( A ) Time course of viability of AS901 , stably overexpressing either wild - type or G842C - mutated PlxnB2 , or controls transduced with an empty vector . Plotted values are mean ± SD of n = 3 independent experiments ( six technical replicates for each ) . statistical significance was assessed by two - way ANOVA with Tukey ' s correction for multiple comparisons . PlxnB2 - G842C vs . controls ( CTRL ) : at 24h * * p = 0 . 0097 , 48h * * p = 0 . 0026 , 72h * * p = 0 . 0079 , 96h * p = 0 . 0226 . PlxnB2 - G842C vs . PlxnB2 - WT : at 48h $ p = 0 . 0418 , 72h $ p = 0 . 0318 . ( B ) growth curves of ADX 901 over - expressing either PlxnB2 - G842C or PlxnB2 - WT , or mock control . Plotted values are mean ± SD ( n = 8 each groups ) . statistical significance was assessed by two - way ANOVA with Tukey ' s correction for multiple comparisons . PlxnB2 - G842C vs control ( CTRL ) : at 25 days * * p = 0 . 0029 , 28 days * * p = 0 . 0028 , 32 days * * p = 0 . 0033 . PlxnB2 - G842C vs PlxnB2 - WT : at 25 days $ p = 0 . 0221 , 28 days $ p = 0 . 0234 , 32 days $ $ p = 0 . 0024 .

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(A) Time course analysis of the cellular viability of AS901, stably overexpressing either wild-type or G842C-mutated PlxnB2, or controls transduced with an empty vector. Plotted values are the mean ± SD of n =3 independent experiments (six technical replicates for each). The statistical significance was assessed by two-way ANOVA with Tukey's correction for multiple comparisons. PlxnB2-G842C vs. controls (CTRL): at 24h **p = 0.0097, 48h **p = 0.0026, 72h **p = 0.0079, 96h *p = 0.0226. PlxnB2-G842C vs. PlxnB2-WT: at 48h $p = 0.0418, 72h $p = 0.0318. (B) Tumor growth curves of ADX 901 over-expressing either PlxnB2-G842C or PlxnB2-WT, or mock control. Plotted values are the mean ± SD (n = 8 each groups). The statistical significance was assessed by two-way ANOVA with Tukey's correction for multiple comparisons. PlxnB2-G842C vs control (CTRL): at 25 days **p = 0.0029, 28 days **p = 0.0028, 32 days **p = 0.0033. PlxnB2-G842C vs PlxnB2-WT: at 25 days $p = 0.0221, 28 days $p = 0.0234, 32 days $$p = 0.0024.


( A ) Western blotting showing basal endogenous or overexpressed PlxnB2 , either WT or G842C - mutated isoforms , in MCF7 . In this wide view of hybridized , it is appreciable presence of full molecules at 250 kDa , as well as processed 80 kDa fragments ( reported in ref . 64 ) . Vinculin provided loading controls .

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(A) Western blotting showing basal endogenous or overexpressed PlxnB2, either WT or G842C-mutated isoforms, in MCF7 cells. In this wide view of the hybridized membrane, it is appreciable the presence of full length molecules at 250 kDa, as well as the processed 80 kDa fragments (reported in ref. 64). Vinculin staining provided protein loading controls.


( B ) graph shows mean ± SD of relative increase in 24 - hour spontaneous migration across Transwell inserts of MCF7 ( analysed above ) over - expressing either wild - type or G842C - mutated PlxnB2 , versus mock - transfected controls ( n = 4 biological replicates ) . statistical significance was verified by one - way ANOVA with Bonferroni correction for multiple comparisons : PlxnB2 - WT vs . mock * p = 0 . 0145 ; PlxnB2 - G842C vs . PlxnB2 - WT * p = 0 . 0277 ; PlxnB2 - G842C vs . mock * * * p = 0 . 0002 .

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(B) The graph shows the mean ± SD of relative increase in 24-hour spontaneous migration across Transwell inserts of MCF7 cells (analysed above) over-expressing either wild-type or G842C-mutated PlxnB2, versus mock-transfected controls (n=4 biological replicates). The statistical significance was verified by one-way ANOVA with Bonferroni correction for multiple comparisons: PlxnB2-WT vs. mock *p=0.0145; PlxnB2-G842C vs. PlxnB2-WT *p=0.0277; PlxnB2-G842C vs. mock ***p=0.0002.


( C ) Wound healing scratch assays on monolayers of MCF7 engineered as described above . graph shows mean ± SD of values obtained in N = 3 independent experiments ( with at least three technical replicates for each ) . statistical significance was verified by one - way ANOVA with Tukey ' s correction for multiple comparisons : * p = 0 . 0350 ; * * p = 0 . 0029 ; * * * p = 0 . 0002 . Representative phase contrast of wounded monolayers are further shown in larger in Appendix Figure S4B .

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(C) Wound healing scratch assays on monolayers of MCF7 cells engineered as described above. The graph shows the mean ± SD of values obtained in N=3 independent experiments (with at least three technical replicates for each). The statistical significance was verified by one-way ANOVA with Tukey's correction for multiple comparisons: *p = 0.0350; **p = 0.0029; ***p = 0.0002. Representative phase contrast images of the wounded monolayers are further shown in larger size in Appendix Figure S4B.


( D ) Western blotting of endogenous overexpressed PlxnB2 ( either WT or G842C - mutated isoforms ) in CUP AS906 ( both full molecules at 250 kDa processed 80 kDa fragments ) . Vinculin provided loading controls .

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(D) Western blotting analysis of endogenous and overexpressed PlxnB2 (either WT or G842C-mutated isoforms) in CUP cells AS906 (both full length molecules at 250 kDa and processed 80 kDa fragments). Vinculin staining provided protein loading controls.


( E ) invasive capacity of CUP AS906 ( shown in previous panel ) , overexpressing WT or G842 - mutated PlxnB2 ( or mock control ) , was assessed across Matrigel - coated transwell inserts . graph shows mean ± SD of values obtained in N = 3 independent experiments ( including technical duplicates , in two cases ) . Below graph , are shown representative fields of semipermeable membranes ( additional larger can be found in appendix Figure S4C ) ; scale bar : 100μm . statistical significance was verified by one - way ANOVA with Bonferroni correction for multiple comparisons : * p = 0 . 0406 ; * * p = 0 . 0045 ; * * * p = 0 . 0003 .

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(E) The invasive capacity of CUP cells AS906 (shown in previous panel), overexpressing WT or G842-mutated PlxnB2 (or mock control), was assessed across Matrigel-coated transwell inserts. The graph shows the mean ± SD of values obtained in N=3 independent experiments (including technical duplicates, in two cases). Below the graph, are shown representative fields of the semipermeable membranes (additional larger size images can be found in appendix Figure S4C); scale bar: 100μm. The statistical significance was verified by one-way ANOVA with Bonferroni correction for multiple comparisons: *p=0.0406; **p=0.0045; ***p=0.0003.


( A ) Phospho - EGFR by western blotting in control PlxnB2 - silenced AS43 . show representative results of n = 3 independent experiments , while graph at bottom shows mean ± SD of normalized band . statistical significance across replicates was verified by unpaired t - : * * * * p < 0 . 0001 . ( B ) Phospho - EGFR by western blotting in AS901 AS906 either mock , or overexpressing WT or G842C - mutated PlxnB2 . show representative results of n = 3 independent experiments , while graphs at bottom show mean ± SD of normalized band . statistical significance across independent replicas was verified by one - way ANOVA followed by Tukey : AS901 PlxnB2 - G842C vs . PlxnB2 - WT * p = 0 . 0434 , PlxnB2 - G842C vs . mock * p = 0 . 0211 ; AS906 PlxnB2 - G842C vs . PlxnB2 - WT * p = 0 . 0436 , PlxnB2 - G842C vs . mock * p = 0 . 0481 .

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(A) Phospho-EGFR analysis by western blotting in control and PlxnB2-silenced AS43 cells. Images show representative results of n=3 independent experiments, while the graph at the bottom shows the mean ± SD of normalized band quantification. The statistical significance across replicates was verified by unpaired t-test: ****p<0.0001. (B) Phospho-EGFR analysis by western blotting in AS901 and AS906 cells either mock, or overexpressing WT or G842C-mutated PlxnB2. Images show representative results of n=3 independent experiments, while the graphs at the bottom show the mean ± SD of normalized band quantification. The statistical significance across independent replicas was verified by one-way ANOVA followed by Tukey test: AS901 PlxnB2-G842C vs. PlxnB2-WT *p=0.0434, PlxnB2-G842C vs. mock *p=0.0211; AS906 PlxnB2-G842C vs. PlxnB2-WT *p=0.0436, PlxnB2-G842C vs. mock *p=0.0481.


( C ) Western blotting of pERK pS6 intracellular signal transducers in indicated agnospheres , after 6 - hour treatment with EGFR inhibitor cetuximab ( 50μg / ml ) . show representative results of n = 3 independent experiments , while graphs at bottom show mean ± SD of normalized band . statistical significance across independent replicas was verified by Sidak ' s multiple comparison between cetuximab - treated untreated conditions , per each group : AS43 * p = 0 . 0153 , * * * p = 0 . 0001 , * * * * p < 0 . 0001 ; AS901 * p = 0 . 0201 .

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(C) Western blotting analysis of pERK and pS6 intracellular signal transducers in the indicated agnospheres, after 6-hour treatment with the EGFR inhibitor cetuximab (50μg/ml). Images show representative results of n=3 independent experiments, while the graphs at the bottom show the mean ± SD of normalized band quantification. The statistical significance across independent replicas was verified by Sidak's multiple comparison test between cetuximab-treated and untreated conditions, per each group: AS43 *p=0.0153, ***p=0.0001, ****p<0.0001; AS901 *p=0.0201.


( A ) viability of indicated agnospheres was assessed after six days in presence or absence of EGFR inhibitor cetuximab ( 50μg / ml ) . In plot , each box represents values from 25th to 75th percentiles , central band indicates mean , whiskers expand from maximum to minimum values of cetuximab - treated experimental points ( n ≥ 9 ) , normalized to average value of untreated within each group . statistical significance across three independent experiments , each comprising ≥ 3 technical replicates , was verified by unpaired t - multiple comparisons of cetuximab - treated untreated conditions , per each group : * * * p = 0 . 0002 ; * * * * p < 0 . 0001 .

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(A) The cellular viability of the indicated agnospheres was assessed after six days in presence or absence of the EGFR inhibitor cetuximab (50μg/ml). In the plot, each box represents values from 25th to 75th percentiles, the central band indicates the mean, and the whiskers expand from maximum to minimum values of individual cetuximab-treated experimental points (n≥9), normalized to the average value of untreated cells within each group. The statistical significance across three independent experiments, each comprising ≥3 technical replicates, was verified by unpaired t-test multiple comparisons of cetuximab-treated and untreated conditions, per each group: ***p=0.0002; ****p<0.0001.


( B ) viability of AS43 , in presence ( or absence ) of cetuximab ( 50μg / ml ) , was assessed over indicated time course , compared to values at T0 . graph shows mean ± SD of two independent experiments ( with quadruplicate technical replicates each ) . statistical significance was verified through multiple comparisons by two - way ANOVA with Bonferroni correction of cetuximab - treated untreated samples , per each time point : * * * * p < 0 . 0001 .

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(B) The viability of AS43, in presence (or absence) of cetuximab (50μg/ml), was assessed over the indicated time course, compared to values at T0. The graph shows the mean ± SD of two independent experiments (with quadruplicate technical replicates each). The statistical significance was verified through multiple comparisons by two-way ANOVA with Bonferroni correction of cetuximab-treated and untreated samples, per each time point: ****p<0.0001.


( D ) invasive capacity of AS906 ( overexpressing WT or G842C - mutated PlxnB2 , or mock transfected ) was assessed as in Fig . 7E , in presence or absence of cetuximab 50μg / ml . show representative results ( scale bar : 100μm ) , while graph on right shows mean ± SD of n = 3 independent experiments . statistical significance across replicates was verified by one - way ANOVA with Bonferroni correction : * * p = 0 . 0010 ; * * * p = 0 . 0005 ; * * * * p < 0 . 0001 .

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(D) The invasive capacity of AS906 cells (overexpressing WT or G842C-mutated PlxnB2, or mock transfected) was assessed as in Fig. 7E, in the presence or absence of cetuximab 50μg/ml. Images show representative results (scale bar: 100μm), while the graph on the right shows the mean ± SD of n=3 independent experiments. The statistical significance across replicates was verified by one-way ANOVA with Bonferroni correction: **p= 0.0010; ***p=0.0005; ****p<0.0001.


( A ) Tandem affinity purification was used to isolate KDF1 associated from SILAC - labelled keratinocytes . Precipitated were resolved by SDS - PAGE subjected to identification with LC - MS / MS . Arrows denote IgG heavy chains . Star denotes KDF1 . Ctrl : control . Kd : kilodalton for molecular markers .

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(A) Tandem affinity purification was used to isolate KDF1 associated proteins from SILAC-labelled keratinocytes. Precipitated proteins were resolved by SDS-PAGE and subjected to identification with LC-MS/MS. Arrows denote IgG heavy and light chains. Star denotes KDF1. Ctrl: control. Kd: kilodalton for molecular weight markers.


( B - C ) HEK293 were transfected with empty vector or plasmids encoding HA tagged KDF1 / or GST - tagged IKKα . Immunoprecipitation ( α - HA or α - GST ) was carried out to determine their interaction . Immunoprecipitates ( IP ) whole lysate ( WCL ) were immunoblotted ( IB ) with different antibodies as indicated .

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(B-C) HEK293 cells were transfected with empty vector or plasmids encoding HA tagged KDF1 and/or GST-tagged IKKα. Immunoprecipitation (α-HA or α-GST) was carried out to determine their interaction. Immunoprecipitates (IP) and whole cell lysate (WCL) were immunoblotted (IB) with different antibodies as indicated.


( D ) Immunoprecipitation was carried out with α - KDF1 antibody in keratinocyte cultured in medium with low or high of calcium ( Ca ) . IPs were blotted with different antibodies as indicated . Star denotes KDF1 .

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


( E ) HEK293 were transfected to co - express HA - tagged KDF1 with GST - tagged IKKα or its kinase dead ( KD ) mutant . IP WCL were blotted with different antibodies as indicated .

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(E) HEK293 cells were transfected to co-express HA-tagged KDF1 with GST-tagged IKKα or its kinase dead (KD) mutant. IP and WCL were blotted with different antibodies as indicated.


( F ) HEK293 were transfected to co - express HA - tagged KDF1 with GST - tagged IKKα or its different truncation mutants ( EV Fig . 1B ) . IP WCL were blotted with different antibodies as indicated . Arrow denotes antibody heavy chain .

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(F) HEK293 cells were transfected to co-express HA-tagged KDF1 with GST-tagged IKKα or its different truncation mutants (EV Fig. 1B). IP and WCL were blotted with different antibodies as indicated. Arrow denotes antibody heavy chain.


( G ) HEK293 were transfected to co - express GST - tagged IKKα with HA - tagged KDF1 or its different truncation mutants ( EV Fig . 1C ) . IP WCL were blotted with different antibodies as indicated .

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(G) HEK293 cells were transfected to co-express GST-tagged IKKα with HA-tagged KDF1 or its different truncation mutants (EV Fig. 1C). IP and WCL were blotted with different antibodies as indicated.


( A ) H / E of E18 . 5 skin sections from WT , IKKα KO , KDF1 KO mice . Dotted lines denote dermal - epidermal boundaries . Epi : epidermis , Der : dermis . Scale bar = 50 μm . ( B ) E18 . 5 skin sections from WT , IKKα KO , KDF1 KO mice were immunostained with different antibodies as indicated ( Krt14 : Keratin 14 , Krt10 : Keratin 10 , Lor : Loricrin , β4 : β4 - integrin , CD104 ) . Dotted lines denote dermal - epidermal boundaries . Epi : epidermis , Der : dermis . Scale bar = 100 μm .

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(A) H/E staining of E18.5 skin sections from WT, IKKα KO, and KDF1 KO mice. Dotted lines denote dermal-epidermal boundaries. Epi: epidermis, Der: dermis. Scale bar=50 μm. (B) E18.5 skin sections from WT, IKKα KO, and KDF1 KO mice were immunostained with different antibodies as indicated (Krt14: Keratin 14, Krt10: Keratin 10, Lor: Loricrin, β4: β4-integrin, CD104). Dotted lines denote dermal-epidermal boundaries. Epi: epidermis, Der: dermis. Scale bar=100 μm.


( C ) Immunoblots of WCL collected at 0 , 1 2 days after calcium shift with different antibodies as indicated .

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(C) Immunoblots of WCL collected at 0, 1 and 2 days after calcium shift with different antibodies as indicated.


A ) Immunoblots of WCL from IKKα KO with rescued expression of HA - tagged IKKα or its mutant using different antibodies as indicated .

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A) Immunoblots of WCL from IKKα KO cells with rescued expression of HA-tagged IKKα or its mutant using different antibodies as indicated.


B ) Immunoblots of WCL from KDF1 KO with rescued expression of KDF1 or its mutant using different antibodies as indicated . Expression f exogenous KDF1 was induced by doxycycline ( DOX ) at different .

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B) Immunoblots of WCL from KDF1 KO cells with rescued expression of KDF1 or its mutant using different antibodies as indicated. Expression f exogenous KDF1 was induced by doxycycline (DOX) at different concentration.


C - D ) Expression of loricrin is determined by immunoblots quantified by densitometry . relative expression level of loricrin upon calcium shift in different types was calculated presented as bar graphs . Statistical is conducted using unpaired Student ' s t - . Error bar represents S . D . ( standard deviation ) . N = 3 ( biological replicates ) . * * * , p < 0 . 001 ; * * , p < 0 . 01 ; * , p < 0 .

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C-D) Expression of loricrin is determined by immunoblots and quantified by densitometry. The relative expression level of loricrin upon calcium shift in different cell types was calculated and presented 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<0.001; **, p<0.01; *, p<0.05


( C - D ) Expression of loricrin is determined by immunoblots quantified by densitometry . relative expression level of loricrin upon calcium shift in different types was calculated presented as bar graphs . Statistical is conducted using unpaired Student ' s t - . Error bar represents S . D . ( standard deviation ) . N = 3 ( biological replicates ) . * * * , p < 0 . 001 ; * * , p < 0 . 01 ; * , p < 0 .

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(C-D) Expression of loricrin is determined by immunoblots and quantified by densitometry. The relative expression level of loricrin upon calcium shift in different cell types was calculated and presented 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<0.001; **, p<0.01; *, p<0.05


E ) Sections of regenerated skin developed from engrafted IKKα KO , KDF1 KO , their rescued were immunostained with different antibodies as indicated . Dotted lines denote dermal - epidermal boundaries . Epi : epidermis , Der : dermis . Scale bar = 50 μm .

E) Sections of regenerated skin developed from engrafted IKKα KO, KDF1 KO, and their rescued cells were immunostained with different antibodies as indicated. Dotted lines denote dermal-epidermal boundaries. Epi: epidermis, Der: dermis. Scale bar=50 μm.


( A ) WCL from WT KDF1 KO before after calcium shift were immunoblotted with different antibodies as indicated . Lo : low calcium ; Hi : high calcium .

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(A) WCL from WT and KDF1 KO cells before and after calcium shift were immunoblotted with different antibodies as indicated. Lo: low calcium; Hi: high calcium.


( B - C ) WT KDF1 KO keratinocyte were treated with 20nM cycloheximide ( CHX ) . WCL was collected at 0 , 30 , 60 , 90 , 120 min post CHX treatment subjected to immunoblotting with IKKα antibody ( B ) . Band is determined by densitometry amount of IKKα is calculated quantified ( C ) . Statistical is conducted using 2 - way ANOVA . N = 3 ( biological replicates ) . * , p < 0 . 05 . Error bar represents S . D . ( standard deviation ) .

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(B-C) WT and KDF1 KO keratinocyte were treated with 20nM cycloheximide (CHX). WCL was collected at 0, 30, 60, 90, 120 min post CHX treatment and subjected to immunoblotting with IKKα antibody (B). Band intensity is determined by densitometry and the amount of IKKα is calculated and quantified (C). Statistical analysis is conducted using 2-way ANOVA. N=3 (biological replicates). *, p<0.05. Error bar represents S.D. (standard deviation).


( D - E ) WT KDF1 KO keratinocyte ( right panels ) or transfected with plasmid encoding exogenous IKKα ( left panels ) were treated with MG132 at 10µM for 6 hours , then were subjected to immunoprecipitation using anti - ubiquitin ( Ub ) antibody . IP WCL were analyzed by immunoblots with α - IKKα antibody ( D ) . Overall of ubiquitinated IKKα bands was determined by densitometry . of ubiquitinated IKKα was quantified presented as bar graphs ( E ) . Statistical is conducted using unpaired Student ' s t - . Error bar represents S . D . ( standard deviation ) . N = 3 ( biological replicates ) . * , p < 0 . 05 . Kd : kilodalton for molecular markers .

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(D-E) WT and KDF1 KO keratinocyte (right panels) or cells transfected with plasmid encoding exogenous IKKα (left panels) were treated with MG132 at 10µM for 6 hours, then were subjected to immunoprecipitation using anti-ubiquitin (Ub) antibody. IP and WCL were analyzed by immunoblots with α-IKKα antibody (D). Overall intensity of all ubiquitinated IKKα bands was determined by densitometry. Ratio of ubiquitinated IKKα was quantified and presented as bar graphs (E). Statistical analysis is conducted using unpaired Student's t-test. Error bar represents S.D. (standard deviation). N=3 (biological replicates). *, p<0.05. Kd: kilodalton for molecular weight markers.


( F ) IKKα level in KDF1 KO keratinocyte expressing WT KDF1 or KDF1 mutant , under both low high calcium conditions , was examined 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 . WCL were collected 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 were transfected to co - express KDF1 with USP7 . IP 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 of calcium ( Ca ) , same as in Fig . 1D . IPs were blotted with different antibodies as indicated . 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 USP7 CRISPR KO 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 level in WT USP7 KO keratinocyte were examined by immunoblotting before 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 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 was determined by densitometry shown as bar graphs . Statistical is conducted using unpaired Student ' s t - . Error bar represents S . D . ( standard deviation ) . N = 3 ( biological replicates ) . * * , p < 0 . 01 . Kd : kilodalton for molecular 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 Loricrin ( Lor ) with WT USP7 KO before after calcium shift ( F ) . Star denotes an unspecific band in α - Loricrin blots . Band was determined by densitometry 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 is conducted using unpaired Student ' s t - . Error bar represents S . D . ( standard deviation ) . N = 3 ( biological replicates ) . * * * * , p < 0 . 0001 ; * * , p < 0 . 01 .

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 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.


image ( B ) distribution of pcMNPs ; average of nanoparticles is represented as mean ± SD of over 250 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 pcMNPs in water . ( D ) magnetic response property of bare MNPs , NH2 - MNPs 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.


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 extracted by different pcMNPs - methods . RT - PCR assays amplifying ( A ) ORF1ab region ( B ) N gene in pseudoviral 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 ORF1ab region in pseudoviral extracted by automated protocol , ( B ) RT - PCR assays amplifying N gene in pseudoviral 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 N gene in pseudoviral following Conventional RT - PCR protocol , ( B ) corresponding calibration curve .

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 N gene in pseudoviral following Direct RT - PCR protocol , ( D ) corresponding calibration curve . red is 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 SOX2 locus . promoter is defined as unmethylated region spanning 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 , ( D ) differentially methyl - ated regions ( DMRs ) between MCF7 - control 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 DMRs identified between ZF - D3A + dox MCF7 - control is : ∆ mCG ( difference in methylation level [ of C base calls to total base calls for CG dinucleotides in region , mCG / CG ] between ZF - D3A + dox 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 ) , 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 ) methylation level in MCF7 - control ZF - D3A + dox flanking ( ± 1 kb , 100 bp bins ) 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 UMRs with an intersection of ≥ 1 bp .

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


( A ) histogram of DMR methylation in each sample . Kernel density estimate smoothed scatterplot comparison of DMR methylation in MCF7 - control ( 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 ) histogram of UMR methylation in each sample . Scatterplot compari - son of UMR methylation in MCF7 - control ( 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 : methylation ( ± 2 . 5 kb ) , differences ( ΔmCG ) , classification as promoter or non - promoter located , CpG dinucleotide density ( ± 2 . 5 kb ) , histone modification ChIP - seq read density ( ± 2 . 5 kb ) ; ( H ) As in ( G ) but for 6 , 000 DMRs with lowest H3K4me3 , ordered by decreasing H3K4me1 read density , 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 ) centre of DMRs with ∆ mCG > 0 . 2 ( top ) , 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 in DMRs with ∆ mCG > 0 . 2 , from H3K4me3 ChIP - seq in ZF - D3A no - dox ZF - D3A + dox . Black represents equal normalized read in ZF - D3A no - dox ZF - D3A + dox ; blue represents a linear regression of 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 difference in H3K4me3 ChIP - seq normalized read between ZF - D3A no - dox ZF - D3A + dox . Blue represents a linear regression of 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 ) methylation of UMRs that intersect with H3K4me3 ChIP - seq peaks in ZF - D3A no - dox ZF - D3A + dox , for genomic or 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 ) methylation of UMRs that intersect with phos - pho - ser5 polymerase II ChIP - seq peaks in ZF - D3A no - dox ZF - D3A + dox , for genomic or immunoprecipitated with an anti - phospho - ser5 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 abundance upon doxycycline induction withdrawal .

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


( B ) Box whisker plots of methylation of 121 CpG sites in DACH1 promoter region through - out doxycycline induction withdrawal . Whiskers indicate 1 . 5 times interquartile range or 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 methylation level at 121 CpG sites in DACH1 promoter region throughout doxycycline induction followed by doxycycline withdrawal cycle inhibition by growth in doxycycline - free media containing different 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 hmCG in MCF7 - control ZF - D3A + dox , ordered by difference in DMR hmCG level ( hmCG / CG ) between MCF7 - control ZF - D3A + dox , representation of H3K4me3 in ZF - D3A + dox flanking ( ± 2 . 5 kb , 50 bp bins ) centre of 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 ) of hmCG flanking ( ± 2 . 5 kb , 50 bp bins ) centre of DMRs , ordered by DMR hmCG level in MCF7 - control .

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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 difference in UMR methylation , versus fold change in mRNA abun - dance of UMR - associated expressed genes , between MCF7 - control ZF - D3A + dox . Point color indicates gene differential expression significance : red indicates FDR < 0 . 01 , black indicates FDR > 0 . 01 . Trend ( green ) was fitted using a generalized additive model , contour lines represent 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 ratios of mRNA between ZF - D3A + dox MCF7 - control , for expressed genes that have an associated UMR with a ∆ mCG > 0 . 3 . Column color indicates class of mRNA abundance ratios between ZF - D3A + dox 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 methylation in ZF - D3A + dox 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.


distribution of stained with - FISH probes targeting ( A ) GAPDH or ( B ) PSME1 mRNA before 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 , ( D ) PSME1 by - seq .

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


methylation of single CpGs at promoter region of ( E ) GAPDH , ( F ) PSME1 , for low ( x axis ) high ( y axis ) expressing sorted 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 GAPDH ( top ) PSME1 ( bottom ) promoter regions showing methylation in MCF7 - control low high expressing sorted 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 ( blue ) for selected ZF - D3A + dox WGBS reads ( ≥ 10 cytosines in CpG context ) that align within robustly methylated DMRs ( DMR mean methylation level [ mCG / CG ] of 0 . 4 - 0 . 6 in ZF - D3A + dox < 0 . 1 in MCF7 - control ) , average CG methylation from ZF - D3A + dox WGBS data of DMR genomic intervals covered by selected reads ( red ) .

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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 . horizontal pink lines represent threshold used in these samples for background correction . Each dot on ddPCR output represents a unique droplet that is either positive or negative for signal . Right : of TRAP activity after normalization to SW39 / TEL + ( n = 3 ) . Data information : Data are presented as mean ± SD . Statistical analyses were performed with Student ' s two - unpaired t - 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 hTERT expression , normalized to ACTB mRNA to SW39 / TEL + . Data information : Data are presented as mean ± SD . Statistical analyses were performed with Student ' s two - unpaired t - 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 . Pol II binding at hTR promoter in SI14 / TEL + , SI24 / ALT + 8G12 / ALT + lines , normalized to H3 ChIP . Control loci with , respectively , high or low transcriptional activity : c - fos promoter subtelomeric 4qHox locus ( n = 3 ) . Statistical analyses were performed using one - way ANOVA with post - hoc Tukey ' s for multiple comparisons . Data information : Data are presented as mean ± SD . Statistical analyses were performed with Student ' s two - unpaired t - 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 H3K27me3 ( left ) or H3K4me3 H3Ac ( right ) densities at hTR promoter in SI14 / TEL + 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 - unpaired t - 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 + IMRB / ALT + normalized to H3 . Control loci with , respectively , high low NFYB binding : hnRNPA1 promoter subtelomeric 4qHox ( n = 3 ) . Data information : Data are presented as mean ± SD . Statistical analyses were performed with Student ' s two - unpaired t - with Welch ' s correction for normality of distribution unless specified otherwise . PTM , post - translational modification .

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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 in TEL + ALT + hybrids . β - Actin is used as loading control . of western blots is shown on right , normalized to β - Actin to SI14 / TEL + ( n = 2 - 4 ) . Statistical analyses were performed using one - way ANOVA with post - hoc Tukey ' s 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 , normalized to ACTB mRNA to SI14 / TEL + ( n = 3 ) . Data information : Data are presented as mean ± SD .

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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 + 8G12 / ALT + lines treated with DMSO or 10 μM MG132 for 6 h . β - Actin served as loading control . Quantifications are shown on right , normalized to β - Actin to SI14 / TEL + ( n = 5 ) . Statistical analyses were performed with Student ' s two - unpaired t - . 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 NHP2 mRNA expression , normalized to ACTB mRNA 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 in NHP2 - overexpressing 8G12 / ALT + , 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 lines . β - Actin served as loading control . SI14 / TEL + are shown as reference for NHP2 expression in hybrids . ( Bottom ) Western blot for NHP2 overexpression in SI14 / TEL + 8G12 / ALT + 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 , hTERT NHP2 mRNA expression , normalized to ACTB mRNA 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 . 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 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 , hTERT NHP2 mRNA expression , normalized to ACTB mRNA to 143B / TEL + , in a series of TEL + ALT + human 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 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 . is shown on right after normalization to β - Actin to 143B / TEL + ( n = 3 ) . Data information : Data are presented as mean ± SD . Statistical analyses were performed using one - way ANOVA with post - hoc Tukey ' s 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.


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

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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 early passages of hTR - expressing IMRB or U2OS , 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 . of doubling time for U2OS , U2OS - hTR , IMRB IMRB - hTR lines ( n = 3 ) . Statistical analyses were performed with Student ' s two - unpaired t - with Welch ' s correction for normality of distribution Data information : Data are shown as mean ± SD , with red marking 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 U2OS - hTR treated with or without Hydroxyurea ( 2 mM for 24 h ) , for indicated . β - Actin 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 for co - localization between p - RPA32S33 ( green ) telomeres ( red FISH probe ) . arrows indicate co - localization events . Scale bars : 5 μm . is shown on right . At least 50 nuclei were scored in each of three independent experiments performed . Each dot represents number of p - RPA32S33 - telomere co - localizations in one nucleus . Statistical analyses were performed with Student ' s two - unpaired t - with Welch ' s correction for normality of distribution . Data information : Data are shown as mean ± SD , with red marking 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 . of live numbers with trypan blue manual counting , 72 h post - transfection with siLuc , sihTR # 1 or sihTR # 2 , measured as of siLuc - treated ( n = 3 ) . On right , representative of U2OS treated with sihTR # 1 for 72 h . Statistical analyses were performed using one - way ANOVA with post - hoc Tukey ' s for multiple comparisons . Data information : Data are shown as mean ± SD , with red marking 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 indicated lines transfected for 48 h with siLuc or sihTR , with indicated antibodies . U2OS treated with Hydroxyurea ( 2 mM for 24 h ) were used as positive 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 B-CONTROLLED_VAR O O O O O O O O O O O O O

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


A . Representative for fragile telomeres using CO - FISH . Lagging strands ( C - probe ) leading strands ( G - probe ) were marked with green PNA probes red LNA probes , respectively . circles arrows indicate " smeary " or " doublet " telomeric signals , respectively . of fragile telomeres is shown on right as of total telomeres counted ( n = 932 - 1073 ) . Comparison of fragile to non - fragile telomere was performed using Χ2 - . Data information : 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 for co - localization between RAD51 ( green ) telomeres ( red FISH probe ) . arrows indicate representative RAD51 - telomere co - localization events . Quantifications are shown on right . A total of 120 nuclei were scored from two independent experiments . Data information : scale bars : 5 μm . Data are shown as mean ± SD . B , red marks mean value . Statistical analyses were performed with Student ' s two - unpaired t - 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 for TIF : co - localization between 53BP1 ( green ) telomeres ( red FISH probe ) in U2OS or IMRB with or without ectopic hTR overexpression . arrows indicate TIF . of TIF is on right . A total of 110 - 170 nuclei were scored from three independent experiments . Each dot represents number of TIF per nucleus . Data information : scale bars : 5 μm . Data are shown as mean ± SD . , C , red marks mean value . Statistical analyses were performed with Student ' s two - unpaired t - 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 for TIF , as in ( A ) , in U2OS or U2OS - hTR , treated as indicated with DMSO or 5 μM ATRi or 5 μM ATMi for 20 h . arrows indicate TIF . Data information : 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 . of TIF from ( D ) . At least 65 nuclei were scored in each of two independent experiments performed . Each dot represents number of TIF per nucleus . Statistical analyses were performed using one - way ANOVA with post - hoc Games - Howell ' s for multiple comparisons . Data information : scale bars : 5 μm . Data are shown as mean ± SD . red marks mean value . Statistical analyses were performed with Student ' s two - unpaired t - with Welch ' s correction for normality of distribution , unless specified otherwise .

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 for co - localization between POLD3 ( red ) RAP1 ( green ) . arrows indicate co - localization events . of co - localization events between POLD3 RAP1 is shown on right . A total of 190 - 230 nuclei were scored from three independent experiments . Data information : scale bars : 5 μm . Data are shown as mean ± SD . F : red marks mean value . Statistical analyses were performed with Student ' s two - unpaired t - 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 using slot - blot hybridized with radioactive C - rich telomeric probe . is shown below ( n = 7 ) . Statistical analyses were performed using non - parametrical Mann Whitney . Data information : scale bars : 5 μm . Data are shown as mean ± SD .

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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 indicated 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 with defective fork reversal activity ( Zhang et al , 2019b ) . Data information : 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 for co - localization between PML ( green ) telomeres ( red FISH probe ) . arrows indicate co - localization events , marking APBs . of is shown on right . A total of 85 - 300 nuclei were scored from four independent experiments . Each dot represents number of events per nucleus : PML foci or APBs . Data information : scale bars : 5 μm . Data are shown as mean ± SD , with red marking mean value . Statistical analyses were performed with Student ' s two - unpaired t - with Welch ' s correction for normality of distribution .

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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 for co - localization between EdU ( green ) telomeres ( red FISH probe ) within PML bodies ( cyan ) in U2OS U2OS - hTR . arrows indicate co - localization events . D , E . of shown in ( C ) . Co - localization events between EdU foci APBs were analyzed ( D ) 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 : scale bars : 5 μm . Data are shown as mean ± SD , with red marking mean value . Statistical analyses were performed with Student ' s two - unpaired t - with Welch ' s correction for normality of distribution .

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

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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 for co - localization between RPA32 or p - RPA32S33 ( green ) telomeres ( red FISH probe ) , in indicated lines treated with siLuc or sihnRNPA1 for 48 h . arrows indicate co - localization events . Data information : scale bars : 5 μm . Data are shown as mean ± SD , with red marking mean value . Statistical analyses were performed with Student ' s two - unpaired t - 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.


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


F . Representative for co - localization between RPA32 ( green ) telomeres ( red FISH probe ) in indicated lines treated with either DMSO or 10 μM NU7026 - PK - inhibitor ( - PK - i ) for 48 h . arrows indicate co - localization events . Quantifications are shown on 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 for multiple comparisons . Data information : scale bars : 5 μm . Data are shown as mean ± SD , with red marking mean value . Statistical analyses were performed with Student ' s two - unpaired t - with Welch ' s correction for normality of distribution unless specified otherwise .

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

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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 in indicated treated with siLuc or siNHP2 , 72 h post - transfection . β - Actin served as loading control .

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

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

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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 U2OS - hTR treated with indicated siRNAs , 72 h post - transfection to detect total using indicated antibodies . β - Actin Vinculin served as loading controls .

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

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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 . of shown in ( F ) . Each dot represents 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 : scale bars : 5 μm . Data are shown as mean ± SD , with red marking mean value .

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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 of YAP phosphorylated YAP ( Ser127 ) in control ( si - Ctrl ) YAP knockdown ( si - YAP ) hOSEs . were detected by Western blotting . β - Actin was used as a loading control . Right panel : proliferation ( number ) in control ( si - Ctrl ) YAP knockdown ( si - YAP ) hOSE .

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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 of YAP phosphorylated YAP ( Ser127 ) in control ( si - Ctrl ) , YAP knockdown ( si - YAP ) , TAZ knockdown ( si - TAZ ) YAP / TAZ double knockdown ( si - YAP / TAZ ) hOSE . β - Actin was used as a loading control . Right panel : proliferation ( number ) of control ( si - Ctrl ) , YAP knockdown ( si - YAP ) , TAZ knockdown ( si - TAZ ) YAP / TAZ double knockdown ( si - YAP & si - TAZ ) hOSE .

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

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

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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 showing morphologic changes of hOSE - MXIV , hOSE - YAP hOSE - YAPS127A at passage 7 . Scale bar : 50 µm .

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


Representative showing expression location of YAP in hOSE - MXIV , hOSE - YAP hOSE - YAPS127A 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 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 showing SA - β - gal in hOSE - MXIV , hOSE - YAP hOSE - YAPS127A 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 of SA - β - gal positive in hOSE - MXIV , hOSE - YAP hOSE - YAPS127A .

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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 associated with cycle arrest in hOSE - MXIV , hOSE - YAP hOSE - YAPS127A analyzed using Western blotting at 7th passage . β - Actin was used as a loading control .

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

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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 RB1 expression in hOSE - MXIV , hOSE - YAP hOSE - YAPS127A in presence or absence of HPV E6 / E7 oncoproteins at 7th passage . β - Actin was used as a loading control .

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 hOSE - YAPS127A in presence of HPV E6 / E7 oncoproteins at 9th passage . Each point represents mean ± of four independent samples .

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

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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 of SA - β - gal positive in hOSE - MXIV , hOSE - YAP hOSE - YAPS127A in 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 showed SA - β - gal in cultured hOSE in presence or absence of HPV E6 / E7 oncoproteins . were collected stained at 13th passage . Scale bar : 25 µm . Right graph showing of SA - β - gal positive in P13 hOSE transfected with empty vectors ( CTRL - P13 ) or vectors expressing E6 / E7 oncoproteins ( E6 / E7 - P13 ) .

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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 expression of YAP RB1 in hOSE - MXIV , hOSE - YAP hOSE - YAPS127A with or without pRB knockdown . were collected at 7th passage were analyzed using Western blotting . β - Actin was used as a loading control .

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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 hOSE - YAPS127A after RB1 knockdown .

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


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

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 YAP in hOSEs at fourth , ninth thirteenth passage . β - Actin was used as a loading control .

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

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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.