F . Quantification of live cell numbers with trypan blue staining and manual counting , 72 h post - transfection with siLuc , sihTR # 1 or sihTR # 2 , measured as the percentage of siLuc - treated cells ( n = 3 ) . On the right , representative images of U2OS cells treated with sihTR # 1 for 72 h . Statistical analyses were performed using one - way ANOVA with post - hoc Tukey ' s test for multiple comparisons . Data information : Data are shown as mean ± SD , with the red line marking the mean value .

O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O B-CELL_LINE O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Quantification of live cell numbers with trypan blue staining and manual counting, 72 h post-transfection with siLuc, sihTR#1 or sihTR#2, measured as the percentage of siLuc-treated cells (n=3). On the right, representative images of U2OS cells treated with sihTR#1 for 72 h. Statistical analyses were performed using one-way ANOVA with post-hoc Tukey's test for multiple comparisons. Data information: Data are shown as mean ± SD, with the red line marking the mean value.


G . Western blot analyses in the indicated cell lines cells transfected for 48 h with siLuc or sihTR , with the indicated antibodies . U2OS cells treated with Hydroxyurea ( 2 mM for 24 h ) were used as positive controls .

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 B-CELL_LINE O O O B-SMALL_MOLECULE O O O O O O O O O O O O O

G. Western blot analyses in the indicated cell lines cells transfected for 48 h with siLuc or sihTR, with the indicated antibodies. U2OS cells treated with Hydroxyurea (2 mM for 24 h) were used as positive controls.


A . Representative images for fragile telomeres using the CO - FISH assay . Lagging strands ( C - probe ) and leading strands ( G - probe ) were marked with green PNA probes and red LNA probes , respectively . White circles and arrows indicate " smeary " or " doublet " telomeric signals , respectively . Quantification of fragile telomeres is shown on the right as the percentage of total telomeres counted ( n = 932 - 1073 ) . Comparison of fragile to non - fragile telomere percentage was performed using Χ2 - test . Data information : All scale bars : 5 μm . Data are shown as mean ± SD .

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

A. Representative images for fragile telomeres using the CO-FISH assay. Lagging strands (C-probe) and leading strands (G-probe) were marked with green PNA probes and red LNA probes, respectively. White circles and arrows indicate "smeary" or "doublet" telomeric signals, respectively. Quantification of fragile telomeres is shown on the right as the percentage of total telomeres counted (n=932-1073). Comparison of fragile to non-fragile telomere percentage was performed using Χ2-test. Data information: All scale bars: 5 μm. Data are shown as mean ± SD.


B . Representative images for co - localization between RAD51 ( green ) and telomeres ( red FISH probe ) . White arrows indicate representative RAD51 - telomere co - localization events . Quantifications are shown on the right . A total of 120 nuclei were scored from two independent experiments . Data information : All scale bars : 5 μm . Data are shown as mean ± SD . B , the red line marks the mean value . Statistical analyses were performed with Student ' s two - tailed unpaired t - tests with Welch ' s correction for normality of distribution , unless specified otherwise .

O O O B-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O B-SUBCELLULAR O O B-EXP_ASSAY O O O O O O O B-GENEPROD O B-SUBCELLULAR B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Representative images for co-localization between RAD51 (green) and telomeres (red FISH probe). White arrows indicate representative RAD51-telomere co-localization events. Quantifications are shown on the right. A total of 120 nuclei were scored from two independent experiments. Data information: All scale bars: 5 μm. Data are shown as mean ± SD. B, the red line marks the mean value. Statistical analyses were performed with Student's two-tailed unpaired t-tests with Welch's correction for normality of distribution, unless specified otherwise.


C . Representative images for TIF quantification : co - localization between 53BP1 ( green ) and telomeres ( red FISH probe ) in U2OS or IMRB cells with or without ectopic hTR overexpression . White arrows indicate TIF . Quantification of TIF is on the right . A total of 110 - 170 nuclei were scored from three independent experiments . Each dot represents the number of TIF per nucleus . Data information : All scale bars : 5 μm . Data are shown as mean ± SD . , C , the red line marks the mean value . Statistical analyses were performed with Student ' s two - tailed unpaired t - tests with Welch ' s correction for normality of distribution , unless specified otherwise .

O O O B-EXP_ASSAY O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O B-SUBCELLULAR O O B-EXP_ASSAY O O O B-CELL_LINE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR 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 O O O O O O O O O O O O O O O O O O O O O O O O O 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. Representative images for TIF quantification: co-localization between 53BP1 (green) and telomeres (red FISH probe) in U2OS or IMRB cells with or without ectopic hTR overexpression. White arrows indicate TIF. Quantification of TIF is on the right. A total of 110-170 nuclei were scored from three independent experiments. Each dot represents the number of TIF per nucleus. Data information: All scale bars: 5 μm. Data are shown as mean ± SD. , C, the red line marks the mean value. Statistical analyses were performed with Student's two-tailed unpaired t-tests with Welch's correction for normality of distribution, unless specified otherwise.


D . Representative images for TIF quantification , as in ( A ) , in U2OS or U2OS - hTR cells , treated as indicated with DMSO or 5 μM ATRi or 5 μM ATMi for 20 h . White arrows indicate TIF . Data information : All scale bars : 5 μm . Data are shown as mean ± SD .

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

D. Representative images for TIF quantification, as in (A), in U2OS or U2OS-hTR cells, treated as indicated with DMSO or 5 μM ATRi or 5 μM ATMi for 20 h. White arrows indicate TIF. Data information: All scale bars: 5 μm. Data are shown as mean ± SD.


E . Quantification of TIF from ( D ) . At least 65 nuclei were scored in each of the two independent experiments performed . Each dot represents the number of TIF per nucleus . Statistical analyses were performed using one - way ANOVA with post - hoc Games - Howell ' s test for multiple comparisons . Data information : All scale bars : 5 μm . Data are shown as mean ± SD . the red line marks the mean value . Statistical analyses were performed with Student ' s two - tailed unpaired t - tests with Welch ' s correction for normality of distribution , unless specified otherwise .

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 O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Quantification of TIF from (D). At least 65 nuclei were scored in each of the two independent experiments performed. Each dot represents the number of TIF per nucleus. Statistical analyses were performed using one-way ANOVA with post-hoc Games-Howell's test for multiple comparisons. Data information: All scale bars: 5 μm. Data are shown as mean ± SD. the red line marks the mean value. Statistical analyses were performed with Student's two-tailed unpaired t-tests with Welch's correction for normality of distribution, unless specified otherwise.


F . Representative images for co - localization between POLD3 ( red ) and RAP1 ( green ) . White arrows indicate co - localization events . Quantification of co - localization events between POLD3 and RAP1 is shown on the right . A total of 190 - 230 nuclei were scored from three independent experiments . Data information : All scale bars : 5 μm . Data are shown as mean ± SD . F : the red line marks the mean value . Statistical analyses were performed with Student ' s two - tailed unpaired t - tests with Welch ' s correction for normality of distribution , unless specified otherwise .

O O O B-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O B-GENEPROD O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O B-GENEPROD O O O O O O 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 O O O O O O O O O O O O O O O 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. Representative images for co-localization between POLD3 (red) and RAP1 (green). White arrows indicate co-localization events. Quantification of co-localization events between POLD3 and RAP1 is shown on the right. A total of 190-230 nuclei were scored from three independent experiments. Data information: All scale bars: 5 μm. Data are shown as mean ± SD. F: the red line marks the mean value. Statistical analyses were performed with Student's two-tailed unpaired t-tests with Welch's correction for normality of distribution, unless specified otherwise.


G . C - circle analysis using slot - blot assay hybridized with radioactive C - rich telomeric probe . Quantification is shown below ( n = 7 ) . Statistical analyses were performed using the non - parametrical Mann Whitney test . Data information : All scale bars : 5 μm . Data are shown as mean ± SD .

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

G. C-circle analysis using slot-blot assay hybridized with radioactive C-rich telomeric probe. Quantification is shown below (n=7). Statistical analyses were performed using the non-parametrical Mann Whitney test. Data information: All scale bars: 5 μm. Data are shown as mean ± SD.


H , I . C - circle analyses in the indicated cell lines 72 h after treatment with siPOLD3 or siLuc used as control for transfection . Quantifications are shown below ( n = 3 ) . siSMARCAL1 was used as positive control for increased C - circle production in cells with defective fork reversal activity ( Zhang et al , 2019b ) . Data information : All scale bars : 5 μm . Data are shown as mean ± SD .

O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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,I. C-circle analyses in the indicated cell lines 72 h after treatment with siPOLD3 or siLuc used as control for transfection. Quantifications are shown below (n=3). siSMARCAL1 was used as positive control for increased C-circle production in cells with defective fork reversal activity (Zhang et al, 2019b). Data information: All scale bars: 5 μm. Data are shown as mean ± SD.


A . Representative images for co - localization between PML ( green ) and telomeres ( red FISH probe ) . White arrows indicate co - localization events , marking APBs . Quantification of images is shown on the right . A total of 85 - 300 nuclei were scored from four independent experiments . Each dot represents the number of events per nucleus : PML foci or APBs . Data information : All scale bars : 5 μm . Data are shown as mean ± SD , with the red line marking the mean value . Statistical analyses were performed with Student ' s two - tailed unpaired t - test with Welch ' s correction for normality of distribution .

O O O B-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-SUBCELLULAR O O O O B-SUBCELLULAR O O B-EXP_ASSAY O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-SUBCELLULAR O O O B-EXP_ASSAY O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O B-SUBCELLULAR O B-SUBCELLULAR 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 O O O O 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. Representative images for co-localization between PML (green) and telomeres (red FISH probe). White arrows indicate co-localization events, marking APBs. Quantification of images is shown on the right. A total of 85-300 nuclei were scored from four independent experiments. Each dot represents the number of events per nucleus: PML foci or APBs. Data information: All scale bars: 5 μm. Data are shown as mean ± SD, with the red line marking the mean value. Statistical analyses were performed with Student's two-tailed unpaired t-test with Welch's correction for normality of distribution.


C . Representative images for co - localization between EdU ( green ) and telomeres ( red FISH probe ) within PML bodies ( cyan ) in U2OS and U2OS - hTR cells . White arrows indicate co - localization events . D , E . Quantification of images shown in ( C ) . Co - localization events between EdU foci and APBs were analyzed ( D ) and the percentage of APBs that are actively engaged in G2 / M telomeric synthesis was calculated ( E ) . A total of at least 50 nuclei were scored from three independent experiments . Data information : All scale bars : 5 μm . Data are shown as mean ± SD , with the red line marking the mean value . Statistical analyses were performed with Student ' s two - tailed unpaired t - test with Welch ' s correction for normality of distribution .

O O O B-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-SMALL_MOLECULE O O O O B-SUBCELLULAR O O B-EXP_ASSAY O O O B-SUBCELLULAR I-SUBCELLULAR O O O O B-CELL_LINE O B-CELL_LINE O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O B-EXP_ASSAY O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-SMALL_MOLECULE O O B-SUBCELLULAR O O O O O O O O O B-SUBCELLULAR O O O O O O O O B-SUBCELLULAR 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 O O O O O O O O O O O O O O O O O O O O O O O O O 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. Representative images for co-localization between EdU (green) and telomeres (red FISH probe) within PML bodies (cyan) in U2OS and U2OS-hTR cells. White arrows indicate co-localization events. D, E. Quantification of images shown in (C). Co-localization events between EdU foci and APBs were analyzed (D) and the percentage of APBs that are actively engaged in G2/M telomeric synthesis was calculated (E). A total of at least 50 nuclei were scored from three independent experiments. Data information: All scale bars: 5 μm. Data are shown as mean ± SD, with the red line marking the mean value. Statistical analyses were performed with Student's two-tailed unpaired t-test with Welch's correction for normality of distribution.


A . Representative images for co - localization between RPA32 ( green ) and single stranded C - rich telomeric sequences ( ss - Telo - C , red FISH probe ) , in G2 / M - synchronized cells . White arrows indicate co - localization events . B . Quantification of images in ( A ) . A total of at least 55 nuclei were scored from three independent experiments . Each dot represents the number of events per nucleus . C . Quantification of images in ( A ) as the percentage of single stranded C - rich telomeric sequences associated with RPA32 to total single stranded C - rich telomeric sequences A total of at least 55 nuclei were scored from three independent experiments . Data information : All scale bars : 5 μm . Data are shown as mean ± SD , with the red line marking the mean value . Statistical analyses were performed with Student ' s two - tailed unpaired t - test with Welch ' s correction for normality of distribution unless specified otherwise .

O O O B-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR 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 I-EXP_ASSAY O O O O O O B-EXP_ASSAY O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O B-EXP_ASSAY O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O B-GENEPROD O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR I-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 O O O O O O O O O O O O O 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. Representative images for co-localization between RPA32 (green) and single stranded C-rich telomeric sequences (ss-Telo-C, red FISH probe), in G2/M-synchronized cells. White arrows indicate co-localization events. B. Quantification of images in (A). A total of at least 55 nuclei were scored from three independent experiments. Each dot represents the number of events per nucleus. C. Quantification of images in (A) as the percentage of single stranded C-rich telomeric sequences associated with RPA32 to total single stranded C-rich telomeric sequences A total of at least 55 nuclei were scored from three independent experiments. Data information: All scale bars: 5 μm. Data are shown as mean ± SD, with the red line marking the mean value. Statistical analyses were performed with Student's two-tailed unpaired t-test with Welch's correction for normality of distribution unless specified otherwise.


D . Representative images for co - localization between RPA32 or p - RPA32S33 ( green ) and telomeres ( red FISH probe ) , in the indicated cell lines treated with siLuc or sihnRNPA1 for 48 h . White arrows indicate co - localization events . Data information : All scale bars : 5 μm . Data are shown as mean ± SD , with the red line marking the mean value . Statistical analyses were performed with Student ' s two - tailed unpaired t - test with Welch ' s correction for normality of distribution unless specified otherwise .

O O O B-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O B-GENEPROD O O O O B-SUBCELLULAR 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 B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Representative images for co-localization between RPA32 or p-RPA32S33 (green) and telomeres (red FISH probe), in the indicated cell lines treated with siLuc or sihnRNPA1 for 48 h. White arrows indicate co-localization events. Data information: All scale bars: 5 μm. Data are shown as mean ± SD, with the red line marking the mean value. Statistical analyses were performed with Student's two-tailed unpaired t-test with Welch's correction for normality of distribution unless specified otherwise.


E . Quantification of images in ( D ) . A total of 85 - 135 nuclei were scored from three independent experiments . Each dot represents the number of events per nucleus . Statistical analyses were performed using one - way ANOVA with post - hoc Games - Howell ' s test for multiple comparisons . Data information : All scale bars : 5 μm . Data are shown as mean ± SD , with the red line marking the mean value . Statistical analyses were performed with Student ' s two - tailed unpaired t - test with Welch ' s correction for normality of distribution unless specified otherwise .

O O O O B-EXP_ASSAY O O O O O O O O O O O B-SUBCELLULAR 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Quantification of images in (D). A total of 85-135 nuclei were scored from three independent experiments. Each dot represents the number of events per nucleus. Statistical analyses were performed using one-way ANOVA with post-hoc Games-Howell's test for multiple comparisons. Data information: All scale bars: 5 μm. Data are shown as mean ± SD, with the red line marking the mean value. Statistical analyses were performed with Student's two-tailed unpaired t-test with Welch's correction for normality of distribution unless specified otherwise.


F . Representative images for co - localization between RPA32 ( green ) and telomeres ( red FISH probe ) in the indicated cell lines treated with either DMSO or 10 μM NU7026 DNA - PK - inhibitor ( DNA - PK - i ) for 48 h . White arrows indicate co - localization events . Quantifications are shown on the right . A total of 53 - 84 nuclei were scored from three independent experiments . Statistical analyses were performed using one - way ANOVA with post - hoc Games - Howell ' s test for multiple comparisons . Data information : All scale bars : 5 μm . Data are shown as mean ± SD , with the red line marking the mean value . Statistical analyses were performed with Student ' s two - tailed unpaired t - test with Welch ' s correction for normality of distribution unless specified otherwise .

O O O B-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O B-SUBCELLULAR O O B-EXP_ASSAY O O O O O O O O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Representative images for co-localization between RPA32 (green) and telomeres (red FISH probe) in the indicated cell lines treated with either DMSO or 10 μM NU7026 DNA-PK-inhibitor (DNA-PK-i) for 48 h. White arrows indicate co-localization events. Quantifications are shown on the right. A total of 53-84 nuclei were scored from three independent experiments. Statistical analyses were performed using one-way ANOVA with post-hoc Games-Howell's test for multiple comparisons. Data information: All scale bars: 5 μm. Data are shown as mean ± SD, with the red line marking the mean value. Statistical analyses were performed with Student's two-tailed unpaired t-test with Welch's correction for normality of distribution unless specified otherwise.


A . hTR and NHP2 mRNA expression in U2OS - hTR cells 72 h post - transfection with siLuc or siNHP2 , normalized to ACTB mRNA and to SI14 / TEL + cells ( n = 2 ) . Data information : All scale bars : 5 μm . Data are shown as mean ± SD , with the red line marking the mean value .

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

A. hTR and NHP2 mRNA expression in U2OS-hTR cells 72 h post-transfection with siLuc or siNHP2, normalized to ACTB mRNA and to SI14/TEL+ cells (n=2). Data information: All scale bars: 5 μm. Data are shown as mean ± SD, with the red line marking the mean value.


B . Western blot showing NHP2 protein levels in the indicated cells treated with siLuc or siNHP2 , 72 h post - transfection . β - Actin served as loading control .

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

B. Western blot showing NHP2 protein levels in the indicated cells treated with siLuc or siNHP2, 72 h post-transfection. β-Actin served as loading control.


C . Representative images for co - localization between p - RPA32S33 ( green ) and telomeres ( red FISH probe ) in U2OS - hTR cells treated with control siLuc or siNHP2 , 72 h post - transfection . White arrows indicate co - localization events . Quantification of images is shown on the right . Each dot indicates the number of co - localization events per nucleus ( n = 2 ) . Statistical analyses were performed with Student ' s two - tailed unpaired t - tests with Welch ' s correction for normality of distribution . Data information : All scale bars : 5 μm . Data are shown as mean ± SD , with the red line marking the mean value .

O O O B-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O O O O B-SUBCELLULAR O O B-EXP_ASSAY O O O B-CELL_LINE O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-EXP_ASSAY O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY 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 O O O O O O O O O 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. Representative images for co-localization between p-RPA32S33 (green) and telomeres (red FISH probe) in U2OS-hTR cells treated with control siLuc or siNHP2, 72 h post-transfection. White arrows indicate co-localization events. Quantification of images is shown on the right. Each dot indicates the number of co-localization events per nucleus (n=2). Statistical analyses were performed with Student's two-tailed unpaired t-tests with Welch's correction for normality of distribution. Data information: All scale bars: 5 μm. Data are shown as mean ± SD, with the red line marking the mean value.


D . Representative images for TIF quantification : co - localization between 53BP1 ( green ) and telomeres ( red FISH probe ) , measured 72 h post - transfection with siLuc or siNHP2 in cells further treated with either DMSO or Doxorubicin ( DOXO , 1 . 5 μM ) for 2 h . White arrows indicate TIF . Quantification of TIF is shown on the right . A total of at least 100 nuclei were scored from two independent experiments . Each dot represents the number of TIF per nucleus . Statistical analyses were performed using one - way ANOVA with post - hoc Games - Howell ' s test for multiple comparisons . Data information : All scale bars : 5 μm . Data are shown as mean ± SD , with the red line marking the mean value .

O O O B-EXP_ASSAY O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O B-SUBCELLULAR O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR 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 O O O O O O O O O O O O O O O O O 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. Representative images for TIF quantification: co-localization between 53BP1 (green) and telomeres (red FISH probe), measured 72 h post-transfection with siLuc or siNHP2 in cells further treated with either DMSO or Doxorubicin (DOXO, 1.5 μM) for 2 h. White arrows indicate TIF. Quantification of TIF is shown on the right. A total of at least 100 nuclei were scored from two independent experiments. Each dot represents the number of TIF per nucleus. Statistical analyses were performed using one-way ANOVA with post-hoc Games-Howell's test for multiple comparisons. Data information: All scale bars: 5 μm. Data are shown as mean ± SD, with the red line marking the mean value.


E . Western blot analyses in U2OS and U2OS - hTR cells treated with the indicated siRNAs , 72 h post - transfection to detect total protein levels using the indicated antibodies . β - Actin and Vinculin served as loading controls .

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

E. Western blot analyses in U2OS and U2OS-hTR cells treated with the indicated siRNAs, 72 h post-transfection to detect total protein levels using the indicated antibodies. β-Actin and Vinculin served as loading controls.


F . Representative images for 53BP1 foci formation in U2OS and U2OS - hTR cells , 72 h post - transfection with siLuc or siNHP2 , exposed to 1 Gy irradiation and fixed at various time points post - irradiation as indicated .

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

F. Representative images for 53BP1 foci formation in U2OS and U2OS-hTR cells, 72 h post-transfection with siLuc or siNHP2, exposed to 1 Gy irradiation and fixed at various time points post-irradiation as indicated.


G . Quantification of images shown in ( F ) . Each dot represents the number of 53BP1 foci per nucleus . A total of 114 - 140 nuclei were scored from two independent experiments . Statistical analyses were performed using two - way ANOVA Mixed effect model . Data information : All scale bars : 5 μm . Data are shown as mean ± SD , with the red line marking the mean value .

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

G. Quantification of images shown in (F). Each dot represents the number of 53BP1 foci per nucleus. A total of 114-140 nuclei were scored from two independent experiments. Statistical analyses were performed using two-way ANOVA Mixed effect model. Data information: All scale bars: 5 μm. Data are shown as mean ± SD, with the red line marking the mean value.


Left panel : Representative blots showing protein levels of YAP and phosphorylated YAP ( Ser127 ) in control ( si - Ctrl ) and YAP knockdown ( si - YAP ) hOSEs . Protein levels were detected by Western blotting . β - Actin was used as a protein loading control . Right panel : Cell proliferation ( cell number ) in control ( si - Ctrl ) and YAP knockdown ( si - YAP ) hOSE cells .

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

Left panel: Representative blots showing protein levels of YAP and phosphorylated YAP (Ser127) in control (si-Ctrl) and YAP knockdown (si-YAP) hOSEs. Protein levels were detected by Western blotting. β-Actin was used as a protein loading control. Right panel: Cell proliferation (cell number) in control (si-Ctrl) and YAP knockdown (si-YAP) hOSE cells.


Left panel : Representative blots showing protein levels of YAP and phosphorylated YAP ( Ser127 ) in control ( si - Ctrl ) , YAP knockdown ( si - YAP ) , TAZ knockdown ( si - TAZ ) and YAP / TAZ double knockdown ( si - YAP / TAZ ) hOSE Cells . β - Actin was used as a protein loading control . Right panel : Cell proliferation ( cell number ) of control ( si - Ctrl ) , YAP knockdown ( si - YAP ) , TAZ knockdown ( si - TAZ ) and YAP / TAZ double knockdown ( si - YAP & si - TAZ ) hOSE cells .

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

Left panel: Representative blots showing protein levels of YAP and phosphorylated YAP (Ser127) in control (si-Ctrl), YAP knockdown (si-YAP), TAZ knockdown (si-TAZ) and YAP/TAZ double knockdown (si-YAP/TAZ) hOSE Cells. β-Actin was used as a protein loading control. Right panel: Cell proliferation (cell number) of control (si-Ctrl), YAP knockdown (si-YAP), TAZ knockdown (si-TAZ) and YAP/TAZ double knockdown (si-YAP&si-TAZ) hOSE cells.


Representative blots showing protein levels of YAP and phosphorylated YAP ( Ser127 ) in control hOSEs ( MXIV ) and hOSEs expressing wild type YAP ( YAP ) or constitutively active YAP ( YAPS127A ) . Cells were collected at passage 4 . β - Actin was used as a protein loading control .

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

Representative blots showing protein levels of YAP and phosphorylated YAP (Ser127) in control hOSEs (MXIV) and hOSEs expressing wild type YAP (YAP) or constitutively active YAP (YAPS127A). Cells were collected at passage 4. β-Actin was used as a protein loading control.


Growth of hOSE - MXIV , hOSE - YAP and hOSE - YAPS127A cells at passage 4 . Each bar represents mean ± SEM of four independent samples . Growth curves of hOSE - MXIV , hOSE - YAP and hOSE - YAPS127A cells at passage 7 .

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

Growth of hOSE-MXIV, hOSE-YAP and hOSE-YAPS127A cells at passage 4. Each bar represents mean ± SEM of four independent samples. Growth curves of hOSE-MXIV, hOSE-YAP and hOSE-YAPS127A cells at passage 7.


Representative images showing the morphologic changes of hOSE - MXIV , hOSE - YAP and hOSE - YAPS127A cells at passage 7 . Scale bar : 50 µm .

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

Representative images showing the morphologic changes of hOSE-MXIV, hOSE-YAP and hOSE-YAPS127A cells at passage 7. Scale bar: 50 µm.


Representative images showing expression and location of YAP in hOSE - MXIV , hOSE - YAP and hOSE - YAPS127A cells at passage 7 . YAP was visualized using an Alexa - 488 ( Green ) conjugated secondary antibody . Nuclei were stained with DAPI . Scale bar : 50 µm .

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

Representative images showing expression and location of YAP in hOSE-MXIV, hOSE-YAP and hOSE-YAPS127A cells at passage 7. YAP was visualized using an Alexa-488 (Green) conjugated secondary antibody. Nuclei were stained with DAPI. Scale bar: 50 µm.


Representative images showing SA - β - gal staining in hOSE - MXIV , hOSE - YAP and hOSE - YAPS127A cells at passage 7 . Scale bar : 50 µm .

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

Representative images showing SA-β-gal staining in hOSE-MXIV, hOSE-YAP and hOSE-YAPS127A cells at passage 7. Scale bar: 50 µm.


Quantitative data showing the ratio of SA - β - gal positive cells in hOSE - MXIV , hOSE - YAP and hOSE - YAPS127A cells .

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

Quantitative data showing the ratio of SA-β-gal positive cells in hOSE-MXIV, hOSE-YAP and hOSE-YAPS127A cells.


Representative blots showing expression of key proteins associated with cell cycle arrest in hOSE - MXIV , hOSE - YAP and hOSE - YAPS127A cells analyzed using Western blotting at the 7th passage . β - Actin was used as a protein loading control .

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

Representative blots showing expression of key proteins associated with cell cycle arrest in hOSE-MXIV, hOSE-YAP and hOSE-YAPS127A cells analyzed using Western blotting at the 7th passage. β-Actin was used as a protein loading control.


Representative images showing expression and location of phosphorylated pRB ( 807 / 811 ) in hOSE - MXIV , hOSE - YAP and hOSE - YAPS127A cells at the 7th passage . Phosphorylated pRB ( 807 / 811 ) was visualized using an Alexa - 488 ( Green ) conjugated secondary antibody . Nuclei were stained with DAPI ( blue ) .

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

Representative images showing expression and location of phosphorylated pRB (807/811) in hOSE-MXIV, hOSE-YAP and hOSE-YAPS127A cells at the 7th passage. Phosphorylated pRB (807/811) was visualized using an Alexa-488 (Green) conjugated secondary antibody. Nuclei were stained with DAPI (blue).


Representative blots showing expression of TP53 and RB1 expression in hOSE - MXIV , hOSE - YAP and hOSE - YAPS127A cells in the presence or absence of HPV E6 / E7 oncoproteins at the 7th passage . β - Actin was used as a protein loading control .

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

Representative blots showing expression of TP53 and RB1 expression in hOSE-MXIV, hOSE-YAP and hOSE-YAPS127A cells in the presence or absence of HPV E6/E7 oncoproteins at the 7th passage. β-Actin was used as a protein loading control.


Growth curves of hOSE - MXIV , hOSE - YAP and hOSE - YAPS127A cells in the presence of HPV E6 / E7 oncoproteins at the 9th passage . Each point represents mean ± SEM of four independent samples .

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

Growth curves of hOSE-MXIV, hOSE-YAP and hOSE-YAPS127A cells in the presence of HPV E6/E7 oncoproteins at the 9th passage. Each point represents mean ± SEM of four independent samples.


Representative images showing SA - β - gal staining in hOSE - MXIV , hOSE - YAP and hOSE - YAPS127A cells in the presence or absence of HPV E6 / E7 oncoproteins at the 7th passage .

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

Representative images showing SA-β-gal staining in hOSE-MXIV, hOSE-YAP and hOSE-YAPS127A cells in the presence or absence of HPV E6/E7 oncoproteins at the 7th passage.


Quantitative data showing the ratio of SA - β - gal positive cells in hOSE - MXIV , hOSE - YAP and hOSE - YAPS127A cells in the presence or absence of HPV E6 / E7 oncoproteins .

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

Quantitative data showing the ratio of SA-β-gal positive cells in hOSE-MXIV, hOSE-YAP and hOSE-YAPS127A cells in the presence or absence of HPV E6/E7 oncoproteins.


Representative images showed SA - β - gal staining in cultured primary hOSE cells in the presence or absence of HPV E6 / E7 oncoproteins . Cells were collected and stained at the 13th passage . Scale bar : 25 µm . Right graph showing the ratio of SA - β - gal positive cells in P13 hOSE cells transfected with empty vectors ( CTRL - P13 ) or vectors expressing E6 / E7 oncoproteins ( E6 / E7 - P13 ) .

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

Representative images showed SA-β-gal staining in cultured primary hOSE cells in the presence or absence of HPV E6/E7 oncoproteins. Cells were collected and stained at the 13th passage. Scale bar: 25 µm. Right graph showing the ratio of SA-β-gal positive cells in P13 hOSE cells transfected with empty vectors (CTRL-P13) or vectors expressing E6/E7 oncoproteins (E6/E7-P13).


Representative blots showing the expression of YAP and RB1 in hOSE - MXIV , hOSE - YAP and hOSE - YAPS127A cells with or without pRB knockdown . Cells were collected at the 7th passage and protein levels were analyzed using Western blotting . β - Actin was used as a protein loading control .

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

Representative blots showing the expression of YAP and RB1 in hOSE-MXIV, hOSE-YAP and hOSE-YAPS127A cells with or without pRB knockdown. Cells were collected at the 7th passage and protein levels were analyzed using Western blotting. β-Actin was used as a protein loading control.


Growth curves of hOSE - MXIV , hOSE - YAP and hOSE - YAPS127A cells after RB1 knockdown .

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

Growth curves of hOSE-MXIV, hOSE-YAP and hOSE-YAPS127A cells after RB1 knockdown.


Representative images showing SA - β - gal staining in the hOSE - MXIV , hOSE - YAP and hOSE - YAPS127A cells with or without RB1 or TP53 knockdown . Cells were collected at the 7th passage .

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

Representative images showing SA-β-gal staining in the hOSE-MXIV, hOSE-YAP and hOSE-YAPS127A cells with or without RB1 or TP53 knockdown. Cells were collected at the 7th passage.


Representative blots showing LATS and YAP protein levels in hOSEs at the fourth , ninth and thirteenth passage . β - Actin was used as a protein loading control .

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

Representative blots showing LATS and YAP protein levels in hOSEs at the fourth, ninth and thirteenth passage. β-Actin was used as a protein loading control.


Representative gel photographs showing mRNA levels of LATS1 and LATS2 in hOSEs with or without LATS2 knockout . CRISPR / Cas - 9 system was used to knock out LATS2 in hOSEs . LATS1 / 2 mRNA levels were analyzed using qRT - PCR at the fourth , ninth and thirteenth passage .

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

Representative gel photographs showing mRNA levels of LATS1 and LATS2 in hOSEs with or without LATS2 knockout. CRISPR/Cas-9 system was used to knock out LATS2 in hOSEs. LATS1/2 mRNA levels were analyzed using qRT-PCR at the fourth, ninth and thirteenth passage.


Representative images showing the expression and location of YAP in control and LATS2 knockout hOSE cells . Cells were collected at the 4th passage and YAP expression was examined by fluorescent immunohistochemistry . YAP protein was visualized using an Alexa - 488 ( Green ) conjugated secondary antibody . Nuclei were stained with DAPI .

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

Representative images showing the expression and location of YAP in control and LATS2 knockout hOSE cells. Cells were collected at the 4th passage and YAP expression was examined by fluorescent immunohistochemistry. YAP protein was visualized using an Alexa-488 (Green) conjugated secondary antibody. Nuclei were stained with DAPI.


Representative images showing YAP protein expression in the 13th passage hOSE Cells with or without LATS2 knockout . YAP protein was examined by fluorescent immunohistochemistry and visualized using an Alexa - 488 ( Green ) conjugated secondary antibody . Nuclei were stained with DAPI ( blue ) .

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

Representative images showing YAP protein expression in the 13th passage hOSE Cells with or without LATS2 knockout. YAP protein was examined by fluorescent immunohistochemistry and visualized using an Alexa-488 (Green) conjugated secondary antibody. Nuclei were stained with DAPI (blue).


Representative images showing morphological change ( E ) in the 13th passage ( P13 ) hOSE cells with ( LATS2 KO ) or without ( CTRL ) LATS2 knockout .

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

Representative images showing morphological change (E) in the 13th passage (P13) hOSE cells with (LATS2 KO) or without (CTRL) LATS2 knockout.


Representative images showing SA - β - gal staining ( F ) in the 13th passage ( P13 ) hOSE cells with ( LATS2 KO ) or without ( CTRL ) LATS2 knockout . Scale bar : 50 µm .

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

Representative images showing SA-β-gal staining (F) in the 13th passage (P13) hOSE cells with (LATS2 KO) or without (CTRL) LATS2 knockout. Scale bar: 50 µm.


Quantitative data showing the ratio of SA - β - gal positive cells in the control ( CTRL ) and LATS2 - knockout ( LATS2KO ) P13 hOSE cells .

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 B-GENEPROD O O B-CELL_TYPE O O

Quantitative data showing the ratio of SA-β-gal positive cells in the control (CTRL) and LATS2-knockout (LATS2KO) P13 hOSE cells.


Representative blots showing expression levels of LATS1 / 2 , CCNE1 , CCND1 and YAP in hOSE - MXIV , hOSE - YAP and hOSE - YAPS127A cells . Cells were collected at passage four ( P4 ) and Passage seven ( P7 ) . Actin was used as internal control .

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

Representative blots showing expression levels of LATS1/2, CCNE1, CCND1 and YAP in hOSE-MXIV, hOSE-YAP and hOSE-YAPS127A cells. Cells were collected at passage four (P4) and Passage seven (P7). Actin was used as internal control.


mRNA levels of LATS1 , LATS2 and YAP in hOSE - MXIV , hOSE - YAP and hOSE - YAPS127A cells . Cells were collected at passage seven and protein levels were analyzed using quantitative real - time PCR .

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

mRNA levels of LATS1, LATS2 and YAP in hOSE-MXIV, hOSE-YAP and hOSE-YAPS127A cells. Cells were collected at passage seven and protein levels were analyzed using quantitative real-time PCR.


Representative gel photographs showing mRNA levels of LATS1 , LATS2 , and YAP in the LATS2 - knockout hOSE - MXIV , hOSE - YAP and hOSE - YAPS127A cells examined using semi - quantitative RT - PCR at passage seven .

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

Representative gel photographs showing mRNA levels of LATS1, LATS2, and YAP in the LATS2-knockout hOSE-MXIV, hOSE-YAP and hOSE-YAPS127A cells examined using semi-quantitative RT-PCR at passage seven.


Representative images showing the morphologic changes and SA - β - gal staining in control and LATS2 - knockout hOSE - MXIV , hOSE - YAP and hOSE - YAPS127A cells .

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Representative images showing the morphologic changes and SA-β-gal staining in control and LATS2-knockout hOSE-MXIV, hOSE-YAP and hOSE-YAPS127A cells.


Quantitative data showing the ratio of SA - β - gal positive cells in the control and LATS2 - knockout hOSE - MXIV , hOSE - YAP and hOSE - YAPS127A cells at the seventh passage .

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Quantitative data showing the ratio of SA-β-gal positive cells in the control and LATS2-knockout hOSE-MXIV, hOSE-YAP and hOSE-YAPS127A cells at the seventh passage.


Growth curves of the control and LATS2 - knockout hOSE - MXIV , hOSE - YAP and hOSE - YAPS127A cells .

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Growth curves of the control and LATS2-knockout hOSE-MXIV, hOSE-YAP and hOSE-YAPS127A cells.


Representative images showing cells with ectopic expression of LATS2 , which is labelled with GFP ( the control is empty vectors ) .

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Representative images showing cells with ectopic expression of LATS2, which is labelled with GFP (the control is empty vectors).


Relative mRNA levels of LATS1 , LATS2 , and YAP in the control and LATS2 - expressing hOSEs at the 4th passage . Relative mRNA levels were determined with RT - PCR .

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Relative mRNA levels of LATS1, LATS2, and YAP in the control and LATS2-expressing hOSEs at the 4th passage. Relative mRNA levels were determined with RT-PCR.


Growth curves of hOSE cells at the 4th passage with or without ectopic expression of LATS2 .

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Growth curves of hOSE cells at the 4th passage with or without ectopic expression of LATS2.


Representative images showing the effects of LATS2 overexpression on the morphology and growth of hOSE cells at the 9th passage .

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Representative images showing the effects of LATS2 overexpression on the morphology and growth of hOSE cells at the 9th passage.


Growth curves of control and LATS2 - overexpressing hOSE cells .

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Growth curves of control and LATS2-overexpressing hOSE cells.


Quantitative data showing the ratio of SA - β - gal positive hOSEs at their 4th passage with or without ectopic expression of LATS2 .

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Quantitative data showing the ratio of SA-β-gal positive hOSEs at their 4th passage with or without ectopic expression of LATS2.


Representative images showing the effect of LATS2 on the morphology and β - galactosidase activities in cultured primary hOSE cells at the 9th passage .

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Representative images showing the effect of LATS2 on the morphology and β-galactosidase activities in cultured primary hOSE cells at the 9th passage.


Quantitative data showing the ratio of SA - β - gal positive hOSEs at their 9th passage with or without ectopic expression of LATS2 .

Quantitative data showing the ratio of SA-β-gal positive hOSEs at their 9th passage with or without ectopic expression of LATS2.


mRNA levels of major components of the DREAM complex ( E2F4 , E2F5 , RBBP4 , TFDP2 ) in the control and LATS2 - overexpressing hOSEs at the 9th passage . Relative mRNA levels were examined by real - time PCR .

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mRNA levels of major components of the DREAM complex (E2F4, E2F5, RBBP4, TFDP2) in the control and LATS2-overexpressing hOSEs at the 9th passage. Relative mRNA levels were examined by real-time PCR.


Growth curves of the hOSE - shRB1 cells and hOSE - shRB1 - LATS2 cells at their 16th passages .

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Growth curves of the hOSE-shRB1 cells and hOSE-shRB1-LATS2 cells at their 16th passages.


Representative images showing cellular morphology and β - galactosidase activities in control and LATS2 - overexpressing hOSEs ( K ) . Scale Bar : 50 µm . Right bar graph ( L ) showing quantitative results of SA - β - gal positive cells in the hOSE - shRB1 cells and hOSE - shRB1 - LATS2 cells .

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Representative images showing cellular morphology and β-galactosidase activities in control and LATS2-overexpressing hOSEs (K). Scale Bar: 50 µm. Right bar graph (L) showing quantitative results of SA-β-gal positive cells in the hOSE-shRB1 cells and hOSE-shRB1-LATS2 cells.


Representative images showing colony formation in hOSE - MXIV , hOSE - YAP and hOSE - YAPS127A cells with or without pRB knockdown or LATS2 knockout . hOSEs at the 7th passage were incubated in the growth medium in a soft agar culture system for 9 days before imaging .

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Representative images showing colony formation in hOSE-MXIV, hOSE-YAP and hOSE-YAPS127A cells with or without pRB knockdown or LATS2 knockout. hOSEs at the 7th passage were incubated in the growth medium in a soft agar culture system for 9 days before imaging.


Quantitative data showing colony numbers in hOSE - MXIV , hOSE - YAP and hOSE - YAPS127A cells with or without pRB knockdown or LATS2 deletion . hOSE cells at the 7th passage were incubated in the growth medium in a soft agar culture system for 9 days before counting colonies under a microscope .

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Quantitative data showing colony numbers in hOSE-MXIV, hOSE-YAP and hOSE-YAPS127A cells with or without pRB knockdown or LATS2 deletion. hOSE cells at the 7th passage were incubated in the growth medium in a soft agar culture system for 9 days before counting colonies under a microscope.


Representative images showing tumorigenesis ( red circle area ) of hOSE - YAPS127A cells with or without pRB knockdown or LATS2 deletion . Tumors formed by hOSE - YAPS127A cells with RB1 knockdown or LATS2 deletion are shown in red circle area . Right panel showing representative tumors formed by hOSE - YAPS127A - LATS2KO and hOSE - YAPS127A - shRB1 cells . Please note that no tumor xenografts formed in hOSE - YAPS127A alone group .

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Representative images showing tumorigenesis (red circle area) of hOSE-YAPS127A cells with or without pRB knockdown or LATS2 deletion. Tumors formed by hOSE-YAPS127A cells with RB1 knockdown or LATS2 deletion are shown in red circle area. Right panel showing representative tumors formed by hOSE-YAPS127A-LATS2KO and hOSE-YAPS127A-shRB1 cells. Please note that no tumor xenografts formed in hOSE-YAPS127A alone group.


Representative images showing expression of YAP , Ki67 and LATS2 in hOSE - LATS2KO - YAPS127A cell - derived tumors examined by immunohistochemistry .

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Representative images showing expression of YAP, Ki67 and LATS2 in hOSE-LATS2KO-YAPS127A cell-derived tumors examined by immunohistochemistry.


( A ) Alcian blue ‐ stained sections of descending colons from control and Atg5VC mice . The area between the two black dashed lines indicates the crypt base where epithelial progenitors and nascent goblet cells reside . Bars = 200 μm .

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(A) Alcian blue‐stained sections of descending colons from control and Atg5VC mice. The area between the two black dashed lines indicates the crypt base where epithelial progenitors and nascent goblet cells reside. Bars=200 μm.


( B ) Quantification of average mucin area / goblet cell in control and Atg5VC mice ( n = 5 - 7 mice / group from 3 independent experiments ; 100 goblet cells measured / mouse ) . * * P0 . 01 as determined by the Student ' s t ‐ test . This analysis was performed on upper crypt goblet cells ( above the upper dashed line in A ) .

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(B) Quantification of average mucin area/goblet cell in control and Atg5VC mice (n=5-7 mice/group from 3 independent experiments; 100 goblet cells measured/mouse). **P0.01 as determined by the Student's t‐test. This analysis was performed on upper crypt goblet cells (above the upper dashed line in A).


( C ) Quantification of the average number of goblet cells per crypt ( n = 3 mice / group ; over 100 crypts measured / mouse ) .

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(C) Quantification of the average number of goblet cells per crypt (n=3 mice/group; over 100 crypts measured/mouse).


( D ) TEM images of upper crypt goblet cells from control and Atg5VC transverse colons . The yellow dashed lines outline the mucin granules in the apex of the cells . Bars = 2 μm .

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(D) TEM images of upper crypt goblet cells from control and Atg5VC transverse colons. The yellow dashed lines outline the mucin granules in the apex of the cells. Bars=2 μm.


( E , F ) Quantification of ( E ) the average granule density and ( F ) the average number of mucin granules / goblet cell ( n = 3 mice / group and 20 goblet cells evaluated / mouse ) . * * * P0 . 001 as determined by the Student ' s t ‐ test .

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(E, F) Quantification of (E) the average granule density and (F) the average number of mucin granules/goblet cell (n=3 mice/group and 20 goblet cells evaluated/mouse). ***P0.001 as determined by the Student's t‐test.


( G , H ) Whole ‐ mount images of the mucosal surface from control and Atg5VC transverse colons . Whole ‐ mount colonic samples were either stained with Ulex europaeus agglutinin ‐ 1 ( UEA ) that labelled mucus ( G ) or analysed by the scanning electron microscopy ( H ) . Yellow arrows indicate the crypt opening orifices . Green arrowheads indicate goblet cells protruding from the surface epithelium of Atg5VC mice ( insets in H show higher power image of the latter ) . Bars = 100 μm .

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(G, H) Whole‐mount images of the mucosal surface from control and Atg5VC transverse colons. Whole‐mount colonic samples were either stained with Ulex europaeus agglutinin‐1 (UEA) that labelled mucus (G) or analysed by the scanning electron microscopy (H). Yellow arrows indicate the crypt opening orifices. Green arrowheads indicate goblet cells protruding from the surface epithelium of Atg5VC mice (insets in H show higher power image of the latter). Bars=100 μm.


( B ) Representative whole ‐ mount images of single spheroids cultured in either 50 % conditioned media ( CM ) or 5 % CM + 5 μM DAPT and stained for Muc2 ( green ) to visualize goblet cells . Bars = 20 μm .

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(B) Representative whole‐mount images of single spheroids cultured in either 50% conditioned media (CM) or 5% CM+5 μM DAPT and stained for Muc2 (green) to visualize goblet cells. Bars=20 μm.


( C , D ) Graph of relative expression ( as measured by qRT ‐ PCR ) of Atoh1 ( C ) and Muc2 ( D ) mRNAs comparing spheroids grown in either 5 % CM or 5 % CM + DAPT to spheroids grown in 50 % CM ( n = 4 - 5 samples / group ) . Error bars indicate s . e . m . * P0 . 05 ; * * P0 . 01 as determined by a two ‐ tailed Student ' s t ‐ test .

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(C, D) Graph of relative expression (as measured by qRT‐PCR) of Atoh1 (C) and Muc2 (D) mRNAs comparing spheroids grown in either 5% CM or 5% CM+ DAPT to spheroids grown in 50% CM (n=4-5 samples/group). Error bars indicate s.e.m. *P0.05; **P0.01 as determined by a two‐tailed Student's t‐test.


( E , F ) Images of colonic epithelial spheroids isolated from control and Atg5VC mice grown in 5 % CM + DAPT and stained for Muc2 ( green : E ) or with the lectin , UEA ( red : F ) . Bars = 20 μm .

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(E, F) Images of colonic epithelial spheroids isolated from control and Atg5VC mice grown in 5% CM+DAPT and stained for Muc2 (green: E) or with the lectin, UEA (red: F). Bars=20 μm.


( G ) Quantification of average mucin area / goblet cell . Error bars indicate s . e . m . ( n = 6 samples / group ; 100 cells were quantified / sample ; * * P0 . 01 as determined by a two ‐ tailed Student ' s t ‐ test ) .

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(G) Quantification of average mucin area/goblet cell. Error bars indicate s.e.m. (n=6 samples/group; 100 cells were quantified/sample; **P0.01 as determined by a two‐tailed Student's t‐test).


( H ) Relative expression of Muc2 mRNA in differentiated spheroids from control and Atg5VC mice as measured by qRT - PCR ( n = 4 - 5 samples / group ) . No statistically significant differences were found in Muc2 expression as determined by the Student ' s t ‐ test .

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(H) Relative expression of Muc2 mRNA in differentiated spheroids from control and Atg5VC mice as measured by qRT-PCR (n=4-5 samples/group). No statistically significant differences were found in Muc2 expression as determined by the Student's t‐test.


( I , K ) Images of FIP200f / f ( I ) and Atg14f / f ( K ) colonic epithelial spheroids treated with recombinant Tat ‐ Cre and stained with UEA ( red ) .

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(I, K) Images of FIP200f/f (I) and Atg14f/f (K) colonic epithelial spheroids treated with recombinant Tat‐Cre and stained with UEA (red).


( J , L ) Quantification of average mucin area / goblet cell . Error bars indicate s . e . m . ( n = 6 samples / group ; 100 cells were quantified / sample ; * * P0 . 01 and * * * P0 . 001 as determined by a two ‐ tailed Student ' s t ‐ test ) .

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(J, L) Quantification of average mucin area/goblet cell. Error bars indicate s.e.m. (n=6 samples/group; 100 cells were quantified/sample; **P0.01 and ***P0.001 as determined by a two‐tailed Student's t‐test).


( A ) Images of colonic sections stained for LC3β ( green ) from control and Atg5VC mice . The white ‐ dashed lines indicate the border between the basal surface of the crypt epitheliumand the mesenchyme . White asterisks denote non ‐ specific plasma cell staining from the mouse monoclonal anti ‐ LC3β antibody . Bars = 50 μm .

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(A) Images of colonic sections stained for LC3β (green) from control and Atg5VC mice. The white‐dashed lines indicate the border between the basal surface of the crypt epitheliumand the mesenchyme. White asterisks denote non‐specific plasma cell staining from the mouse monoclonal anti‐LC3β antibody. Bars=50 μm.


( B ) Immunogold transmission electron microscopic image for LC3β in a colonic goblet cell from a wild ‐ type mouse . The inset is a low power image of the entire goblet cell . The black box within the inset indicates the area shown in this panel . Note this area is not associated with mucin granules . Bar = 500 nm .

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(B) Immunogold transmission electron microscopic image for LC3β in a colonic goblet cell from a wild‐type mouse. The inset is a low power image of the entire goblet cell. The black box within the inset indicates the area shown in this panel. Note this area is not associated with mucin granules. Bar=500 nm.


( C ) Enriched KEGG pathways for Atg5 ‐ deficient epithelial cells obtained from a microarray comparing control and Atg5VC colonic crypt base epithelium . P ‐ values were determined by modified Fisher ' s exact test .

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(C) Enriched KEGG pathways for Atg5‐deficient epithelial cells obtained from a microarray comparing control and Atg5VC colonic crypt base epithelium. P‐values were determined by modified Fisher's exact test.


( D - G ) Double label immunogold transmission electron microscopic images from wild ‐ type mice for LC3β ( 12 nm particles ) and EEA1 ( D ) , Rab7 ( E ) , Rab11 ( F ) , or Lamp1 ( G ) ( 18 nm particles ) . Bars = 200 nm .

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(D-G) Double label immunogold transmission electron microscopic images from wild‐type mice for LC3β (12 nm particles) and EEA1 (D), Rab7 (E), Rab11 (F), or Lamp1 (G) (18 nm particles). Bars=200 nm.


( B ) Colonic epithelial monolayers from wild ‐ type mice treated with either vehicle or 100 μM Dynasore and stained for LC3β ( green ) and EEA1 ( red ) . Bars = 5 μm .

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(B) Colonic epithelial monolayers from wild‐type mice treated with either vehicle or 100 μM Dynasore and stained for LC3β (green) and EEA1 (red). Bars=5 μm.


( C , D ) Quantification of average number of LC3β dots per cell ( C ) and the percentage of LC3β dots that co ‐ localized with EEA1 per cell ( D ) ( n = 3 experiments / group ; 25 - 30 cells / experiment ) .

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(C, D) Quantification of average number of LC3β dots per cell (C) and the percentage of LC3β dots that co‐localized with EEA1 per cell (D) (n=3 experiments/group; 25-30 cells/experiment).


( E ) LC3 immunoblots of epithelial spheroids treated with 100 μM Dynasore and 100 nM BafA1 as indicated . GAPDH is a loading control . Representative image is shown from n = 3 experiments .

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(E) LC3 immunoblots of epithelial spheroids treated with 100 μM Dynasore and 100 nM BafA1 as indicated. GAPDH is a loading control. Representative image is shown from n=3 experiments.


( F ) Images of wild ‐ type colonic epithelial spheroids labelled with TRITC ‐ UEA ( red ) after treatment with either vehicle or 100 μM Dynasore . Bars = 20 μm .

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(F) Images of wild‐type colonic epithelial spheroids labelled with TRITC‐UEA (red) after treatment with either vehicle or 100 μM Dynasore. Bars=20 μm.


( G ) Quantification of average mucin area / goblet cell ( n = 6 samples / group ; 150 cells were quantified / sample ) . Error bars indicate s . e . m . * * * P0 . 001 as determined by the Student ' s t ‐ test .

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(G) Quantification of average mucin area/goblet cell (n=6 samples/group; 150 cells were quantified/sample). Error bars indicate s.e.m. ***P0.001 as determined by the Student's t‐test.


( A ) Colonic epithelial monolayers from a wild ‐ type and p22 mut mouse stained with LC3β ( green ) and EEA1 ( red ) . Bar = 5 μm .

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(A) Colonic epithelial monolayers from a wild‐type and p22 mut mouse stained with LC3β (green) and EEA1 (red). Bar=5 μm.


( B , C ) Quantification of average number of LC3β dots per cell ( B ) and the percentage of LC3β dots that co ‐ localized with EEA1 per cell ( C ) ( n = 3 experiments / group ; 25 - 30 cells / experiment ) .

(B, C) Quantification of average number of LC3β dots per cell (B) and the percentage of LC3β dots that co‐localized with EEA1 per cell (C) (n=3 experiments/group; 25-30 cells/experiment).


( D ) LC3 immunoblots of either wild ‐ type or p22 mut epithelial spheroids treated with 100 nM BafA1 as indicated . Representative image is shown from n = 3 experiments .

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(D) LC3 immunoblots of either wild‐type or p22 mut epithelial spheroids treated with 100 nM BafA1 as indicated. Representative image is shown from n=3 experiments.


( E ) Double label immunogold transmission electron microscopic images for LC3β ( 12μnm particles ) and p22phox ( 18 nm particles ) showing a p22phox ‐ positive single membrane cytoplasmic vesicle ( D ) and a p22phox ‐ , LC3β ‐ positive vacuole ( E ) . Bars = 200μnm .

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(E) Double label immunogold transmission electron microscopic images for LC3β (12μnm particles) and p22phox (18 nm particles) showing a p22phox‐positive single membrane cytoplasmic vesicle (D) and a p22phox‐, LC3β‐positive vacuole (E). Bars=200μnm.


( F ) Colonic epithelial monolayers from a wild ‐ type mouse stained for LC3β ( green ) and p22phox ( red ) and quantification of percent LC3β dots co ‐ localized with p22phox per cell , Bar = 5 μm .

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(F) Colonic epithelial monolayers from a wild‐type mouse stained for LC3β (green) and p22phox (red) and quantification of percent LC3β dots co‐localized with p22phox per cell, Bar=5 μm.


( G ) Colonic epithelial cellspheroids from control and p22 mut mice stained with TRITC ‐ UEA to label goblet cellmucin . Bars = 50 μm .

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(G) Colonic epithelial cellspheroids from control and p22 mut mice stained with TRITC‐UEA to label goblet cellmucin. Bars=50 μm.


( H ) Quantification of average mucin area / goblet cell ( n = 6 samples / group ; 150 cells were quantified / sample ) . Error bars indicate s . e . m . * * P0 . 01 ; * * * * P0 . 0001 as determined by the Student ' s t ‐ test .

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(H) Quantification of average mucin area/goblet cell (n=6 samples/group; 150 cells were quantified/sample). Error bars indicate s.e.m. **P0.01; ****P0.0001 as determined by the Student's t‐test.


( A ) Colonic epithelial cell spheroids from p22phox − / − , Atg5VC and wild ‐ type mice . Wild ‐ type spheroids were treated with vehicle ( control ) , 10 μM DPI ( NADPH oxidase inhibitor ) or 100 μM Dynasore . Intracellular ROS in live cells were labelled by 5 μM DCF . Bars = 100 μm .

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(A) Colonic epithelial cell spheroids from p22phox−/−, Atg5VC and wild‐type mice. Wild‐type spheroids were treated with vehicle (control), 10 μM DPI (NADPH oxidase inhibitor) or 100 μM Dynasore. Intracellular ROS in live cells were labelled by 5 μM DCF. Bars=100 μm.


( B ) Quantification of fluorescent DCF normalized to control ( n = 3 independent experiments / group ; 30 spheroids quantified / experiment ) .

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(B) Quantification of fluorescent DCF normalized to control (n=3 independent experiments/group; 30 spheroids quantified/experiment).


( C ) Quantification of average area of cytoplasmic mucin from wild ‐ type mice treated with 3 % NAC as compared to controls ( n = 6 - 7 mice / group ) . Error bars indicate s . e . m . * P0 . 05 ; * * P0 . 01 ; * * * * P0 . 0001 as determined by the Student ' s t ‐ test .

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(C) Quantification of average area of cytoplasmic mucin from wild‐type mice treated with 3% NAC as compared to controls (n=6-7 mice/group). Error bars indicate s.e.m. *P0.05; **P0.01; ****P0.0001 as determined by the Student's t‐test.


( D ) TEM images of colonic epithelial monolayers treated with cerium chloride . The presence of electron dense precipitate is indicative of ROS production . Bar = 500 nm .

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(D) TEM images of colonic epithelial monolayers treated with cerium chloride. The presence of electron dense precipitate is indicative of ROS production. Bar=500 nm.


Exogeneous ROS can restore mucingranule accumulation defects . ( A ) Quantification of fluorescent DCF normalized to controls ( n = 3 independent experiments / group ; 10 - 30 spheroids quantified / culture ) . * P0 . 05 as determined by ANOVA with Tukey ' s multiple post ‐ test

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Exogeneous ROS can restore mucingranule accumulation defects. (A) Quantification of fluorescent DCF normalized to controls (n=3 independent experiments/group; 10-30 spheroids quantified/culture). *P0.05 as determined by ANOVA with Tukey's multiple post‐test comparisons


( B ) Images of control and Atg5VC colonic epithelial spheroids labelled with TRITC ‐ UEA ( red ) after treatment with either vehicle or 300 μM H2O2 . Bars = 20 μm . ( C ) Quantification of average mucin area / goblet cell ( n = 6 samples / group ; 150 cells were quantified / sample ) . * * * P0 . 001 , * * * * P . 0001 as determined by ANOVA with Tukey ' s multiple post ‐ test comparisons .

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(B) Images of control and Atg5VC colonic epithelial spheroids labelled with TRITC‐UEA (red) after treatment with either vehicle or 300 μM H2O2. Bars=20 μm. (C) Quantification of average mucin area/goblet cell (n=6 samples/group; 150 cells were quantified/sample). ***P0.001, ****P.0001 as determined by ANOVA with Tukey's multiple post‐test comparisons.


( D ) Relative expression of Muc2 mRNA in differentiated spheroids from control and Atg5VC mice treated with either vehicle or 300 μM H2O2 as measured by qRT - PCR . No statistically significant differences were found in either group as determined by ANOVA .

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(D) Relative expression of Muc2 mRNA in differentiated spheroids from control and Atg5VC mice treated with either vehicle or 300 μM H2O2 as measured by qRT-PCR. No statistically significant differences were found in either group as determined by ANOVA.


( E , G ) Images of p22 mut or Dynasore ‐ treated colonic epithelial spheroids ( mucin is labelled with TRITC ‐ UEA ; red ) after treatment with either vehicle or 300 μM H2O2 . Bars = 20 μm . ( F , H ) Quantification of average mucin area / goblet cell ( n = 6 sample / group ; 30 - 60 cells were quantified / sample ) corresponding to experiments performed using p22 mut ( E ) and Dynasore ‐ treated wild ‐ type spheroids ( G )

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(E, G) Images of p22 mut or Dynasore‐treated colonic epithelial spheroids (mucin is labelled with TRITC‐UEA; red) after treatment with either vehicle or 300 μM H2O2. Bars=20 μm. (F, H) Quantification of average mucin area/goblet cell (n=6 sample/group; 30-60 cells were quantified/sample) corresponding to experiments performed using p22 mut (E) and Dynasore‐treated wild‐type spheroids (G)


Atg5VC and p22 mut spheroids were treated with 300 μM H2O2 plus or minus 100 μM BAPTA ‐ AM ( I ) or PMA / ionomycin ( J ) . ( I , J ) Quantification of average mucin area / goblet cell ( n = 5 sample / group ; 150 cells were quantified / sample ) . Error bars indicate s . e . m . * * * * P0 . 0001 as determined by the Student ' s t ‐ test ( I ) or ANOVA with Tukey ' s multiple post ‐ test comparisons ( J ) .

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Atg5VC and p22 mut spheroids were treated with 300 μM H2O2 plus or minus 100 μM BAPTA‐AM (I) or PMA/ionomycin (J). (I, J) Quantification of average mucin area/goblet cell (n=5 sample/group; 150 cells were quantified/sample). Error bars indicate s.e.m. ****P0.0001 as determined by the Student's t‐test (I) or ANOVA with Tukey's multiple post‐test comparisons (J).


B . In vitro modification reactions containing the six fragments and HeLa NE were performed . After nuclease P1 digestion , the products were analyzed by 1D - TLC with pU and pψ as controls .

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B. In vitro modification reactions containing the six fragments and HeLa NE were performed. After nuclease P1 digestion, the products were analyzed by 1D-TLC with pU and pψ as controls.


C . WT and mutant 7SK fragment 5 containing U250G were analyzed as in B .

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C. WT and mutant 7SK fragment 5 containing U250G were analyzed as in B.


E . 7SK RNA , generated by either in vitro transcription ( IVT ) or purified from HeLa total RNA and then site - specifically labeled at the indicated positions by the procedure in D , was analyzed by 1D - TLC .

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E. 7SK RNA, generated by either in vitro transcription (IVT) or purified from HeLa total RNA and then site-specifically labeled at the indicated positions by the procedure in D, was analyzed by 1D-TLC.


F . Cyber Gold staining of 7SK obtained by either IVT or immunoprecipitation ( IP ) with the indicated antibodies .

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F. Cyber Gold staining of 7SK obtained by either IVT or immunoprecipitation (IP) with the indicated antibodies.


G . 2D - TLC analysis of 32p - labeled mononucleotides derived from IVT or CDK9 - bound 7SK RNA , with the first panel depicting a schematic migration pattern of the indicated 5 ' - phosphorylated mononucleotides .

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G. 2D-TLC analysis of 32p-labeled mononucleotides derived from IVT or CDK9-bound 7SK RNA, with the first panel depicting a schematic migration pattern of the indicated 5'-phosphorylated mononucleotides.


H . 7SK RNA , obtained by either IVT or anti - CDK9 IP and then site - specifically labeled at the indicated positions by the procedure in D , was analyzed by 1D - TLC .

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H. 7SK RNA, obtained by either IVT or anti-CDK9 IP and then site-specifically labeled at the indicated positions by the procedure in D, was analyzed by 1D-TLC.


C - F ; H - K ; and N - Q . The tagged WT or mutant 7SK RNA was co - transfected with the indicated Flag - tagged proteins into HeLa cells . Anti - Flag or immunoprecipitates ( IP ) were analyzed by immunoblotting for the indicated proteins and primer extension for the bound 7SK RNA .

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C-F; H-K; and N-Q. The tagged WT or mutant 7SK RNA was co-transfected with the indicated Flag-tagged proteins into HeLa cells. Anti-Flag or immunoprecipitates (IP) were analyzed by immunoblotting for the indicated proteins and primer extension for the bound 7SK RNA.


C - F ; H - K ; and N - Q . The tagged WT or mutant 7SK RNA was co - transfected with the indicated Flag - - tagged proteins into HeLa cells . Anti - Flag immunoprecipitates ( IP ) were analyzed by immunoblotting for the indicated proteins and primer extension for the bound 7SK RNA .

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C-F; H-K; and N-Q. The tagged WT or mutant 7SK RNA was co-transfected with the indicated Flag--tagged proteins into HeLa cells. Anti-Flag immunoprecipitates (IP) were analyzed by immunoblotting for the indicated proteins and primer extension for the bound 7SK RNA.


C - F ; H - K ; and N - Q . The tagged WT or mutant 7SK RNA was co - transfected with the indicated Flag - tagged proteins into HeLa cells . Anti - Flag immunoprecipitates ( IP ) were analyzed by immunoblotting for the indicated proteins and primer extension for the bound 7SK RNA .

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C-F; H-K; and N-Q. The tagged WT or mutant 7SK RNA was co-transfected with the indicated Flag-tagged proteins into HeLa cells. Anti-Flag immunoprecipitates (IP) were analyzed by immunoblotting for the indicated proteins and primer extension for the bound 7SK RNA.


C - F ; H - K ; and N - Q . The tagged WT or mutant 7SK RNA was co - transfected with the indicated HA - tagged proteins into HeLa cells . Anti - HA immunoprecipitates ( IP ) were analyzed by immunoblotting for the indicated proteins and primer extension for the bound 7SK RNA .

C-F; H-K; and N-Q. The tagged WT or mutant 7SK RNA was co-transfected with the indicated HA-tagged proteins into HeLa cells. Anti-HA immunoprecipitates (IP) were analyzed by immunoblotting for the indicated proteins and primer extension for the bound 7SK RNA.


R . The indicated 7SK were in vitro transcribed in the presence of 32p - UTP and subjected to in vitro modification reactions . The nuclease P1 - digested products were analyzed by 1D - TLC with pU and pΨ as controls .

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R. The indicated 7SK were in vitro transcribed in the presence of 32p-UTP and subjected to in vitro modification reactions. The nuclease P1-digested products were analyzed by 1D-TLC with pU and pΨ as controls.


A . Anti - V5immunoprecipitates ( IP ) from HeLa cells expressing nothing ( - ) or V5 - DKC1 were analyzed by immunoblotting for V5 - DKC1 and qRT - PCR for the bound 7SK RNA . The error bars represent mean ± SD from three independent experiments .

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A. Anti-V5immunoprecipitates (IP) from HeLa cells expressing nothing (-) or V5-DKC1 were analyzed by immunoblotting for V5-DKC1 and qRT-PCR for the bound 7SK RNA. The error bars represent mean ± SD from three independent experiments.


B . Anti - FlagIP from NE of HeLa or the HeLa - derived F1C2 cells expressing CDK9 - F were analyzed by Western blotting ( WB ) for the indicated proteins and primer extension ( PEx ) for 7SK RNA .

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B. Anti-FlagIP from NE of HeLa or the HeLa-derived F1C2 cells expressing CDK9-F were analyzed by Western blotting (WB) for the indicated proteins and primer extension (PEx) for 7SK RNA.


C . WB analysis of DKC1 and CDK9 levels in two HeLa clones inducibly expressing shDKC1 upon doxycycline ( DOX ) treatment for 5 days .

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C. WB analysis of DKC1 and CDK9 levels in two HeLa clones inducibly expressing shDKC1 upon doxycycline (DOX) treatment for 5 days.


D . The 7SK RNA levels in these two clones were detected by qRT - PCR before and after the DOX treatment and normalized to the GAPDH mRNA levels . The error bars represent mean ± SD from three independent experiments .

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D. The 7SK RNA levels in these two clones were detected by qRT-PCR before and after the DOX treatment and normalized to the GAPDH mRNA levels. The error bars represent mean ± SD from three independent experiments.


E . 1D - TLC analysis of Ψ250 in 7SK RNA from inducible DKC1 KD clone 1 before and after the DOX treatment .

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E. 1D-TLC analysis of Ψ250 in 7SK RNA from inducible DKC1 KD clone 1 before and after the DOX treatment.


F . NE and anti - CDK9IP from NE of inducible DKC1 KD clone 1 , which was untreated or treated with DOX , were analyzed by WB for the indicated proteins and PEx for 7SK RNA .

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F. NE and anti-CDK9IP from NE of inducible DKC1 KD clone 1, which was untreated or treated with DOX, were analyzed by WB for the indicated proteins and PEx for 7SK RNA.


G . Clone 1 was treated with DOX for 3 days and then transfected with the Tat - Flag cDNA ( 20 ug / 150 - mm dish ) . Two days later , NE and anti - FlagIP from NE were analyzed as in F .

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G. Clone 1 was treated with DOX for 3 days and then transfected with the Tat-Flag cDNA (20 ug/150-mm dish). Two days later, NE and anti-FlagIP from NE were analyzed as in F.


A . Luciferase activities were measured in HeLa cells transfected with the indicated reporter constructs , an empty vector ( - ) or the Tat cDNA , and either siDKC1 or a non - target siRNA ( siNT ) . For each reporter , the activity in cells expressing siNT was set to 1 .

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A. Luciferase activities were measured in HeLa cells transfected with the indicated reporter constructs, an empty vector (-) or the Tat cDNA, and either siDKC1 or a non-target siRNA (siNT). For each reporter, the activity in cells expressing siNT was set to 1.


B , C , and E . The mRNA levels of luciferase ( B ) or Tat ( C E ) in cells expressing the indicated siRNAs were determined by qRT - PCR and normalized to the GAPDH mRNA and plotted .

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B, C, and E. The mRNA levels of luciferase (B) or Tat (C E) in cells expressing the indicated siRNAs were determined by qRT-PCR and normalized to the GAPDH mRNA and plotted.


D . NH1 cells containing the integrated HIV - 1 LTR - luciferase reporter and expressing Tat were transfected with siDKC1 or siNT . ChIP - qPCR analysis was performed to determine the levels of the indicated factors bound to the HIV - 1 promoter . The signals were normalized to those of input and plotted .

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D. NH1 cells containing the integrated HIV-1 LTR-luciferase reporter and expressing Tat were transfected with siDKC1 or siNT. ChIP-qPCR analysis was performed to determine the levels of the indicated factors bound to the HIV-1 promoter. The signals were normalized to those of input and plotted.


B , C , and E . The mRNA levels of Tat ( C E ) in cells expressing the indicated siRNAs were determined by qRT - PCR and normalized to the GAPDH mRNA and plotted .

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B, C, and E. The mRNA levels of Tat (C E) in cells expressing the indicated siRNAs were determined by qRT-PCR and normalized to the GAPDH mRNA and plotted.


F . Western analysis of DKC1 and CDK9 levels in a Jurkat 2D10 - derived clone inducibly expressing shDKC1 upon exposure to DOX .

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F. Western analysis of DKC1 and CDK9 levels in a Jurkat 2D10-derived clone inducibly expressing shDKC1 upon exposure to DOX.


G and H . The inducible DKC1 KD clone , treated or untreated with DOX for 4 days , was incubated with the indicated concentrations of JQ1 ( G ) or prostratin ( H ) for 24 h . The GFP expression was measured by flow cytometry and expressed as percentages of GFP ( + ) cells of the entire population . The error bars in all panels represent mean ± SD from three independent experiments , and the p values determined by the Student ' s t - test .

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G and H. The inducible DKC1 KD clone, treated or untreated with DOX for 4 days, was incubated with the indicated concentrations of JQ1 (G) or prostratin (H) for 24 h. The GFP expression was measured by flow cytometry and expressed as percentages of GFP(+) cells of the entire population. The error bars in all panels represent mean ± SD from three independent experiments, and the p values determined by the Student's t-test.


A . Western blot analysis of supernatants and whole cell lysates collected from WT and IQGAP1 KO YAMC cells with and without LPS stimulation for 4 hours or LPS plus ATP ( 4 hours plus 30 minutes ) treatment as indicated .

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A. Western blot analysis of supernatants and whole cell lysates collected from WT and IQGAP1 KO YAMC cells with and without LPS stimulation for 4 hours or LPS plus ATP (4 hours plus 30 minutes) treatment as indicated.


B . Nanoparticle tracking analysis of supernatant of WT and IQGAP1 KO YAMC cells treated with LPS plus ATP . * * , P < 0 . 01 by unpaired two - tailed t test ( n = 3 biological repeats in one experiments ) .

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B. Nanoparticle tracking analysis of supernatant of WT and IQGAP1 KO YAMC cells treated with LPS plus ATP. **, P<0.01 by unpaired two-tailed t test (n=3 biological repeats in one experiments).


C . Size distributions for vesicles as analyzed in D .

C. Size distributions for vesicles as analyzed in D.


D . Western blot analysis of enriched extracellular vesicles from supernatants WT and IQGAP1 KO YAMC cells .

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D. Western blot analysis of enriched extracellular vesicles from supernatants WT and IQGAP1 KO YAMC cells.


F . Flow cytometry analysis for CD63 + exosomes from supernatants of LPS plus ATP treated WT and IQGAP1 KO supernatants . Quantification was done by mean fluorescence intensity . * * , P < 0 . 01 by unpaired two - tailed t test ( n = 4 ) .

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F. Flow cytometry analysis for CD63+ exosomes from supernatants of LPS plus ATP treated WT and IQGAP1 KO supernatants. Quantification was done by mean fluorescence intensity. **, P<0.01 by unpaired two-tailed t test (n=4).


B . Co - immunoprecipitation of GSDMD with full - length and deletion mutants of IQGAP1 tested in HEK293 cells .

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B. Co-immunoprecipitation of GSDMD with full-length and deletion mutants of IQGAP1 tested in HEK293 cells.


C . Co - immunoprecipitation of GSDMD cleavage mutant D276A with IQGAP1 overexpressed in HEK293 cells .

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C. Co-immunoprecipitation of GSDMD cleavage mutant D276A with IQGAP1 overexpressed in HEK293 cells.


D . Co - immunoprecipitation of GSDMD with IQGAP1 in WT and cleavage mutant ( D276A ) restored GSDMD KO YAMC cells . Cells were treated as indicated .

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D. Co-immunoprecipitation of GSDMD with IQGAP1 in WT and cleavage mutant (D276A) restored GSDMD KO YAMC cells. Cells were treated as indicated.