannel ) were analyzed as in D .

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F Oocytes were injected with either WT, K114R or K114Q KCC4 and incubated with vehicle (V), NAD+ or NAM for 48 h, and KCC4 protein levels (upper pannel), and activity (lower pannel) were analyzed as in D.


A , B KCC4 activity ( A ) , and protein levels ( B ) were evaluated in Xenopus oocytes expressing SIRT7 and KCC4 that were incubated for the indicated times with NAD + . Non - injected oocytes ( UI ) were used as a control .

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A, B KCC4 activity (A), and protein levels (B) were evaluated in Xenopus oocytes expressing SIRT7 and KCC4 that were incubated for the indicated times with NAD+. Non-injected oocytes (UI) were used as a control.


C KCC4 activity was evaluated in Xenopus oocytes injected with cRNA encoding KCC4 and coinjected with either wild - type ( SIRT7 WT ) or the catalytically inactive SIRT7 ( SIRT7 H188Y ) for 48 h .

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C KCC4 activity was evaluated in Xenopus oocytes injected with cRNA encoding KCC4 and coinjected with either wild-type (SIRT7 WT) or the catalytically inactive SIRT7 (SIRT7 H188Y) for 48 h.


D Oocytes were injected with either KCC4 WT , K114R or K114Q for 48 h and then incubated with vehicle ( V ) , NAD + or NAM for 24 h , and KCC4 and SIRT7 protein levels were analyzed by SDS - PAGE / immunoblotting .

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D Oocytes were injected with either KCC4 WT, K114R or K114Q for 48 h and then incubated with vehicle (V), NAD+ or NAM for 24 h, and KCC4 and SIRT7 protein levels were analyzed by SDS-PAGE/immunoblotting.


E Oocytes were injected with either KCC4 WT , K114R or K114Q with and without SIRT7 for 48 h and then incubated with vehicle ( V ) or NAM for 4 h . After lysis , KCC4 was immunoprecipitated using an anti - KCC4 antibody , and the acetylation status of KCC4 was determined by SDS - PAGE / Western blotting using and anti - acetyl lysine antibody .

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E Oocytes were injected with either KCC4 WT, K114R or K114Q with and without SIRT7 for 48 h and then incubated with vehicle (V) or NAM for 4 h. After lysis, KCC4 was immunoprecipitated using an anti-KCC4 antibody, and the acetylation status of KCC4 was determined by SDS-PAGE/Western blotting using and anti-acetyl lysine antibody.


F , G FLAG - KCC4 and SIRT7 were transfected into HEK293 cells and treated for the indicated times with NAD + ( F ) or NAM ( G ) and then analyzed by SDS - PAGE / immunoblotting using an anti - KCC4 or anti - SIRT7 antibody . Non - transfected cells ( UT ) were used as a control .

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F, G FLAG-KCC4 and SIRT7 were transfected into HEK293 cells and treated for the indicated times with NAD+ (F) or NAM (G) and then analyzed by SDS-PAGE/immunoblotting using an anti-KCC4 or anti-SIRT7 antibody. Non-transfected cells (UT) were used as a control.


H FLAG - KCC4 and SIRT7 were transfected into HEK293 cells and treated for the indicated times with NAD + and NAM . After lysis , KCC4 immunoprecipitation using an anti - FLAG antibody was performed , and the interaction with SIRT7 was determined by SDS - PAGE / Western blotting .

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H FLAG-KCC4 and SIRT7 were transfected into HEK293 cells and treated for the indicated times with NAD+ and NAM. After lysis, KCC4 immunoprecipitation using an anti-FLAG antibody was performed, and the interaction with SIRT7 was determined by SDS-PAGE/Western blotting.


I HEK293 cells expressing KCC4 were incubated with NAD + 200 μM and cycloheximide ( CHX ) for 0 , 3 , 6 and 9 h . KCC4 protein levels are shown at the indicated times in cells transfected with a scramble shRNA ( + SIRT7 ) or a shRNA against SIRT7 ( - SIRT7 ) .

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I HEK293 cells expressing KCC4 were incubated with NAD+ 200 μM and cycloheximide (CHX) for 0, 3, 6 and 9 h. KCC4 protein levels are shown at the indicated times in cells transfected with a scramble shRNA (+SIRT7) or a shRNA against SIRT7 (-SIRT7).


A KCC4 acetylation at the indicated pH in HEK293 transfected with KCC4 . After lysis , KCC4 was immunoprecipitated using an anti - FLAG antibody , and then analyzed by SDS - PAGE / Western blotting using an anti - acetylated - lysine ( AcK ) or anti - KCC4 antibody . Non - transfected cells ( UT ) were used as a control .

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A KCC4 acetylation at the indicated pH in HEK293 transfected with KCC4. After lysis, KCC4 was immunoprecipitated using an anti-FLAG antibody, and then analyzed by SDS-PAGE/Western blotting using an anti-acetylated-lysine (AcK) or anti-KCC4 antibody. Non-transfected cells (UT) were used as a control.


B HEK293 cells were transfected with KCC4 together with SIRT7 with a scramble shRNA ( - ) or a shRNA against SIRT7 ( + ) , and KCC4 activity and protein level were determined at the indicated pH . SIRT7 expression in these conditions is presented in C .

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B HEK293 cells were transfected with KCC4 together with SIRT7 with a scramble shRNA (-) or a shRNA against SIRT7 (+), and KCC4 activity and protein level were determined at the indicated pH. SIRT7 expression in these conditions is presented in C.


C Nuclear and cytoplasmic SIRT7 levels at different pH values in HEK293 cells . At a lower pH , SIRT7 appears to translocate to the cytoplasm .

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C Nuclear and cytoplasmic SIRT7 levels at different pH values in HEK293 cells. At a lower pH, SIRT7 appears to translocate to the cytoplasm.


A , B Sirt7 mRNA ( A ) and protein ( B ) abundance in microdissected renal tubules normalized to Rpl26 or Ponceau , respectively . Enrichment for each segment was previously validated by immunoblotting against segment - specific markers as indicated in material and methods ( Spirli et al , 2019 ) .

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A, B Sirt7 mRNA (A) and protein (B) abundance in microdissected renal tubules normalized to Rpl26 or Ponceau, respectively. Enrichment for each segment was previously validated by immunoblotting against segment-specific markers as indicated in material and methods (Spirli et al, 2019).


C Immunolocalization of SIRT7 and ATPase in the kidneys of C57BL6 mice with metabolic acidosis ( SIRT7 , red ; ATPase , green ; DAPI , Blue ) . Scale bar : 20 μm . Controls of the specificity of antibodies using kidney of SIRT7 - deficient mice are provided in Appendix Fig S4 .

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C Immunolocalization of SIRT7 and ATPase in the kidneys of C57BL6 mice with metabolic acidosis (SIRT7, red; ATPase, green; DAPI, Blue). Scale bar: 20 μm. Controls of the specificity of antibodies using kidney of SIRT7-deficient mice are provided in Appendix Fig S4.


D KCC4 and SIRT7 protein levels in kidneys from control mice or mice with metabolic acidosis . Values are the mean ± SEM .

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D KCC4 and SIRT7 protein levels in kidneys from control mice or mice with metabolic acidosis. Values are the mean ± SEM.


A KCC4 protein levels were determined in the kidneys of wild - type ( SIRT7 + / + ) and SIRT7 - deficient mice ( SIRT7 - / - ) . SIRT7 protein expression are absent in the kidneys of SIRT7 - deficient mice .

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A KCC4 protein levels were determined in the kidneys of wild-type (SIRT7+/+) and SIRT7-deficient mice (SIRT7-/-). SIRT7 protein expression are absent in the kidneys of SIRT7-deficient mice.


B KCC4 protein levels , acetylation status of KCC4 , and interaction with SIRT7 in kidneys of SIRT7 + / + and SIRT7 - / - mice under normal conditions ( Control ) or with metabolic acidosis .

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B KCC4 protein levels, acetylation status of KCC4, and interaction with SIRT7 in kidneys of SIRT7+/+ and SIRT7-/-mice under normal conditions (Control) or with metabolic acidosis.


C Immunolocalization of KCC4 and H - ATPase in the kidneys of SIRT7 + / + and SIRT7 - / - mice under normal conditions ( Control ) or with metabolic acidosis ( NH4Cl ) . ( KCC4 , red ; H - ATPase , green ) . Scale bar : 20 μm . Cell count was performed automatically using the Gen5 software to report the percentage of KCC4 + cells among H - ATPase + cells ( lower panel ) .

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C Immunolocalization of KCC4 and H-ATPase in the kidneys of SIRT7+/+ and SIRT7-/- mice under normal conditions (Control) or with metabolic acidosis (NH4Cl). (KCC4, red; H-ATPase, green). Scale bar: 20 μm. Cell count was performed automatically using the Gen5 software to report the percentage of KCC4+ cells among H-ATPase+ cells (lower panel).


D NCC and NKCC2 protein levels in the kidney of SIRT7 + / + and SIRT7 - / - mice under basal conditions .

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D NCC and NKCC2 protein levels in the kidney of SIRT7+/+ and SIRT7-/- mice under basal conditions.


HeLa cell lines were treated with the small - molecule Bcl - 2 / Bcl - XL - inhibitor ABT - 737 for 72 h . Cytokines were measured by ELISA . ( A ) Concentrations of IL - 6 were determined ( CTRL , n = 9 ; Bax / Bak , n = 6 ; Bcl - XL , n = 3 ; STING , n = 3 ) .

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HeLa cell lines were treated with the small-molecule Bcl-2/Bcl-XL-inhibitor ABT-737 for 72 h. Cytokines were measured by ELISA. (A) Concentrations of IL-6 were determined (CTRL, n=9; Bax/Bak, n=6; Bcl-XL, n=3; STING, n=3).


HeLa cell lines were treated with the small - molecule Bcl - 2 / Bcl - XL - inhibitor ABT - 737 for 72 h . Cytokines were measured by ELISA . ( B ) Concentrations of IL - 8 were determined ( CTRL , n = 7 ; Bax / Bak , n = 4 ) .

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HeLa cell lines were treated with the small-molecule Bcl-2/Bcl-XL-inhibitor ABT-737 for 72 h. Cytokines were measured by ELISA. (B) Concentrations of IL-8 were determined (CTRL, n=7; Bax/Bak, n=4).


HeLa cell lines were treated with the small - molecule Bcl - 2 / Bcl - XL - inhibitor ABT - 737 for 72 h . Cytokines were measured by ELISA . ( C ) Concentrations of CXCL1 were determined ( CTRL , n = 8 ; Bax / Bak , n = 5 ) .

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HeLa cell lines were treated with the small-molecule Bcl-2/Bcl-XL-inhibitor ABT-737 for 72 h. Cytokines were measured by ELISA. (C) Concentrations of CXCL1 were determined (CTRL, n=8; Bax/Bak, n=5).


HeLa cell lines were treated with the small - molecule Bcl - 2 / Bcl - XL - inhibitor ABT - 737 for 72 h . Cytokines were measured by ELISA . ( D ) Concentrations of FGF - 2 were determined ( CTRL , n = 7 ; Bax / Bak , n = 4 ) .

HeLa cell lines were treated with the small-molecule Bcl-2/Bcl-XL-inhibitor ABT-737 for 72 h. Cytokines were measured by ELISA. (D) Concentrations of FGF-2 were determined (CTRL, n=7; Bax/Bak, n=4).


HeLa cell lines were treated with the Mcl - 1 - inhibitor S63845 for 72 h . Cytokines were measured by ELISA . ( E ) HeLa CTRL cells and Bax / Bak - deficient cells were treated with the Mcl - 1 - inhibitor S63845 . Concentrations of IL - 6 in supernatants are shown ( CTRL , n = 8 ; Bax / Bak , n = 7 ) .

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HeLa cell lines were treated with the Mcl-1-inhibitor S63845 for 72 h. Cytokines were measured by ELISA. (E) HeLa CTRL cells and Bax/Bak-deficient cells were treated with the Mcl-1-inhibitor S63845. Concentrations of IL-6 in supernatants are shown (CTRL, n=8; Bax/Bak, n=7).


F 1205Lu human melanoma cells or 1205Lu melanoma cells over - expressing Bcl - XL were treated with ABT - 737 . Concentrations of IL - 6 in the supernatants were measured after 48 h ( n = 5 ) .

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F 1205Lu human melanoma cells or 1205Lu melanoma cells over-expressing Bcl-XL were treated with ABT-737. Concentrations of IL-6 in the supernatants were measured after 48 h (n=5).


G 1205Lu human melanoma cells or 1205Lu melanoma cells over - expressing mouse Bcl - XL were treated with various concentrations of ABT - 737 . To some aliquots the caspase - inhibitor zVAD - fmk ( 50 μM ) was added . Relative numbers of apoptotic cells were determined as in EV 1B after 48 h . Data are means / SEM of at least three independent experiments . ( 1205Lu , n = 4 ; 1205Lu 5 µM ABT - 737 , n = 3 ; 1205Lu / Bcl - XL and 1205Lu + zVAD , n = 3 ) .

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G 1205Lu human melanoma cells or 1205Lu melanoma cells over-expressing mouse Bcl-XL were treated with various concentrations of ABT-737. To some aliquots the caspase-inhibitor zVAD-fmk (50 μM) was added. Relative numbers of apoptotic cells were determined as in EV 1B after 48 h. Data are means/SEM of at least three independent experiments. (1205Lu, n=4; 1205Lu 5 µM ABT-737, n=3; 1205Lu/Bcl-XL and 1205Lu + zVAD, n=3).


H HeLa - UL12 . 5 cells untreated or treated with tamoxifen ( 100 nM , 24 h ) were stained with Picogreen ( 3 µl / ml ) to detect mitochondrial DNA . Scale bare , 10

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H HeLa-UL12.5 cells untreated or treated with tamoxifen (100 nM, 24 h) were stained with Picogreen (3 µl/ml) to detect mitochondrial DNA. Scale bare, 10 µm


I HeLa CTRL cells and HeLa - UL12 . 5 cells were treated with ABT - 737 ( 10µM ) . Concentration of IL - 6 in the supernatant were measured after 72h ( n = 3 ) .

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I HeLa CTRL cells and HeLa-UL12.5 cells were treated with ABT-737 (10µM). Concentration of IL-6 in the supernatant were measured after 72h (n=3).


A , The caspase - 3 - reporter cell line ( labelled DEVD ) and the control cell line ( DEVG ; see methods ) were infected and analysed at various time points post - infection by flow cytometry ( Chlamydia trachomatis ( Ctr ; MOI = 1 , 30 h ) ; Salmonella Typhimurium ( STy ; MOI = 50 , 24h ) ; Toxoplasma gondii ( MOI = 5 , 16 h ) ; MVA ( MOI = 10 , 16h ) ; Influenza A virus ( IAV ; MOI = 1 , 16h ) ; Herpes simplex virus 1 ( HSV - 1 ; MOI = 1 , 16h ) . Staurosporine treated cells ( 1 μM , 3h ) were used as a positive control . Percentages of cells positive for caspase - 3 - activation ( FRET - loss ) were determined . Data are means / SEM of three independent experiments . ( n = 3 )

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A, The caspase-3-reporter cell line (labelled DEVD) and the control cell line (DEVG; see methods) were infected and analysed at various time points post-infection by flow cytometry (Chlamydia trachomatis (Ctr; MOI=1, 30 h); Salmonella Typhimurium (STy; MOI=50, 24h); Toxoplasma gondii (MOI=5, 16 h); MVA (MOI=10, 16h); Influenza A virus (IAV; MOI=1, 16h); Herpes simplex virus 1 (HSV-1; MOI=1, 16h). Staurosporine treated cells (1 μM, 3h) were used as a positive control. Percentages of cells positive for caspase-3-activation (FRET-loss) were determined. Data are means/SEM of three independent experiments. (n=3)


B , C Cells were infected with modified vaccinia virus Ankara ( MVA ) ( MOI = 10 ) . The caspase - inhibitor zVAD - fmk was used at 100µM ; cells were analysed 16 h post - infection .

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B, C Cells were infected with modified vaccinia virus Ankara (MVA) (MOI=10). The caspase-inhibitor zVAD-fmk was used at 100µM; cells were analysed 16 h post-infection.


D , E Cells were infected with influenza A virus ( IAV ) ( MOI = 1 ; analysis was 16 h post - infection ) . D , detection of viral M2 protein was used as infection control .

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D, E Cells were infected with influenza A virus (IAV) (MOI=1; analysis was 16 h post-infection). D, detection of viral M2 protein was used as infection control.


F , G Cells were infected with Herpes simplex virus 1 ( HSV - 1 ) ( MOI = 1 ; analysis was 16 h post - infection ) .

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F, G Cells were infected with Herpes simplex virus 1 (HSV-1) (MOI=1; analysis was 16 h post-infection).


H Cells were infected with Chlamydia trachomatis ( Ctr ) ( MOI = 1 ; analysis was 30 h post - infection ) .

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H Cells were infected with Chlamydia trachomatis (Ctr) (MOI=1; analysis was 30 h post-infection).


I Cells were infected with Salmonella Typhimurium ( Sty ) ( MOI = 50 ; analysis was 24 h post - infection ) .

I Cells were infected with Salmonella Typhimurium (Sty) (MOI=50; analysis was 24 h post-infection).


J Cells were infected with the protozoan parasite Toxoplasma gondii ( MOI = 5 ; analysis was 16 h post - infection ) .

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J Cells were infected with the protozoan parasite Toxoplasma gondii (MOI=5; analysis was 16 h post-infection).


A Infection with MVA ( MOI = 10 for 16 h ; IL - 6 ) .

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A Infection with MVA (MOI=10 for 16 h; IL-6).


B infection with HSV - 1 ( MOI = 1 for 16 h ; IL - 6 ) .

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B infection with HSV-1 (MOI=1 for 16 h; IL-6).