( F ) IKKα protein level in KDF1 KO keratinocyte expressing WT KDF1 or KDF1 mutant , under both low and high calcium conditions , was examined and quantified by western

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(F) IKKα protein level in KDF1 KO keratinocyte expressing WT KDF1 or KDF1 mutant, under both low and high calcium conditions, was examined and quantified by western blotting


( G ) PiggyBac transposon was used to ectopically express HA - tagged IKKα in KDF1 deficient cells . WCL were collected and analyzed by immunoblots with different antibodies as indicated .

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(G) PiggyBac transposon was used to ectopically express HA-tagged IKKα in KDF1 deficient cells. WCL were collected and analyzed by immunoblots with different antibodies as indicated.


( H ) Overexpression of IKKα in KDF1 KO keratinocyte can restore normal skin stratification . Skin sections from grafted tissue were immunostained with different antibodies as indicated . Dotted lines denote dermal - epidermal boundaries . Epi : epidermis , Der : dermis . Scale bars = 50 µm .

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(H) Overexpression of IKKα in KDF1 KO keratinocyte can restore normal skin stratification. Skin sections from grafted tissue were immunostained with different antibodies as indicated. Dotted lines denote dermal-epidermal boundaries. Epi: epidermis, Der: dermis. Scale bars = 50 µm.


A ) HEK293 cells were transfected to co - express KDF1 with USP7 . IP and WCL were blotted with different antibodies as indicated .

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A) HEK293 cells were transfected to co-express KDF1 with USP7. IP and WCL were blotted with different antibodies as indicated.


B ) Immunoprecipitation was carried out with α - KDF1 antibody in keratinocyte cultured in medium with low or high concentration of calcium ( Ca ) , same as in Fig . 1D . IPs were blotted with different antibodies as indicated . The star denotes KDF1 .

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B) Immunoprecipitation was carried out with α-KDF1 antibody in keratinocyte cultured in medium with low or high concentration of calcium (Ca), same as in Fig. 1D. IPs were blotted with different antibodies as indicated. The star denotes KDF1.


C ) WCL from WT and USP7 CRISPR KO cells were subjected to immunoblotting with different antibodies as indicated .

C) WCL from WT and USP7 CRISPR KO cells were subjected to immunoblotting with different antibodies as indicated.


D ) IKKα protein level in WT and USP7 KO keratinocyte were examined by immunoblotting before and after calcium shift .

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D) IKKα protein level in WT and USP7 KO keratinocyte were examined by immunoblotting before and after calcium shift.


E ) WT and USP7 KO keratinocyte were treated with MG132 at 10μM for 6 hours , then subjected to immunoprecipitation using anti - ubiquitin antibody . Precipitated product was analyzed by immunoblotting with IKKα antibody . Band intensity was determined by densitometry and shown as bar graphs . Statistical analysis is conducted using unpaired Student ' s t - test . Error bar represents S . D . ( standard deviation ) . N = 3 ( biological replicates ) . * * , p < 0 . 01 . Kd : kilodalton for molecular weight markers .

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E) WT and USP7 KO keratinocyte were treated with MG132 at 10μM for 6 hours, then subjected to immunoprecipitation using anti-ubiquitin antibody. Precipitated product was analyzed by immunoblotting with IKKα antibody. Band intensity was determined by densitometry and shown as bar graphs. Statistical analysis is conducted using unpaired Student's t-test. Error bar represents S.D. (standard deviation). N=3 (biological replicates). **, p<0.01. Kd: kilodalton for molecular weight markers.


F - G ) Immunoblot of Krt10 and Loricrin ( Lor ) with WT and USP7 KO cells before and after calcium shift ( F ) . Star denotes an unspecific band in α - Loricrin blots . Band intensity was determined by densitometry and shown as bar

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F-G) Immunoblot of Krt10 and Loricrin (Lor) with WT and USP7 KO cells before and after calcium shift (F). Star denotes an unspecific band in α-Loricrin blots. Band intensity was determined by densitometry and shown as bar graphs


Statistical analysis is conducted using unpaired Student ' s t - test . Error bar represents S . D . ( standard deviation ) . N = 3 ( biological replicates ) . * * * * , p < 0 . 0001 ; * * , p < 0 . 01 .

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Statistical analysis is conducted using unpaired Student's t-test. Error bar represents S.D. (standard deviation). N=3 (biological replicates). ****, p<0.0001; **, p<0.01.


H ) Sections of engrafted skin developed from USP7 KO or control WT cells were immunostained with different antibodies as indicated . Dotted lines denote dermal - epidermal boundaries . Epi : epidermis , Der : dermis . Scale bar = 100μm .

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H) Sections of engrafted skin developed from USP7 KO or control WT cells were immunostained with different antibodies as indicated. Dotted lines denote dermal-epidermal boundaries. Epi: epidermis, Der: dermis. Scale bar=100μm.


TEM image and ( B ) size distribution of pcMNPs ; The average size of nanoparticles is represented as mean ± SD of over 250 individual particles .

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TEM image and (B) size distribution of pcMNPs; The average size of nanoparticles is represented as mean ± SD of over 250 individual particles.


Zeta potentials of bare MNPs , NH2 - MNPs and pcMNPs in water . ( D ) The magnetic response property of bare MNPs , NH2 - MNPs and MBs in 30 s .

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Zeta potentials of bare MNPs, NH2-MNPs and pcMNPs in water. (D) The magnetic response property of bare MNPs, NH2-MNPs and MBs in 30 s.


RNA binding affinity of pcMNPs analysed by native PAGE ;

RNA binding affinity of pcMNPs analysed by native PAGE;


Dispersion stability of pcMNPs in a high - salt solution ( 50 mM NaCl ) .

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Dispersion stability of pcMNPs in a high-salt solution (50 mM NaCl).


RT - PCR assays towards viral RNA extracted by different pcMNPs - based methods . RT - PCR assays amplifying ( A ) the ORF1ab region and ( B ) the N gene in pseudoviral RNA extracted by a manual protocol .

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RT-PCR assays towards viral RNA extracted by different pcMNPs-based methods.RT-PCR assays amplifying (A) the ORF1ab region and (B) the N gene in pseudoviral RNA extracted by a manual protocol.


RT - PCR assays amplifying the ORF1ab region in pseudoviral RNA extracted by automated protocol , and ( B ) RT - PCR assays amplifying the N gene in pseudoviral RNA extracted by an automated protocol .

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RT-PCR assays amplifying the ORF1ab region in pseudoviral RNA extracted by automated protocol, and (B) RT-PCR assays amplifying the N gene in pseudoviral RNA extracted by an automated protocol.


RT - PCR assays amplifying the N gene in pseudoviral RNA following the Conventional RT - PCR protocol , and ( B ) corresponding calibration curve .

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RT-PCR assays amplifying the N gene in pseudoviral RNA following the Conventional RT-PCR protocol, and (B) corresponding calibration curve.


RT - PCR assays amplifying the N gene in pseudoviral RNA following the Direct RT - PCR protocol , and ( D ) corresponding calibration curve . The red line is the linear regression fit ( R2 = 0 . 998 ) .

RT-PCR assays amplifying the N gene in pseudoviral RNA following the Direct RT-PCR protocol, and (D) corresponding calibration curve. The red line is the linear regression fit (R2 = 0.998).


( B ) Genome browser representation of the SOX2 locus . The promoter is defined as the unmethylated region spanning the SOX2 transcriptional start site in MCF7 - control .

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(B) Genome browser representation of the SOX2 locus. The promoter is defined as the unmethylated region spanning the SOX2 transcriptional start site in MCF7-control.


Genomic distribution of ( C ) identified ZF - D3A binding sites , and ( D ) differentially methyl - ated regions ( DMRs ) between MCF7 - control and ZF - D3A + dox .

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Genomic distribution of (C) identified ZF-D3A binding sites, and (D) differentially methyl- ated regions (DMRs) between MCF7-control and ZF-D3A +dox.


( E ) Shown for all DMRs identified between ZF - D3A + dox and MCF7 - control is : ∆ mCG ( difference in the methylation level [ ratio of C base calls to total base calls for all CG dinucleotides in region , mCG / CG ] between ZF - D3A + dox and MCF7 - control ) , ZF - D3A ChIP - seq signal ( normalized ChIP - seq read density , ± 1kb flanking DMR center ) , CpG dinucleotide density ( CpG / 100bp , ± 1kb flanking DMR center ) , and classification of each DMR as promoter or non - promoter located .

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(E) Shown for all DMRs identified between ZF-D3A +dox and MCF7-control is: ∆mCG (difference in the methylation level [ratio of C base calls to total base calls for all CG dinucleotides in region, mCG/CG] between ZF-D3A +dox and MCF7-control), ZF-D3A ChIP-seq signal (normalized ChIP-seq read density, ±1kb flanking DMR center), CpG dinucleotide density (CpG/100bp, ±1kb flanking DMR center), and classification of each DMR as promoter or non-promoter located.


( F ) DNA methylation level in MCF7 - control and ZF - D3A + dox cells flanking ( ± 1 kb , 100 bp bins ) the center of ZF - D3A ChIP - seq peaks identified in ZF - D3A + dox .

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(F) DNA methylation level in MCF7-control and ZF-D3A +dox cells flanking (±1 kb, 100 bp bins) the center of ZF-D3A ChIP-seq peaks identified in ZF-D3A +dox.


( G ) Genomic distribution of unmethylated regions ( UMRs ) in MCF7 - control . ( H ) DMRs and UMRs with an intersection of ≥ 1 bp .

(G) Genomic distribution of unmethylated regions (UMRs) in MCF7-control. (H) DMRs and UMRs with an intersection of ≥1 bp.


( A ) Line histogram of DMR methylation levels in each sample . Kernel density estimate smoothed scatterplot comparison of DMR methylation levels in MCF7 - control and ( B ) ZF - D3A + dox , or ( C ) ZF - D3A dox - wd .

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(A) Line histogram of DMR methylation levels in each sample. Kernel density estimate smoothed scatterplot comparison of DMR methylation levels in MCF7-control and (B) ZF-D3A +dox, or (C) ZF-D3A dox-wd.


( D ) Line histogram of UMR methylation levels in each sample . Scatterplot compari - son of UMR methylation levels in MCF7 - control and ( E ) ZF - D3A + dox , or ( F ) ZF - D3A dox - wd .

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(D) Line histogram of UMR methylation levels in each sample. Scatterplot compari- son of UMR methylation levels in MCF7-control and (E) ZF-D3A +dox, or (F) ZF-D3A dox-wd.


( G ) Heat - maps of DMRs ( ordered by decreasing H3K4me3 read density ) showing : DNA methylation levels ( ± 2 . 5 kb ) , differences ( ΔmCG ) , classification as promoter or non - promoter located , CpG dinucleotide density ( ± 2 . 5 kb ) , and histone modification ChIP - seq read density ( ± 2 . 5 kb ) ; ( H ) As in ( G ) but for the 6 , 000 DMRs with the lowest H3K4me3 levels , ordered by decreasing H3K4me1 read density , and DMR classification as enhancer or non - enhancer in MCF7 ( ENCODE ) .

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(G) Heat- maps of DMRs (ordered by decreasing H3K4me3 read density) showing: DNA methylation levels (±2.5 kb), differences (ΔmCG), classification as promoter or non-promoter located, CpG dinucleotide density (±2.5 kb), and histone modification ChIP-seq read density (±2.5 kb); (H) As in (G) but for the 6,000 DMRs with the lowest H3K4me3 levels, ordered by decreasing H3K4me1 read density, and DMR classification as enhancer or non-enhancer in MCF7 (ENCODE).


( A ) H3K4me3 ChIP - seq read density flanking ( ± 1 kb , 50 bp bins ) the centre of DMRs with ∆ mCG > 0 . 2 ( top ) , and H3K4me3 ChIP - seq peaks with ∆ mCG < 0 . 2 ( bottom ) .

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(A) H3K4me3 ChIP-seq read density flanking (±1 kb, 50 bp bins) the centre of DMRs with ∆mCG >0.2 (top), and H3K4me3 ChIP-seq peaks with ∆mCG <0.2 (bottom).


( B ) H3K4me3 ChIP - seq normalized read counts in DMRs with ∆ mCG > 0 . 2 , from H3K4me3 ChIP - seq in ZF - D3A no - dox and ZF - D3A + dox cells . Black line represents equal normalized read counts in ZF - D3A no - dox and ZF - D3A + dox cells ; blue line represents a linear regression of the data .

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(B) H3K4me3 ChIP-seq normalized read counts in DMRs with ∆mCG >0.2, from H3K4me3 ChIP-seq in ZF-D3A no-dox and ZF-D3A +dox cells. Black line represents equal normalized read counts in ZF-D3A no-dox and ZF-D3A +dox cells; blue line represents a linear regression of the data.


( C ) Comparison of UMR ∆ mCG and difference in H3K4me3 ChIP - seq normalized read counts between ZF - D3A no - dox and ZF - D3A + dox . Blue line represents a linear regression of the data .

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(C) Comparison of UMR ∆mCG and difference in H3K4me3 ChIP-seq normalized read counts between ZF-D3A no-dox and ZF-D3A +dox. Blue line represents a linear regression of the data.


( D ) DNA methylation levels of UMRs that intersect with H3K4me3 ChIP - seq peaks in ZF - D3A no - dox and ZF - D3A + dox cells , for genomic DNA or DNA immunoprecipitated with an anti - H3K4me3 antibody .

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(D) DNA methylation levels of UMRs that intersect with H3K4me3 ChIP-seq peaks in ZF-D3A no-dox and ZF-D3A +dox cells, for genomic DNA or DNA immunoprecipitated with an anti-H3K4me3 antibody.


( E ) DNA methylation levels of UMRs that intersect with phos - pho - ser5 RNA polymerase II ChIP - seq peaks in ZF - D3A no - dox and ZF - D3A + dox cells , for genomic DNA or DNA immunoprecipitated with an anti - phospho - ser5 RNA polymerase II antibody .

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(E) DNA methylation levels of UMRs that intersect with phos- pho-ser5 RNA polymerase II ChIP-seq peaks in ZF-D3A no-dox and ZF-D3A +dox cells, for genomic DNA or DNA immunoprecipitated with an anti-phospho-ser5 RNA polymerase II antibody.


( A ) Western blot measurement of ZF - D3A protein abundance upon doxycycline induction and withdrawal .

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(A) Western blot measurement of ZF-D3A protein abundance upon doxycycline induction and withdrawal.


( B ) Box and whisker plots of the DNA methylation levels of the 121 CpG sites in the DACH1 promoter region through - out doxycycline induction and withdrawal . Whiskers indicate 1 . 5 times the interquartile range or the most extreme data point , whichever is lower .

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(B) Box and whisker plots of the DNA methylation levels of the 121 CpG sites in the DACH1 promoter region through- out doxycycline induction and withdrawal. Whiskers indicate 1.5 times the interquartile range or the most extreme data point, whichever is lower.


( C ) Average DNA methylation level at the 121 CpG sites in the DACH1 promoter region throughout doxycycline induction followed by doxycycline withdrawal and cell cycle inhibition by growth in doxycycline - free media containing different cell cycle inhibitors ( Lovastatin , RO - 3306 , thymidine ) .

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(C) Average DNA methylation level at the 121 CpG sites in the DACH1 promoter region throughout doxycycline induction followed by doxycycline withdrawal and cell cycle inhibition by growth in doxycycline-free media containing different cell cycle inhibitors (Lovastatin, RO-3306, thymidine).


( D ) DMR mCG and hmCG levels in MCF7 - control and ZF - D3A + dox cells , ordered by the difference in DMR hmCG level ( hmCG / CG ) between MCF7 - control and ZF - D3A + dox cells , and heatmap representation of H3K4me3 levels in ZF - D3A + dox cells flanking ( ± 2 . 5 kb , 50 bp bins ) the centre of the DMRs .

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(D) DMR mCG and hmCG levels in MCF7-control and ZF-D3A +dox cells, ordered by the difference in DMR hmCG level (hmCG/CG) between MCF7-control and ZF-D3A +dox cells, and heatmap representation of H3K4me3 levels in ZF-D3A +dox cells flanking (±2.5 kb, 50 bp bins) the centre of the DMRs.


( E ) Heatmap of hmCG levels flanking ( ± 2 . 5 kb , 50 bp bins ) the centre of the DMRs , ordered by DMR hmCG level in MCF7 - control cells .

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(E) Heatmap of hmCG levels flanking (±2.5 kb, 50 bp bins) the centre of the DMRs, ordered by DMR hmCG level in MCF7-control cells.


( A ) Scatter plot of the difference in UMR DNA methylation levels , versus the fold change in mRNA abun - dance of UMR - associated expressed genes , between MCF7 - control and ZF - D3A + dox . Point color indicates the gene differential expression significance : red indicates FDR < 0 . 01 , black indicates FDR > 0 . 01 . Trend line ( green ) was fitted using a generalized additive model , and contour lines represent the relative density of UMRs .

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(A) Scatter plot of the difference in UMR DNA methylation levels, versus the fold change in mRNA abun- dance of UMR-associated expressed genes, between MCF7-control and ZF-D3A +dox. Point color indicates the gene differential expression significance: red indicates FDR <0.01, black indicates FDR >0.01. Trend line (green) was fitted using a generalized additive model, and contour lines represent the relative density of UMRs.


( B ) Distribution of the ratios of mRNA levels between ZF - D3A + dox and MCF7 - control , for expressed genes that have an associated UMR with a ∆ mCG > 0 . 3 . Column color indicates the class of mRNA abundance ratios between ZF - D3A + dox and MCF7 - control : blue = no decrease ; green = small decrease ; red = decrease .

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(B) Distribution of the ratios of mRNA levels between ZF-D3A +dox and MCF7-control, for expressed genes that have an associated UMR with a ∆mCG >0.3. Column color indicates the class of mRNA abundance ratios between ZF-D3A +dox and MCF7-control: blue = no decrease; green = small decrease; red = decrease.


( C ) Representative genes with high induction of promoter DNA methylation in ZF - D3A + dox and no reduction in mRNA abundance .

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(C) Representative genes with high induction of promoter DNA methylation in ZF-D3A +dox and no reduction in mRNA abundance.


Fluorescence distribution of cells stained with RNA - FISH probes targeting ( A ) GAPDH or ( B ) PSME1 mRNA before and after FACS .

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Fluorescence distribution of cells stained with RNA-FISH probes targeting (A) GAPDH or (B) PSME1 mRNA before and after FACS.


mRNA quantitation after FACS sorting of ( C ) GAPDH by RT - qPCR , and ( D ) PSME1 by RNA - seq .

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mRNA quantitation after FACS sorting of (C) GAPDH by RT-qPCR, and (D) PSME1 by RNA-seq.


DNA methylation levels of single CpGs at the promoter region of ( E ) GAPDH , and ( F ) PSME1 , for the low ( x axis ) and high ( y axis ) expressing sorted cell populations .

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DNA methylation levels of single CpGs at the promoter region of (E) GAPDH, and (F) PSME1, for the low (x axis) and high (y axis) expressing sorted cell populations.


( G ) Genome browser representation of the GAPDH ( top ) and PSME1 ( bottom ) promoter regions showing DNA methylation in MCF7 - control and low and high expressing sorted cell populations .

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(G) Genome browser representation of the GAPDH (top) and PSME1 (bottom) promoter regions showing DNA methylation in MCF7-control and low and high expressing sorted cell populations.


( H ) Distribution of per - read methylation levels ( blue line ) for selected ZF - D3A + dox WGBS reads ( ≥ 10 cytosines in the CpG context ) that align within robustly methylated DMRs ( DMR mean methylation level [ mCG / CG ] of 0 . 4 - 0 . 6 in ZF - D3A + dox and < 0 . 1 in MCF7 - control ) , and average CG methylation levels from ZF - D3A + dox WGBS data of the DMR genomic intervals covered by the selected reads ( red line ) .

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(H) Distribution of per-read methylation levels (blue line) for selected ZF-D3A +dox WGBS reads (≥10 cytosines in the CpG context) that align within robustly methylated DMRs (DMR mean methylation level [mCG/CG] of 0.4-0.6 in ZF-D3A +dox and <0.1 in MCF7-control), and average CG methylation levels from ZF-D3A +dox WGBS data of the DMR genomic intervals covered by the selected reads (red line).


A . Left : Telomerase activity measured using ddTRAP assay . The horizontal pink lines represent the threshold used in these samples for background correction . Each dot on the ddPCR output represents a unique droplet that is either positive or negative for fluorescent signal . Right : Quantification of TRAP activity after normalization to SW39 / TEL + cells ( n = 3 ) . Data information : Data are presented as mean ± SD . Statistical analyses were performed with Student ' s two - tailed unpaired t - test with Welch ' s correction for normality of distribution unless specified otherwise . PTM , post - translational modification .

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A. Left: Telomerase activity measured using ddTRAP assay. The horizontal pink lines represent the threshold used in these samples for background correction. Each dot on the ddPCR output represents a unique droplet that is either positive or negative for fluorescent signal. Right: Quantification of TRAP activity after normalization to SW39/TEL+ cells (n=3). Data information: Data are presented as mean ± SD. Statistical analyses were performed with Student's two-tailed unpaired t-test with Welch's correction for normality of distribution unless specified otherwise. PTM, post-translational modification.


B . hTR and hTERT expression , normalized to ACTB mRNA and to SW39 / TEL + . Data information : Data are presented as mean ± SD . Statistical analyses were performed with Student ' s two - tailed unpaired t - test with Welch ' s correction for normality of distribution unless specified otherwise . PTM , post - translational modification .

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B. hTR and hTERT expression, normalized to ACTB mRNA and to SW39/TEL+. Data information: Data are presented as mean ± SD. Statistical analyses were performed with Student's two-tailed unpaired t-test with Welch's correction for normality of distribution unless specified otherwise. PTM, post-translational modification.


C . RNA Pol II binding at hTR promoter in SI14 / TEL + , SI24 / ALT + and 8G12 / ALT + cell lines , normalized to H3 ChIP . Control loci with , respectively , high or low transcriptional activity : c - fos promoter and subtelomeric 4qHox locus ( n = 3 ) . Statistical analyses were performed using one - way ANOVA with post - hoc Tukey ' s test for multiple comparisons . Data information : Data are presented as mean ± SD . Statistical analyses were performed with Student ' s two - tailed unpaired t - test with Welch ' s correction for normality of distribution unless specified otherwise . PTM , post - translational modification .

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C. RNA Pol II binding at hTR promoter in SI14/TEL+, SI24/ALT+ and 8G12/ALT+ cell lines, normalized to H3 ChIP. Control loci with, respectively, high or low transcriptional activity: c-fos promoter and subtelomeric 4qHox locus (n=3). Statistical analyses were performed using one-way ANOVA with post-hoc Tukey's test for multiple comparisons. Data information: Data are presented as mean ± SD. Statistical analyses were performed with Student's two-tailed unpaired t-test with Welch's correction for normality of distribution unless specified otherwise. PTM, post-translational modification.


D . H3K9me3 and H3K27me3 ( left ) or H3K4me3 and H3Ac ( right ) densities at hTR promoter in SI14 / TEL + and SI24 / ALT + , normalized to H3 ( n = 3 ) . Control loci as in C . Data information : Data are presented as mean ± SD . Statistical analyses were performed with Student ' s two - tailed unpaired t - test with Welch ' s correction for normality of distribution unless specified otherwise . PTM , post - translational modification .

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D. H3K9me3 and H3K27me3 (left) or H3K4me3 and H3Ac (right) densities at hTR promoter in SI14/TEL+ and SI24/ALT+, normalized to H3 (n=3). Control loci as in C. Data information: Data are presented as mean ± SD. Statistical analyses were performed with Student's two-tailed unpaired t-test with Welch's correction for normality of distribution unless specified otherwise. PTM, post-translational modification.


E . NFYB binding at hTR promoter in SI14 / TEL + , SI24 / ALT + , SW39 / TEL + and IMRB / ALT + cells normalized to H3 . Control loci with , respectively , high and low NFYB binding : hnRNPA1 promoter and subtelomeric 4qHox ( n = 3 ) . Data information : Data are presented as mean ± SD . Statistical analyses were performed with Student ' s two - tailed unpaired t - test with Welch ' s correction for normality of distribution unless specified otherwise . PTM , post - translational modification .

E. NFYB binding at hTR promoter in SI14/TEL+, SI24/ALT+, SW39/TEL+ and IMRB/ALT+ cells normalized to H3. Control loci with, respectively, high and low NFYB binding: hnRNPA1 promoter and subtelomeric 4qHox (n=3). Data information: Data are presented as mean ± SD. Statistical analyses were performed with Student's two-tailed unpaired t-test with Welch's correction for normality of distribution unless specified otherwise. PTM, post-translational modification.


A . Western blot for hTR chaperone proteins in TEL + and ALT + hybrids . β - Actin is used as loading control . Quantification of western blots is shown on the right , normalized to β - Actin and to SI14 / TEL + cells ( n = 2 - 4 ) . Statistical analyses were performed using one - way ANOVA with post - hoc Tukey ' s test for multiple comparisons . Data information : Data are presented as mean ± SD .

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A. Western blot for hTR chaperone proteins in TEL+ and ALT+ hybrids. β-Actin is used as loading control. Quantification of western blots is shown on the right, normalized to β-Actin and to SI14/TEL+ cells (n=2-4). Statistical analyses were performed using one-way ANOVA with post-hoc Tukey's test for multiple comparisons. Data information: Data are presented as mean ± SD.


B . NHP2 mRNA expression levels , normalized to ACTB mRNA and to SI14 / TEL + cells ( n = 3 ) . Data information : Data are presented as mean ± SD .

B. NHP2 mRNA expression levels, normalized to ACTB mRNA and to SI14/TEL+ cells (n=3). Data information: Data are presented as mean ± SD.


C . Western blot for NHP2 in SI14 / TEL + , SI24 / ALT + and 8G12 / ALT + cell lines treated with DMSO or 10 μM MG132 for 6 h . β - Actin served as loading control . Quantifications are shown on the right , normalized to β - Actin and to SI14 / TEL + cells ( n = 5 ) . Statistical analyses were performed with Student ' s two - tailed unpaired t - test . Data information : Data are presented as mean ± SD .

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C. Western blot for NHP2 in SI14/TEL+, SI24/ALT+ and 8G12/ALT+ cell lines treated with DMSO or 10 μM MG132 for 6 h. β-Actin served as loading control. Quantifications are shown on the right, normalized to β-Actin and to SI14/TEL+ cells (n=5). Statistical analyses were performed with Student's two-tailed unpaired t-test. Data information: Data are presented as mean ± SD.


D . hTR RNA and NHP2 mRNA expression , normalized to ACTB mRNA and to SI14 / TEL + . Data information : Data are presented as mean ± SD .

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D. hTR RNA and NHP2 mRNA expression, normalized to ACTB mRNA and to SI14/TEL+. Data information: Data are presented as mean ± SD.


E . Telomerase activity measured with ddTRAP assay in NHP2 - overexpressing 8G12 / ALT + cells , normalized to NHP2 - overexpressing SI14 / TEL + . Data information : Data are presented as mean ± SD .

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E. Telomerase activity measured with ddTRAP assay in NHP2-overexpressing 8G12/ALT+cells, normalized to NHP2-overexpressing SI14/TEL+. Data information: Data are presented as mean ± SD.


F . ( Top ) Western blot for NHP2 overexpression in 8G12 / ALT + clonal cell lines . β - Actin served as loading control . SI14 / TEL + cells are shown as reference for NHP2 expression in hybrids . ( Bottom ) Western blot for NHP2 overexpression in SI14 / TEL + and 8G12 / ALT + cells compared to empty vector control . β - Actin served as loading control . Data information : Data are presented as mean ± SD .

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F. (Top) Western blot for NHP2 overexpression in 8G12/ALT+ clonal cell lines. β-Actin served as loading control. SI14/TEL+ cells are shown as reference for NHP2 expression in hybrids. (Bottom) Western blot for NHP2 overexpression in SI14/TEL+ and 8G12/ALT+ cells compared to empty vector control. β-Actin served as loading control. Data information: Data are presented as mean ± SD.


G . hTR RNA , hTERT and NHP2 mRNA expression , normalized to ACTB mRNA and to SI14 / TEL + ( n = 3 ) . Data information : Data are presented as mean ± SD .

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G. hTR RNA, hTERT and NHP2 mRNA expression, normalized to ACTB mRNA and to SI14/TEL+ (n=3). Data information: Data are presented as mean ± SD.


H . RNA Pol II binding at hTR promoter , normalized to H3 ChIP ( n = 3 ) . See Fig . 1C legend for control loci . Statistical analyses were performed using one - way ANOVA with post - hoc Tukey ' s test for multiple comparisons . Data information : Data are presented as mean ± SD .

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H. RNA Pol II binding at hTR promoter, normalized to H3 ChIP (n=3). See Fig. 1C legend for control loci. Statistical analyses were performed using one-way ANOVA with post-hoc Tukey's test for multiple comparisons. Data information: Data are presented as mean ± SD.


A . hTR RNA , hTERT and NHP2 mRNA expression , normalized to ACTB mRNA and to 143B / TEL + cells , in a series of TEL + and ALT + human cell lines as indicated ( n = 3 ) . Data information : Data are presented as mean ± SD . Statistical analyses were performed using one - way ANOVA with post - hoc Tukey ' s test for multiple comparisons .

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A. hTR RNA, hTERT and NHP2 mRNA expression, normalized to ACTB mRNA and to 143B/TEL+ cells, in a series of TEL+ and ALT+ human cell lines as indicated (n=3). Data information: Data are presented as mean ± SD. Statistical analyses were performed using one-way ANOVA with post-hoc Tukey's test for multiple comparisons.


B . Western blot for NHP2 expression . β - Actin served as loading control . Quantification is shown on the right after normalization to β - Actin and to 143B / TEL + cells ( n = 3 ) . Data information : Data are presented as mean ± SD . Statistical analyses were performed using one - way ANOVA with post - hoc Tukey ' s test for multiple comparisons .

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B. Western blot for NHP2 expression. β-Actin served as loading control. Quantification is shown on the right after normalization to β-Actin and to 143B/TEL+ cells (n=3). Data information: Data are presented as mean ± SD. Statistical analyses were performed using one-way ANOVA with post-hoc Tukey's test for multiple comparisons.


A . hTR RNA expression , normalized to ACTB mRNA and to U2OS cells ( n = 3 ) . Data information : Data are shown as mean ± SD , with the red line marking the mean value .

A. hTR RNA expression, normalized to ACTB mRNA and to U2OS cells (n=3). Data information: Data are shown as mean ± SD, with the red line marking the mean value.


B . Western blot for NHP2 expression in the early passages of hTR - expressing IMRB or U2OS cells , with β - Actin as loading control .

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B. Western blot for NHP2 expression in the early passages of hTR-expressing IMRB or U2OS cells, with β-Actin as loading control.


C . Quantification of doubling time for U2OS , U2OS - hTR , IMRB and IMRB - hTR cell lines ( n = 3 ) . Statistical analyses were performed with Student ' s two - tailed unpaired t - test with Welch ' s correction for normality of distribution Data information : Data are shown as mean ± SD , with the red line marking the mean value .

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C. Quantification of doubling time for U2OS, U2OS-hTR, IMRB and IMRB-hTR cell lines (n=3). Statistical analyses were performed with Student's two-tailed unpaired t-test with Welch's correction for normality of distribution Data information: Data are shown as mean ± SD, with the red line marking the mean value.


D . Western blot in U2OS and U2OS - hTR cells treated with or without Hydroxyurea ( 2 mM for 24 h ) , for the indicated proteins . β - Actin and Vinculin served as loading controls .

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D. Western blot in U2OS and U2OS-hTR cells treated with or without Hydroxyurea (2 mM for 24 h), for the indicated proteins. β-Actin and Vinculin served as loading controls.


E . Representative images for co - localization between p - RPA32S33 ( green ) and telomeres ( red FISH probe ) . White arrows indicate co - localization events . Scale bars : 5 μm . Quantification is shown on the right . At least 50 nuclei were scored in each of the three independent experiments performed . Each dot represents the number of p - RPA32S33 - telomere co - localizations in one nucleus . Statistical analyses were performed with Student ' s two - tailed unpaired t - test with Welch ' s correction for normality of distribution . Data information : Data are shown as mean ± SD , with the red line marking the mean value .

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E. Representative images for co-localization between p-RPA32S33 (green) and telomeres (red FISH probe). White arrows indicate co-localization events. Scale bars: 5 μm. Quantification is shown on the right. At least 50 nuclei were scored in each of the three independent experiments performed. Each dot represents the number of p-RPA32S33-telomere co-localizations in one nucleus. Statistical analyses were performed with Student's two-tailed unpaired t-test with Welch's correction for normality of distribution. Data information: Data are shown as mean ± SD, with the red line marking the mean value.


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 .

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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 .

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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 .

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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 .

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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 .

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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 .

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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 .

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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 .

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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 .

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 .

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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 .

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

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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 .

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 O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 .

O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 .

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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 .

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 ) .

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 .

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 .

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 .

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 .

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 .

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 .

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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 .

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 .

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 .

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 .

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 .

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 .

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 .

(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 .

(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 .

(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).