A Atf7ip KO cells show increased expression of SETDB1 - regulated ERVs and the provirus reporter , MSCV - GFP as evidenced by RT - qPCR analysis . RNA expression was normalized to Hprt expression and is shown relative to the level in WT cells . Data are mean ± SD ; n = 3 , technical replicates .

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A Atf7ip KO cells show increased expression of SETDB1-regulated ERVs and the provirus reporter, MSCV-GFP as evidenced by RT-qPCR analysis. RNA expression was normalized to Hprt expression and is shown relative to the level in WT cells. Data are mean ± SD; n=3, technical replicates.


B Atf7ip KO mESCs ( TT # 2 - 12 ) show decreased H3K9me3 at the LTR of the SETDB1 - regulated reporter and the ERVs , as evidenced by Native ChIP followed by qPCR analysis . Gapdh was used as a negative control . Data are mean ± SEM ; n = 5 , independent experiments . * P < 0 . 05 and * * P < 0 . 01 by paired t - test .

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B Atf7ip KO mESCs (TT#2-12) show decreased H3K9me3 at the LTR of the SETDB1-regulated reporter and the ERVs, as evidenced by Native ChIP followed by qPCR analysis. Gapdh was used as a negative control. Data are mean ± SEM; n=5, independent experiments. *P < 0.05 and **P < 0.01 by paired t-test.


C SETDB1 cellular amount is mostly maintained in Atf7ip KO mESCs . Endogenous SETDB1 - ATF7IP interaction is validated by anti - ATF7IP antibody co - IP experiment with anti - ATF7IP antibody .

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C SETDB1 cellular amount is mostly maintained in Atf7ip KO mESCs. Endogenous SETDB1-ATF7IP interaction is validated by anti-ATF7IP antibody co-IP experiment with anti-ATF7IP antibody.


A IF analysis shows preferential cytoplasmic localization of SETDB1 in Atf7ip KO mESCs . Representative projected images are shown , and the quantitative analyses are shown in B - D . Scale bar : 10 µm . B SETDB1 signal in the nucleus that was determined by DAPI staining was calculated . The mean from three independent experiments is shown as a bar - graph with jittered points indicating the average % intensity of each experiment . Over 100 cells were analysed per sample per experiment . * * * P < 0 . 001 by unpaired Student ' s t - test . C , D SETDB1 foci in the nucleus that was determined by DAPI staining were calculated . Violin plot for SETDB1 ' s foci numbers from all the cells analysed in three independent experiments is shown in c . The mean from the three independent experiments is shown as a bar with jittered points indicating the average number of each experiment in D . Over 100 cells were analysed per sample per experiment . * P < 0 . 05 by unpaired Student ' s t - test .

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

A IF analysis shows preferential cytoplasmic localization of SETDB1 in Atf7ip KO mESCs. Representative projected images are shown, and the quantitative analyses are shown in B-D. Scale bar: 10 µm. B SETDB1 signal in the nucleus that was determined by DAPI staining was calculated. The mean from three independent experiments is shown as a bar-graph with jittered points indicating the average % intensity of each experiment. Over 100 cells were analysed per sample per experiment. ***P < 0.001 by unpaired Student's t-test. C, D SETDB1 foci in the nucleus that was determined by DAPI staining were calculated. Violin plot for SETDB1's foci numbers from all the cells analysed in three independent experiments is shown in c. The mean from the three independent experiments is shown as a bar with jittered points indicating the average number of each experiment in D. Over 100 cells were analysed per sample per experiment. *P < 0.05 by unpaired Student's t-test.


E , IF analysis shows preferential cytoplasmic localization of SETDB1 in Atf7ip KO mESCs . Representative projected images are shown . Arrows indicate the region used for line - plot analysis . Cells were treated with 10 ng / mL LMB or mock for 5 h before analysis . Scale bar : 10 µm . F The line - plot analysis for E . Relative intensities of SETDB1 signal were measured by Image J . G The quantitative analysis for E is shown . SETDB1 signal in the nucleus that was determined by DAPI staining was calculated . The mean from three independent experiments is shown as a bar - graph with jittered points indicating the average % intensity of each experiment . Over 100 cells were analysed per sample per experiment . NS : P > 0 . 05 , * * P < 0 . 01 versus " Mock , WT " group by Dunnett ' s test .

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

E, IF analysis shows preferential cytoplasmic localization of SETDB1 in Atf7ip KO mESCs. Representative projected images are shown. Arrows indicate the region used for line-plot analysis. Cells were treated with 10 ng/mL LMB or mock for 5 h before analysis. Scale bar: 10 µm. F The line-plot analysis for E. Relative intensities of SETDB1 signal were measured by Image J. G The quantitative analysis for E is shown. SETDB1 signal in the nucleus that was determined by DAPI staining was calculated. The mean from three independent experiments is shown as a bar-graph with jittered points indicating the average % intensity of each experiment. Over 100 cells were analysed per sample per experiment. NS: P > 0.05, **P < 0.01 versus "Mock, WT" group by Dunnett's test.


A ATF7IP KO HEK293T cells shows endogenous SETDB1 cytoplasmic localization phenotype . Endogenous SETDB1 in HEK293T cells shows cytoplasm + nuclear localization profile . Scale bar : 10 µm .

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A ATF7IP KO HEK293T cells shows endogenous SETDB1 cytoplasmic localization phenotype. Endogenous SETDB1 in HEK293T cells shows cytoplasm+nuclear localization profile. Scale bar: 10 µm.


B Over - production of exogenous mouse SETDB1 induces endogenous SETDB1 nuclear localization in both WT and ATF7IP KO HEK293T cells . The representative data of two independent experiments is shown . Scale bar : 10 µm .

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B Over-production of exogenous mouse SETDB1 induces endogenous SETDB1 nuclear localization in both WT and ATF7IP KO HEK293T cells. The representative data of two independent experiments is shown. Scale bar: 10 µm.


C LMB treatment enhances SETDB1 nuclear accumulation in WT HEK293T cells , but no significant impact for ATF7IP KO HEK293T cells . Cells were treated with 10ng / ml of LMB for 5 h and then analysed with immunofluorescent staining . The representative data of three independent experiments is shown . Scale bar : 10 µm . D SETDB1 signal in the nucleus shown in C was calculated . The mean from three independent experiments is shown as a bar - graph ± SEM ; n = 3 . Over 50 cells were analysed per sample per experiment . * * P < 0 . 01 by Student ' s t - test ( two - sided test ) .

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

C LMB treatment enhances SETDB1 nuclear accumulation in WT HEK293T cells, but no significant impact for ATF7IP KO HEK293T cells. Cells were treated with 10ng/ml of LMB for 5 h and then analysed with immunofluorescent staining. The representative data of three independent experiments is shown. Scale bar: 10 µm. D SETDB1 signal in the nucleus shown in C was calculated. The mean from three independent experiments is shown as a bar-graph ± SEM; n=3. Over 50 cells were analysed per sample per experiment. **P < 0.01 by Student's t-test (two-sided test).


E 5 h of proteasome inhibitor bortezomib treatment do not have much impact on SETDB1 relative nuclear accumulation in ATF7IP KO HEK293T cells . The representative data of three independent experiments is shown . Scale bar : 10 µm . F SETDB1 signal in the nucleus shown in E was calculated . The mean from three independent experiments is shown as a bar - graph ± SEM ; n = 3 . Over 50 cells were analysed per sample per experiment . Statistics comparison was only shown between without and with drug treatment in WT and ATF7IP KO cells . * P < 0 . 05 and * * P < 0 . 01 by Dunnett ' s method .

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

E 5 h of proteasome inhibitor bortezomib treatment do not have much impact on SETDB1 relative nuclear accumulation in ATF7IP KO HEK293T cells. The representative data of three independent experiments is shown. Scale bar: 10 µm. F SETDB1 signal in the nucleus shown in E was calculated. The mean from three independent experiments is shown as a bar-graph ± SEM; n=3. Over 50 cells were analysed per sample per experiment. Statistics comparison was only shown between without and with drug treatment in WT and ATF7IP KO cells. *P < 0.05 and **P < 0.01 by Dunnett's method.


A HEK293T cells were transfected with V5 - SETDB1 and either empty or 3xF - ATF7IP expressing vector . Over 50 transfected cells analysed per sample were analyzed . The representative data of two independent experiments is shown . Scale bar : 10 µm .

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A HEK293T cells were transfected with V5-SETDB1 and either empty or 3xF-ATF7IP expressing vector. Over 50 transfected cells analysed per sample were analyzed. The representative data of two independent experiments is shown. Scale bar: 10 µm.


B HEK293T cells were transfected with V5 - SETDB1 d1 mutant and either empty or 3xF - ATF7IP expressing vector . Over 50 transfected cells per sample were analysed . The representative data of two independent experiments was shown . Scale bar : 10 µm . The yellow arrowheads indicate the cells that show nuclear ( and cytoplasmic ) - localization of introduced exogenous mSETDB1 .

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B HEK293T cells were transfected with V5-SETDB1 d1 mutant and either empty or 3xF-ATF7IP expressing vector. Over 50 transfected cells per sample were analysed. The representative data of two independent experiments was shown. Scale bar: 10 µm. The yellow arrowheads indicate the cells that show nuclear (and cytoplasmic)-localization of introduced exogenous mSETDB1.


A Co - transfection with ATF7IP augments the K885 ubiquitination of SETDB1 . HEK293T cells were transfected After the transfection , V5 - SETDB1 was immune - purified with anti - V5 antibody and used for WB analysis .

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A Co-transfection with ATF7IP augments the K885 ubiquitination of SETDB1. HEK293T cells were transfected After the transfection, V5-SETDB1 was immune-purified with anti-V5 antibody and used for WB analysis.


B ATF7IP regulates the ubiquitination of SETDB1 in mESCs . Endogenous SETDB1 was immune - purified from WT or Atf7ip KO mESCs cells and used for WB analysis .

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B ATF7IP regulates the ubiquitination of SETDB1 in mESCs. Endogenous SETDB1 was immune-purified from WT or Atf7ip KO mESCs cells and used for WB analysis.


C LMB treatment increased the relative intensity of the upper ubiquitinated upper band of SETDB1 in Atf7ip KO mESCs . Cells were treated with 10 ng / mL LMB or mock for 5 h before analysis .

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C LMB treatment increased the relative intensity of the upper ubiquitinated upper band of SETDB1 in Atf7ip KO mESCs. Cells were treated with 10 ng/mL LMB or mock for 5 h before analysis.


D HEK293T cells were transfected with either 3xF - or 3xF - NLS - SETDB1 . Over 50 transfected cells per sample were analysed . The representative data of two independent experiments is shown . Scale bar : 10 µm .

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D HEK293T cells were transfected with either 3xF- or 3xF-NLS-SETDB1. Over 50 transfected cells per sample were analysed. The representative data of two independent experiments is shown. Scale bar: 10 µm.


E Nuclear localization itself promotes the ubiquitination of SETDB1 . HEK293T cells were transfected with 3xFLAG - or 3xFLAG - NLS - SETDB1 . After FLAG - IP , the purified SETDB1s were analyzed by WB .

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E Nuclear localization itself promotes the ubiquitination of SETDB1. HEK293T cells were transfected with 3xFLAG- or 3xFLAG-NLS-SETDB1. After FLAG-IP, the purified SETDB1s were analyzed by WB.


F , G IF analysis was performed with Atf7ip KO mESCs stably expressing 3xF - or 3xF - NLS - SETDB1 . Representative images are shown in F , and the quantitative analysis is shown in G . Scale bar : 10 µm . Addition of NLS increases nuclear localization of SETDB1 in Atf7ip KO mESCs . The mean from three independent experiments is shown as a bar - graph with jittered points indicating the average % intensity of each experiment . Over 50 cells were analysed per sample per experiment . * * * P < 0 . 001 by unpaired Student ' s t - test .

O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O B-CELL_TYPE O O O O O O O O O B-GENEPROD O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O 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, G IF analysis was performed with Atf7ip KO mESCs stably expressing 3xF- or 3xF-NLS-SETDB1. Representative images are shown in F, and the quantitative analysis is shown in G. Scale bar: 10 µm. Addition of NLS increases nuclear localization of SETDB1 in Atf7ip KO mESCs. The mean from three independent experiments is shown as a bar-graph with jittered points indicating the average % intensity of each experiment. Over 50 cells were analysed per sample per experiment. ***P < 0.001 by unpaired Student's t-test.


H Nuclear localization promotes the ubiquitination of SETDB1 in Atf7ip KO mESCs . Atf7ip KO mESCs expressing 3xF - or 3xF - NLS - SETDB were used for FLAG - IP , and the purified SETDB1s were analyzed by WB .

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H Nuclear localization promotes the ubiquitination of SETDB1 in Atf7ip KO mESCs. Atf7ip KO mESCs expressing 3xF- or 3xF-NLS-SETDB were used for FLAG-IP, and the purified SETDB1s were analyzed by WB.


I , J . Normal ciliary formation of Rabl2 - / - MEFs . MEFs derived from E12 . 5 mouse embryos were serum - starved for 48 h to induce primary cilia ( I ) . Statistical results ( J ) were from three independent experiments . Ciliogenesis of at least 200 cells was scored in each experiment and condition . Ciliary lengths of 80 cells were measured in each experiment and condition and the results of the same genotypes were pooled together . K , L . Rabl2 deficiency did not repress ciliary localization of IFT - B . Ciliated MEFs were immunostained for Ift81 and Ift57 , two IFT - B subunits ( K ; see Fig 4F for diagram of IFT - B ) . Relative intensity of ciliary Ift81 ( L ) , presented in arbitrary unit , was obtained by normalizing to that of corresponding Ac - tub . Four MEF strains prepared respectively from two embryos per genotype were used for quantification . 50 cilia were quantified from each MEF strain and results of the same genotypes were pooled together .

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

I, J. Normal ciliary formation of Rabl2-/- MEFs. MEFs derived from E12.5 mouse embryos were serum-starved for 48 h to induce primary cilia (I). Statistical results (J) were from three independent experiments. Ciliogenesis of at least 200 cells was scored in each experiment and condition. Ciliary lengths of 80 cells were measured in each experiment and condition and the results of the same genotypes were pooled together. K, L. Rabl2 deficiency did not repress ciliary localization of IFT-B. Ciliated MEFs were immunostained for Ift81 and Ift57, two IFT-B subunits (K; see Fig 4F for diagram of IFT-B). Relative intensity of ciliary Ift81 (L), presented in arbitrary unit, was obtained by normalizing to that of corresponding Ac-tub. Four MEF strains prepared respectively from two embryos per genotype were used for quantification. 50 cilia were quantified from each MEF strain and results of the same genotypes were pooled together.


I , J . Normal ciliary formation of Rabl2 - / - MEFs . MEFs derived from E12 . 5 mouse embryos were serum - starved for 48 h to induce primary cilia ( I ) . Statistical results ( J ) were from three independent experiments . Ciliogenesis of at least 200 cells was scored in each experiment and condition . Ciliary lengths of 80 cells were measured in each experiment and condition and the results of the same genotypes were pooled together .

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

I, J. Normal ciliary formation of Rabl2-/- MEFs. MEFs derived from E12.5 mouse embryos were serum-starved for 48 h to induce primary cilia (I). Statistical results (J) were from three independent experiments. Ciliogenesis of at least 200 cells was scored in each experiment and condition. Ciliary lengths of 80 cells were measured in each experiment and condition and the results of the same genotypes were pooled together.


D , E . Increased Gli3 FL / R ratios in E11 . 5 hind limb buds and E10 . 5 mouse embryos . Relative band intensities of the full - length ( FL ) and the repressor ( R ) forms of Gli3 were quantified ( red ) . FL / R ratios were from four different sets of limb bud preparations ( D ) and three different sets of embryo preparations ( E ) .

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

D, E. Increased Gli3 FL/R ratios in E11.5 hind limb buds and E10.5 mouse embryos. Relative band intensities of the full-length (FL) and the repressor (R) forms of Gli3 were quantified (red). FL/R ratios were from four different sets of limb bud preparations (D) and three different sets of embryo preparations (E).


Rabl2Q80L inhibited Hh signaling . MEFs from Rabl2 - / - E12 . 5 embryos infected with lentivirus to express GFP , GFP - Rabl2 or GFP - Rabl2Q80L were serum - starved for 24 h and treated with DMSO ( mock ) or 100 nM SAG for an additional 24 h . Uninfected MEFs from the wildtype littermate embryos served as the positive control . The samples were immunostained to examine ciliary Gli2 The white arrowheads ( G ) point to ciliary tips . Quantification results were from three independent experiments , each using MEFs from different Rabl2 - deficient embryos . Gli2 intensities , presented in arbitrary unit , were measured from at least 22 cilia in each experiment and condition and pooled together .

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

Rabl2Q80L inhibited Hh signaling. MEFs from Rabl2-/- E12.5 embryos infected with lentivirus to express GFP, GFP-Rabl2 or GFP-Rabl2Q80L were serum-starved for 24 h and treated with DMSO (mock) or 100 nM SAG for an additional 24 h. Uninfected MEFs from the wildtype littermate embryos served as the positive control. The samples were immunostained to examine ciliary Gli2 The white arrowheads (G) point to ciliary tips. Quantification results were from three independent experiments, each using MEFs from different Rabl2-deficient embryos. Gli2 intensities, presented in arbitrary unit, were measured from at least 22 cilia in each experiment and condition and pooled together.


H . Rabl2Q80L inhibited Hh signaling . MEFs from Rabl2 - / - E12 . 5 embryos infected with lentivirus to express GFP , GFP - Rabl2 or GFP - Rabl2Q80L were serum - starved for 24 h and treated with DMSO ( mock ) or 100 nM SAG for an additional 24 h . Uninfected MEFs from the wildtype littermate embryos served as the positive control . The samples were immunoblotted to examine levels of Gli1 and Gli3 In the samples for immunoblotting ( H ) , GFP - positive cells occupied 82 . 3 % ( GFP ) , 87 . 3 % ( GFP - Rabl2 ) , and 81 . 2 % ( GFP - Rabl2Q80L ) , respectively , when 260 cells in each sample were scored . Relative band intensities are shown in red .

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

H. Rabl2Q80L inhibited Hh signaling. MEFs from Rabl2-/- E12.5 embryos infected with lentivirus to express GFP, GFP-Rabl2 or GFP-Rabl2Q80L were serum-starved for 24 h and treated with DMSO (mock) or 100 nM SAG for an additional 24 h. Uninfected MEFs from the wildtype littermate embryos served as the positive control. The samples were immunoblotted to examine levels of Gli1 and Gli3 In the samples for immunoblotting (H), GFP-positive cells occupied 82.3% (GFP), 87.3% (GFP-Rabl2), and 81.2% (GFP-Rabl2Q80L), respectively, when 260 cells in each sample were scored. Relative band intensities are shown in red.


I . Association of endogenous Rabl2 - GTP with IFT - B . Mouse testis lysates with or without 1 mM GTPγS were subjected to sucrose gradient ultracentrifugation . Fractions were collected from top ( low MW ) to bottom ( high MW ) . Note the obvious enrichment of Rabl2 in fraction # 16 in the presence of GTPγS .

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I. Association of endogenous Rabl2-GTP with IFT-B. Mouse testis lysates with or without 1 mM GTPγS were subjected to sucrose gradient ultracentrifugation. Fractions were collected from top (low MW) to bottom (high MW). Note the obvious enrichment of Rabl2 in fraction #16 in the presence of GTPγS.


D , E . Ciliary accumulation of BBS proteins in Rabl2Q80L - expressing RPE1 cells . The white arrows ( D ) point to positions of cilia . Quantification results ( E ) were from three independent experiments . At least 100 GFP - positive cells were scored in each experiment and condition .

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D, E. Ciliary accumulation of BBS proteins in Rabl2Q80L-expressing RPE1 cells. The white arrows (D) point to positions of cilia. Quantification results (E) were from three independent experiments. At least 100 GFP-positive cells were scored in each experiment and condition.


H , I . BBSomes and IFT - B were able to reach the ciliary base through retrograde IFT in the presence of Rabl2Q80L . Ciliary 3NG - Bbs5 ( H ) or Ift27 - GFP ( I ) in IMCD3 cells co - expressing RFP - Rabl2 or RFP - Rabl2Q80L was live imaged at 4 fps . Snapshots for the ciliary RFP proteins , the first frames from Movies EV2 - EV5 , corresponding kymographs , and quantification results from the indicated numbers of cilia are presented . For processivity analysis ( I ) , only traceable puncta were scored .

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

H, I. BBSomes and IFT-B were able to reach the ciliary base through retrograde IFT in the presence of Rabl2Q80L. Ciliary 3NG-Bbs5 (H) or Ift27-GFP (I) in IMCD3 cells co-expressing RFP-Rabl2 or RFP-Rabl2Q80L was live imaged at 4 fps. Snapshots for the ciliary RFP proteins, the first frames from Movies EV2-EV5, corresponding kymographs, and quantification results from the indicated numbers of cilia are presented. For processivity analysis (I), only traceable puncta were scored.


E , F . Rabl2D73GQ80L did not repress ciliary translocation of SMO and GPR161 . RPE1 cells expressing GFP - tagged Rabl2Q80L or Rabl2D73GQ80L were serum - starved for 24 h and treated with DMSO ( mock ) or SAG for an additional 24 h prior to immunostaining ( E ) . The white arrows ( E ) point to positions of cilia . In quantification results ( F ) , at least 100 cells were scored in each experiment and condition .

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E, F. Rabl2D73GQ80L did not repress ciliary translocation of SMO and GPR161. RPE1 cells expressing GFP-tagged Rabl2Q80L or Rabl2D73GQ80L were serum-starved for 24 h and treated with DMSO (mock) or SAG for an additional 24 h prior to immunostaining (E). The white arrows (E) point to positions of cilia. In quantification results (F), at least 100 cells were scored in each experiment and condition.


G . Ciliary Rabl2D73GQ80L did not repress the BBSome export . FLIP assays were performed in RPE1 cells co - expressing 3NG - Bbs5 with RFP - tagged Rabl2Q80L or Rabl2D73GQ80L as depicted in Fig 5F . Photobleaching was executed at t = 0 . Quantification results on half - life ( t1 / 2 ) were from 10 cilia for each condition .

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G. Ciliary Rabl2D73GQ80L did not repress the BBSome export. FLIP assays were performed in RPE1 cells co-expressing 3NG-Bbs5 with RFP-tagged Rabl2Q80L or Rabl2D73GQ80L as depicted in Fig 5F. Photobleaching was executed at t = 0. Quantification results on half-life (t1/2) were from 10 cilia for each condition.


F , G . Endogenous Rabl2Q80L entered ciliary shaft to repress the export of the BBSome ( Bbs5 ) and its cargo GPCR ( Smo ) . mEPCs cultured from Rabl2KI / KI and wildtype neonatal mice were fixed at day 7 post serum starvation . Anti - Rabl2 antibody was used to visualize Rabl2 and Rabl2Q80L . Ac - tub or Arl13b served as ciliary marker . Representative micrographs of multicilia ( F ) and primary cilia ( G ) are shown . The white arrows point to positions of cilia .

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F, G. Endogenous Rabl2Q80L entered ciliary shaft to repress the export of the BBSome (Bbs5) and its cargo GPCR (Smo). mEPCs cultured from Rabl2KI/KI and wildtype neonatal mice were fixed at day 7 post serum starvation. Anti-Rabl2 antibody was used to visualize Rabl2 and Rabl2Q80L. Ac-tub or Arl13b served as ciliary marker. Representative micrographs of multicilia (F) and primary cilia (G) are shown. The white arrows point to positions of cilia.


A qRT - PCR analysis of LINC00941 repression during keratinocyte differentiation in primary keratinocyte cultures from three different donors compared to undifferentiated keratinocytes ( D0 ) ( n = 6 biological replicates ) . Data is presented as mean ± SD .

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A qRT-PCR analysis of LINC00941 repression during keratinocyte differentiation in primary keratinocyte cultures from three different donors compared to undifferentiated keratinocytes (D0) (n = 6 biological replicates). Data is presented as mean ± SD.


siPool mediated LINC00941 knockdown ( B ) in calcium - induced keratinocyte differentiation ( n = 3 biological replicates / knockdown group ) . Data is presented as mean ± SD . Statistical significance was tested by an unpaired t - test and corrected for multiple testing after Bonferroni ( * = adj . p - value < 0 . 05 , * * = adj . p - value < 0 . 01 , * * * = adj . p ‑ value < 0 . 001 , and n . s . = not significant ) .

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siPool mediated LINC00941 knockdown (B) in calcium-induced keratinocyte differentiation (n = 3 biological replicates/knockdown group). Data is presented as mean ± SD. Statistical significance was tested by an unpaired t-test and corrected for multiple testing after Bonferroni (* = adj. p-value < 0.05, ** = adj. p-value < 0.01, *** = adj. p‑value < 0.001, and n.s. = not significant).


siPool mediated LINC00941 knockdown in calcium - induced keratinocyte differentiation , results in increased abundance of early and late differentiation marker transcripts on day 3 of differentiation ( C ) ( n = 3 biological replicates / knockdown group ) . Data is presented as mean ± SD . Statistical significance was tested by an unpaired t - test and corrected for multiple testing after Bonferroni ( * = adj . p - value < 0 . 05 , * * = adj . p - value < 0 . 01 , * * * = adj . p ‑ value < 0 . 001 , and n . s . = not significant ) .

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siPool mediated LINC00941 knockdown in calcium-induced keratinocyte differentiation, results in increased abundance of early and late differentiation marker transcripts on day 3 of differentiation (C) (n = 3 biological replicates/knockdown group). Data is presented as mean ± SD. Statistical significance was tested by an unpaired t-test and corrected for multiple testing after Bonferroni (* = adj. p-value < 0.05, ** = adj. p-value < 0.01, *** = adj. p‑value < 0.001, and n.s. = not significant).


LINC00941 knockdown in day 3 ( D3 ) organotypic epidermal tissue cultures ( D ) ( n = 3 - 4 tissue cultures / knockdown group ) . Data is presented as mean ± SD . Statistical significance was tested by an unpaired t - test and corrected for multiple testing after Bonferroni ( * = adj . p - value < 0 . 05 , * * = adj . p - value < 0 . 01 , * * * = adj . p ‑ value < 0 . 001 , and n . s . = not significant ) .

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LINC00941 knockdown in day 3 (D3) organotypic epidermal tissue cultures (D) (n = 3-4 tissue cultures/knockdown group). Data is presented as mean ± SD. Statistical significance was tested by an unpaired t-test and corrected for multiple testing after Bonferroni (* = adj. p-value < 0.05, ** = adj. p-value < 0.01, *** = adj. p‑value < 0.001, and n.s. = not significant).


LINC00941 knockdown in day 3 ( D3 ) organotypic epidermal tissue cultures results in increased expression of key differentiation genes ( E ) ( n = 3 - 4 tissue cultures / knockdown group ) . Data is presented as mean ± SD . Statistical significance was tested by an unpaired t - test and corrected for multiple testing after Bonferroni ( * = adj . p - value < 0 . 05 , * * = adj . p - value < 0 . 01 , * * * = adj . p ‑ value < 0 . 001 , and n . s . = not significant ) .

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LINC00941 knockdown in day 3 (D3) organotypic epidermal tissue cultures results in increased expression of key differentiation genes (E) (n = 3-4 tissue cultures/knockdown group). Data is presented as mean ± SD. Statistical significance was tested by an unpaired t-test and corrected for multiple testing after Bonferroni (* = adj. p-value < 0.05, ** = adj. p-value < 0.01, *** = adj. p‑value < 0.001, and n.s. = not significant).


F Immunofluorescence analysis shows increased levels of early and late differentiation proteins . Collagen VII ( col VII , green ) stain indicates the basement membrane , nuclei are shown in blue and the differentiation proteins Keratin 10 and Loricrin are shown in red ( n = 3 - 4 tissue cultures / knockdown group ; one exemplary picture for each group is depicted ) . Scale bar : 50 µm .

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F Immunofluorescence analysis shows increased levels of early and late differentiation proteins. Collagen VII (col VII, green) stain indicates the basement membrane, nuclei are shown in blue and the differentiation proteins Keratin 10 and Loricrin are shown in red (n = 3-4 tissue cultures/knockdown group; one exemplary picture for each group is depicted). Scale bar: 50 µm.


B LINC00941 knockdown efficiency ( siLINC00941 ) in epidermal tissue on day 3 of differentiation as obtained by qRT - PCR measurements ( n = 4 - 5 epidermal tissue cultures / knockdown group ) . Data is presented as mean ± SD . Statistical significance was tested by an unpaired t - test and corrected for multiple testing after Bonferroni ( * = adj . p - value < 0 . 05 , * * = adj . p - value < 0 . 01 , * * * = adj . p ‑ value < 0 . 001 , and n . s . = not significant ) .

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B LINC00941 knockdown efficiency (siLINC00941) in epidermal tissue on day 3 of differentiation as obtained by qRT-PCR measurements (n = 4-5 epidermal tissue cultures/knockdown group). Data is presented as mean ± SD. Statistical significance was tested by an unpaired t-test and corrected for multiple testing after Bonferroni (* = adj. p-value < 0.05, ** = adj. p-value < 0.01, *** = adj. p‑value < 0.001, and n.s. = not significant).


C Heatmap of differentially expressed genes ( padj < 0 . 05 and - 1 > log2FC > 1 ) upon LINC00941 depletion on day 3 in organotypic epidermal tissue with marked keratinocyte differentiation genes ( n = 4 - 5 epidermal tissue cultures / knockdown group ) .

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C Heatmap of differentially expressed genes (padj < 0.05 and -1 > log2FC > 1) upon LINC00941 depletion on day 3 in organotypic epidermal tissue with marked keratinocyte differentiation genes (n = 4-5 epidermal tissue cultures/knockdown group).


A Differentially expressed genes in day 3 LINC00941 depleted epidermal tissue cluster within the epidermal differentiation complex ( EDC ) ( n = 4 - 5 biological replicates / knockdown group ) .

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A Differentially expressed genes in day 3 LINC00941 depleted epidermal tissue cluster within the epidermal differentiation complex (EDC) (n = 4-5 biological replicates/knockdown group).


B LINC00941 knockdown on day 3 in calcium - induced keratinocyte differentiation cultures results in increased expression of SPRR5 ( FC = fold change ) ( n = 3 biological replicates / knockdown group ) . Data is presented as mean ± SD . Statistical significance was tested by an unpaired t - test and corrected for multiple testing after Bonferroni ( * = adj . p - value < 0 . 05 , * * = adj . p - value < 0 . 01 , * * * = adj . p ‑ value < 0 . 001 , and n . s . = not significant ) .

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B LINC00941 knockdown on day 3 in calcium-induced keratinocyte differentiation cultures results in increased expression of SPRR5 (FC = fold change) (n = 3 biological replicates/knockdown group). Data is presented as mean ± SD. Statistical significance was tested by an unpaired t-test and corrected for multiple testing after Bonferroni (* = adj. p-value < 0.05, ** = adj. p-value < 0.01, *** = adj. p‑value < 0.001, and n.s. = not significant).


C Detected fragments per million ( FPM ) for SPRR5 in control ( siCtrl ) as well as LINC00941 deficient ( siLINC00941 ) epidermal tissue for all biological replicates on day 3 ( D3 ) of differentiation as obtained by DeSeq2 ( FC = fold change ) ( n = 4 ‑ 5 epidermal tissue cultures / knockdown group ) .

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C Detected fragments per million (FPM) for SPRR5 in control (siCtrl) as well as LINC00941 deficient (siLINC00941) epidermal tissue for all biological replicates on day 3 (D3) of differentiation as obtained by DeSeq2 (FC = fold change) (n = 4‑5 epidermal tissue cultures/knockdown group).


SPRR5 induction during the course of keratinocyte ( KC ) differentiation is shown by northern blot ( D ) for four different primary keratinocyte isolates and compared to undifferentiated keratinocytes ( n = 8 biological replicates ) . Data is presented as mean ± SD .

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SPRR5 induction during the course of keratinocyte (KC) differentiation is shown by northern blot (D) for four different primary keratinocyte isolates and compared to undifferentiated keratinocytes (n = 8 biological replicates). Data is presented as mean ± SD.


SPRR5 induction during the course of keratinocyte ( KC ) differentiation is shown by qRT - PCR analysis ( E ) for four different primary keratinocyte isolates and compared to undifferentiated keratinocytes ( n = 8 biological replicates ) . Data is presented as mean ± SD .

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SPRR5 induction during the course of keratinocyte (KC) differentiation is shown by qRT-PCR analysis (E) for four different primary keratinocyte isolates and compared to undifferentiated keratinocytes (n = 8 biological replicates). Data is presented as mean ± SD.


F p63 knockdown on day 3 of keratinocyte differentiation leads to decreased SPRR5 transcript levels ( FC = fold change ) ( n = 3 - 4 biological replicates / knockdown group ) . Data is presented as mean ± SD . Statistical significance was tested by an unpaired t - test and corrected for multiple testing after Bonferroni ( * = adj . p - value < 0 . 05 , * * = adj . p - value < 0 . 01 , * * * = adj . p ‑ value < 0 . 001 , and n . s . = not significant ) .

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F p63 knockdown on day 3 of keratinocyte differentiation leads to decreased SPRR5 transcript levels (FC = fold change) (n = 3-4 biological replicates /knockdown group). Data is presented as mean ± SD. Statistical significance was tested by an unpaired t-test and corrected for multiple testing after Bonferroni (* = adj. p-value < 0.05, ** = adj. p-value < 0.01, *** = adj. p‑value < 0.001, and n.s. = not significant).


siPool - mediated SPRR5 knockdown on day 4 to 6 ( D4 - D6 ) of calcium - induced keratinocyte differentiation ( A ) ( n = 3 - 5 cultures / knockdown group ) . Data is presented as mean ± SD . Statistical significance was tested by an unpaired t - test and corrected for multiple testing after Bonferroni ( * = adj . p - value < 0 . 05 , * * = adj . p - value < 0 . 01 , * * * = adj . p ‑ value < 0 . 001 , and n . s . = not significant ) .

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siPool-mediated SPRR5 knockdown on day 4 to 6 (D4-D6) of calcium-induced keratinocyte differentiation (A) (n = 3-5 cultures/knockdown group). Data is presented as mean ± SD. Statistical significance was tested by an unpaired t-test and corrected for multiple testing after Bonferroni (* = adj. p-value < 0.05, ** = adj. p-value < 0.01, *** = adj. p‑value < 0.001, and n.s. = not significant).


siPool - mediated SPRR5 knockdown on day 4 to 6 ( D4 - D6 ) of calcium - induced keratinocyte differentiation leads to decreased expression of key differentiation markers as shown by qRT - PCR ( B ) ( FC = fold change ) ( n = 3 - 5 cultures / knockdown group ) . Data is presented as mean ± SD . Statistical significance was tested by an unpaired t - test and corrected for multiple testing after Bonferroni ( * = adj . p - value < 0 . 05 , * * = adj . p - value < 0 . 01 , * * * = adj . p ‑ value < 0 . 001 , and n . s . = not significant ) .

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siPool-mediated SPRR5 knockdown on day 4 to 6 (D4-D6) of calcium-induced keratinocyte differentiation leads to decreased expression of key differentiation markers as shown by qRT-PCR (B) (FC = fold change) (n = 3-5 cultures/knockdown group). Data is presented as mean ± SD. Statistical significance was tested by an unpaired t-test and corrected for multiple testing after Bonferroni (* = adj. p-value < 0.05, ** = adj. p-value < 0.01, *** = adj. p‑value < 0.001, and n.s. = not significant).


siPool - mediated SPRR5 knockdown on day 4 to 6 ( D4 - D6 ) of calcium - induced keratinocyte differentiation leads to decreased expression of key differentiation markers as shown western blot analysis ( C ) ( n = 3 - 5 cultures / knockdown group ) . Data is presented as mean ± SD . Statistical significance was tested by an unpaired t - test and corrected for multiple testing after Bonferroni ( * = adj . p - value < 0 . 05 , * * = adj . p - value < 0 . 01 , * * * = adj . p ‑ value < 0 . 001 , and n . s . = not significant ) .

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siPool-mediated SPRR5 knockdown on day 4 to 6 (D4-D6) of calcium-induced keratinocyte differentiation leads to decreased expression of key differentiation markers as shown western blot analysis (C) (n = 3-5 cultures/knockdown group). Data is presented as mean ± SD. Statistical significance was tested by an unpaired t-test and corrected for multiple testing after Bonferroni (* = adj. p-value < 0.05, ** = adj. p-value < 0.01, *** = adj. p‑value < 0.001, and n.s. = not significant).


D SPRR5 knockdown efficiency in day 3 ( D3 ) organotypic epidermal tissue ( n = 4 epidermal tissue cultures / knockdown group ) . Data is presented as mean ± SD . Statistical significance was tested by an unpaired t - test and corrected for multiple testing after Bonferroni ( * = adj . p - value < 0 . 05 , * * = adj . p - value < 0 . 01 , * * * = adj . p ‑ value < 0 . 001 , and n . s . = not significant ) .

O B-GENEPROD O O O O O O O O O B-TISSUE I-TISSUE O O O O B-TISSUE I-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 SPRR5 knockdown efficiency in day 3 (D3) organotypic epidermal tissue (n = 4 epidermal tissue cultures/knockdown group). Data is presented as mean ± SD. Statistical significance was tested by an unpaired t-test and corrected for multiple testing after Bonferroni (* = adj. p-value < 0.05, ** = adj. p-value < 0.01, *** = adj. p‑value < 0.001, and n.s. = not significant).


E SPRR5 depletion in regenerated organotypic epidermal tissue results in drastically reduced expression of differentiation proteins as shown by immunofluorescence analysis . Collagen VII ( Col VII , green ) stain indicates the basement membrane , nuclei are shown in blue and the differentiation proteins filaggrin and loricrin are shown in red ( n = 4 tissue cultures / knockdown group ; one exemplary picture for each group is depicted ) . Scale bar : 50 µm .

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E SPRR5 depletion in regenerated organotypic epidermal tissue results in drastically reduced expression of differentiation proteins as shown by immunofluorescence analysis. Collagen VII (Col VII, green) stain indicates the basement membrane, nuclei are shown in blue and the differentiation proteins filaggrin and loricrin are shown in red (n = 4 tissue cultures/knockdown group; one exemplary picture for each group is depicted). Scale bar: 50 µm.


F SPRR5 knockdown in regenerated epidermal tissue results in reduced transcript levels of key differentiation genes as obtained by qRT - PCR ( n = 4 epidermal tissue cultures / knockdown group ) . Data is presented as mean ± SD . Statistical significance was tested by an unpaired t - test and corrected for multiple testing after Bonferroni ( * = adj . p - value < 0 . 05 , * * = adj . p - value < 0 . 01 , * * * = adj . p ‑ value < 0 . 001 , and n . s . = not significant ) .

O B-GENEPROD O O O B-TISSUE I-TISSUE O O O O B-EXP_ASSAY O O B-EXP_ASSAY O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-TISSUE I-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 SPRR5 knockdown in regenerated epidermal tissue results in reduced transcript levels of key differentiation genes as obtained by qRT-PCR (n = 4 epidermal tissue cultures/knockdown group). Data is presented as mean ± SD. Statistical significance was tested by an unpaired t-test and corrected for multiple testing after Bonferroni (* = adj. p-value < 0.05, ** = adj. p-value < 0.01, *** = adj. p‑value < 0.001, and n.s. = not significant).


B Overview of SPRR5 levels in control ( siCtrl ) and SPRR5 deficient ( siSPRR5 ) epidermal tissue on day 3 and day 4 as obtained by qRT - PCR analysis ( n = 3 - 5 epidermal tissue cultures / knockdown group ) . Data is presented as mean ± SD . Statistical significance was tested by an unpaired t - test and corrected for multiple testing after Bonferroni ( * = adj . p - value < 0 . 05 , * * = adj . p - value < 0 . 01 , * * * = adj . p ‑ value < 0 . 001 , and n . s . = not significant ) .

O O O B-GENEPROD B-EXP_ASSAY O O O O O O B-GENEPROD O O O O B-TISSUE I-TISSUE O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-TISSUE I-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Overview of SPRR5 levels in control (siCtrl) and SPRR5 deficient (siSPRR5) epidermal tissue on day 3 and day 4 as obtained by qRT-PCR analysis (n = 3-5 epidermal tissue cultures/knockdown group). Data is presented as mean ± SD. Statistical significance was tested by an unpaired t-test and corrected for multiple testing after Bonferroni (* = adj. p-value < 0.05, ** = adj. p-value < 0.01, *** = adj. p‑value < 0.001, and n.s. = not significant).


Heatmaps of differentially expressed genes ( padj < 0 . 05 and - 0 . 5 > log2FC > 0 . 5 ) upon SPRR5 depletion on day 3 ( C ) in organotypic epidermal tissue ( n = 3 - 5 epidermal tissue cultures / knockdown group ) .

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

Heatmaps of differentially expressed genes (padj < 0.05 and -0.5 > log2FC > 0.5) upon SPRR5 depletion on day 3 (C) in organotypic epidermal tissue (n = 3-5 epidermal tissue cultures/knockdown group).


Heatmaps of differentially expressed genes ( padj < 0 . 05 and - 0 . 5 > log2FC > 0 . 5 ) upon SPRR5 depletion on day 4 ( D ) in organotypic epidermal tissue ( n = 3 - 5 epidermal tissue cultures / knockdown group ) .

Heatmaps of differentially expressed genes (padj < 0.05 and -0.5 > log2FC > 0.5) upon SPRR5 depletion on day 4 (D) in organotypic epidermal tissue (n = 3-5 epidermal tissue cultures/knockdown group).


A HeLa - H2B / tub cells were transfected with control or TIAR siRNAs for 48 hours and synchronized by double thymidine ( TT ) block . After release from the block , mitotic cells were counted by fluorescence microscopy ( mean ± SD ; n = 3 ) . Data information : Statistical significance was determined by unpaired Student ' s t - test ; * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 .

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A HeLa-H2B/tub cells were transfected with control or TIAR siRNAs for 48 hours and synchronized by double thymidine (TT) block. After release from the block, mitotic cells were counted by fluorescence microscopy (mean ± SD; n = 3). Data information: Statistical significance was determined by unpaired Student's t-test; *, p < 0.05; **, p < 0.01; ***, p < 0.001.


B Time from S - to M - phase was measured in si - RNA transfected HeLa - H2B / tub cells by time - lapse microscopy following release from TT block ( 4 repeat experiments ; all measurements depicted ) . Data information : Statistical significance was determined by unpaired Student ' s t - test ; * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 .

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B Time from S- to M-phase was measured in si-RNA transfected HeLa-H2B/tub cells by time-lapse microscopy following release from TT block (4 repeat experiments; all measurements depicted). Data information: Statistical significance was determined by unpaired Student's t-test; *, p < 0.05; **, p < 0.01; ***, p < 0.001.


Western blot analysis of unsynchronized HeLa - H2B / tub cells was carried out to monitor expression of p ( S10 ) - H3 , total H3 and TIAR in control and TIAR - depleted cells ( mean ± SD ; n = 3 ) . Data information : Statistical significance was determined by unpaired Student ' s t - test ; * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 .

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

Western blot analysis of unsynchronized HeLa-H2B/tub cells was carried out to monitor expression of p(S10)-H3, total H3 and TIAR in control and TIAR-depleted cells (mean ± SD; n = 3). Data information: Statistical significance was determined by unpaired Student's t-test; *, p < 0.05; **, p < 0.01; ***, p < 0.001.


D HeLa cells were transfected with control or TIAR si - RNAs , alone or together with Cdc25B siRNA . 72 hours after transfection , p ( S10 ) - H3 - positive cells were quantified by flow cytometry ( mean ± SD ; n = 3 ) . Data information : Statistical significance was determined by unpaired Student ' s t - test ; * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 .

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

D HeLa cells were transfected with control or TIAR si-RNAs, alone or together with Cdc25B siRNA. 72 hours after transfection, p(S10)-H3-positive cells were quantified by flow cytometry (mean ± SD; n = 3). Data information: Statistical significance was determined by unpaired Student's t-test; *, p < 0.05; **, p < 0.01; ***, p < 0.001.


HeLadox - YFP - TIARr cells were transfected with control or TIAR siRNAs , and 24h hours later cell were cultured in the abscence or presence of 1 μg / ml doxocycline for 48 hours . The expression of p ( S10 ) - H3 , total H3 and TIAR was measured by Westen blot analysis . The graph shows the quantification of p - H3 levels normalized to total H3 ( mean ± SD , n = 4 ) . Data information : Statistical significance was determined by unpaired Student ' s t - test ; * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 .

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

HeLadox-YFP-TIARr cells were transfected with control or TIAR siRNAs, and 24h hours later cell were cultured in the abscence or presence of 1 μg/ml doxocycline for 48 hours. The expression of p(S10)-H3, total H3 and TIAR was measured by Westen blot analysis. The graph shows the quantification of p-H3 levels normalized to total H3 (mean ± SD, n = 4). Data information: Statistical significance was determined by unpaired Student's t-test; *, p < 0.05; **, p < 0.01; ***, p < 0.001.


F HeLadox - YFP - TIARr - RRM123m cells were analyzed as in panel ( E ) ( mean ± SD , n = 4 ) . Data information : Statistical significance was determined by unpaired Student ' s t - test ; * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 .

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F HeLadox-YFP-TIARr-RRM123m cells were analyzed as in panel (E) (mean ± SD, n = 4). Data information: Statistical significance was determined by unpaired Student's t-test; *, p < 0.05; **, p < 0.01; ***, p < 0.001.


G HeLadox - YFP - TIARr - dQRD cells were analyzed as in panel ( E ) ( mean ± SD , n = 4 ) . Data information : Statistical significance was determined by unpaired Student ' s t - test ; * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 .

G HeLadox-YFP-TIARr-dQRD cells were analyzed as in panel (E) (mean ± SD, n = 4). Data information: Statistical significance was determined by unpaired Student's t-test; *, p < 0.05; **, p < 0.01; ***, p < 0.001.


A Following transfection of HeLa cells with control or TIAR siRNAs for 72 hours , mitotic cells were analyzed by IF microscopy and scored for chromatin ( mean ± SE , n = 3 ) . B HeLa cells were analyzed as in ( A ) and scored for spindle defects ( mean ± SE , n = 3 ) . Data information : statistical significance was determined by unpaired Student ' s t - test ; * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 .

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

A Following transfection of HeLa cells with control or TIAR siRNAs for 72 hours, mitotic cells were analyzed by IF microscopy and scored for chromatin (mean ± SE, n = 3). B HeLa cells were analyzed as in (A) and scored for spindle defects (mean ± SE, n = 3). Data information: statistical significance was determined by unpaired Student's t-test; *, p < 0.05; **, p < 0.01; ***, p < 0.001.


C HeLa cells were transfected with control or TIAR siRNAs for 4 days , and subjected to colcemid for 2 hours prior to preparation of metaphase spreads . Chromosomes were stained with Hoechst ( red ) and anti - CENP - A antibody ( green ) ; the yellow arrowhead marks a chromosome break .

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

C HeLa cells were transfected with control or TIAR siRNAs for 4 days, and subjected to colcemid for 2 hours prior to preparation of metaphase spreads. Chromosomes were stained with Hoechst (red) and anti-CENP-A antibody (green); the yellow arrowhead marks a chromosome break.


D Metaphase spreads were prepared as in ( C ) , and the frequency of cells with chromosomal breaks was quantified ( mean ± SD , n = 3 independent experiments , approximately 30 metaphase spreads were assessed per experiment and condition ) . E Metaphase spreads were prepared as in ( C ) , and the frequency of cells with scattered chromosomes was quantified ( mean ± SD , n = 3 ) . Data information statistical significance was determined by unpaired Student ' s t - test ; * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 .

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

D Metaphase spreads were prepared as in (C), and the frequency of cells with chromosomal breaks was quantified (mean ± SD, n = 3 independent experiments, approximately 30 metaphase spreads were assessed per experiment and condition). E Metaphase spreads were prepared as in (C), and the frequency of cells with scattered chromosomes was quantified (mean ± SD, n = 3). Data information statistical significance was determined by unpaired Student's t-test; *, p < 0.05; **, p < 0.01; ***, p < 0.001.


F HeLa cells were transfected with control or TIAR si - RNAs , alone or together with Cdc25B siRNA , and 4 days later subjected to colcemid for 2 hours prior to preparation of metaphase spreads . The frequency of cells with scattered chromatids was quantified ( mean ± SD , n = 3 independent experiments , approximately 30 metaphase spreads were assessed per experiment and condition ) . G Metaphase spreads were prepared as in ( F ) , and the frequency of cells with chromosomal breaks was quantified ( mean ± SD , n = 3 ) . Data information , statistical significance was determined by unpaired Student ' s t - test ; * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 .

O B-CELL_LINE O O O O O O B-GENEPROD O O O O O O O O B-GENEPROD O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 HeLa cells were transfected with control or TIAR si-RNAs, alone or together with Cdc25B siRNA, and 4 days later subjected to colcemid for 2 hours prior to preparation of metaphase spreads. The frequency of cells with scattered chromatids was quantified (mean ± SD, n = 3 independent experiments, approximately 30 metaphase spreads were assessed per experiment and condition). G Metaphase spreads were prepared as in (F), and the frequency of cells with chromosomal breaks was quantified (mean ± SD, n = 3). Data information , statistical significance was determined by unpaired Student's t-test; *, p < 0.05; **, p < 0.01; ***, p < 0.001.


A HeLa cells were transfected with control or TIAR siRNAs for 48 hours prior to treatment with 0 . 4 µM APH . Cells were fixed at regular time intervals , and p ( S10 ) - H3 - positive cells were quantified by flow cytometry ( mean ± SE , n = 3 ) .

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

A HeLa cells were transfected with control or TIAR siRNAs for 48 hours prior to treatment with 0.4 µM APH. Cells were fixed at regular time intervals, and p(S10)-H3-positive cells were quantified by flow cytometry (mean ± SE, n = 3).


B HeLa cells transfected with control or TIAR siRNAs were treated with APH for 24 hours . Pan - nuclear γH2AX signals were quantified by HTM ( n = 3 , 2000 cells examined per experiment and condition ) . Each dot represents the signal from one cell , horizontal lines indicate mean values , and the blue area delineates cells above an arbitrarily chosen threshold . Data information : statistical significance was determined by Wilcoxon rank - sum test . ; * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 ; * * * * , p < 0 . 0001 .

O B-CELL_LINE O O O O O B-GENEPROD O O O O B-SMALL_MOLECULE O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR B-GENEPROD O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 HeLa cells transfected with control or TIAR siRNAs were treated with APH for 24 hours. Pan-nuclear γH2AX signals were quantified by HTM (n = 3, 2000 cells examined per experiment and condition). Each dot represents the signal from one cell, horizontal lines indicate mean values, and the blue area delineates cells above an arbitrarily chosen threshold. Data information: statistical significance was determined by Wilcoxon rank-sum test. ; *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001.


C Chromatin - bound RPA2 signals were quantified by HTM HeLa cells transfected with control or TIAR siRNAs ( n = 3 , 600 cells examined per experiment and condition ) .

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C Chromatin-bound RPA2 signals were quantified by HTM HeLa cells transfected with control or TIAR siRNAs (n = 3, 600 cells examined per experiment and condition).


D HeLa cells were transfected with control or TIAR siRNAs for 72 hours prior to treatment with ATRi ( 5 μM . ) alone or together with Ro3306 ( 5 μM ) for 12 hours . Pan - nuclear γH2AX signals were quantified by HTM ( n = 3 , 1000 cells examined per experiment and condition ) . Data information : statistical significance was determined by Wilcoxon rank - sum test . ; * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 ; * * * * , p < 0 . 0001 .

O B-CELL_LINE O O O O O O B-GENEPROD O O O O O O O O B-GENEPROD O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR B-GENEPROD O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 HeLa cells were transfected with control or TIAR siRNAs for 72 hours prior to treatment with ATRi (5 μM.) alone or together with Ro3306 (5 μM) for 12 hours. Pan-nuclear γH2AX signals were quantified by HTM (n = 3, 1000 cells examined per experiment and condition). Data information: statistical significance was determined by Wilcoxon rank-sum test. ; *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001.


E HeLa cells were transfected with control or TIAR siRNAs were treated with ATRi for 48 hours , and the sub - G1 population was quantified by flow cytometry following propidium iodide staining ( mean ± SD , n = 3 ) . Data information : statistical significance was determined by unpaired Student ' s t - test ; * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 ; * * * * , p < 0 . 0001 .

O B-CELL_LINE O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-SMALL_MOLECULE I-SMALL_MOLECULE B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 HeLa cells were transfected with control or TIAR siRNAs were treated with ATRi for 48 hours, and the sub-G1 population was quantified by flow cytometry following propidium iodide staining (mean ± SD, n = 3). Data information: statistical significance was determined by unpaired Student's t-test; *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001.


F HeLa cells transfected with control or TIAR siRNAs were treated with APH , alone or in combination with Gö6976 ( 1µg / ml ) or UCN - 01 ( 300 nM ) . After 24 hours , p ( S10 ) - H3 - positive cells were quantified by flow cytometry , and expressed relative to the values in the untreated condition ( mean ± SE , n = 3 ) . Data information : statistical significance was determined by unpaired Student ' s t - test ; * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 ; * * * * , p < 0 . 0001 .

O B-CELL_LINE O O O O O B-GENEPROD O O O O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O B-GENEPROD O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 HeLa cells transfected with control or TIAR siRNAs were treated with APH, alone or in combination with Gö6976 (1µg/ml) or UCN-01 (300 nM). After 24 hours, p(S10)-H3-positive cells were quantified by flow cytometry, and expressed relative to the values in the untreated condition (mean ± SE, n = 3). Data information: statistical significance was determined by unpaired Student's t-test; *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001.


A HeLa cells , untreated or treated for 24 hours with 0 . 4 µM APH , were fixed with methanol and analyzed by IF microscopy after staining with anti - p ( S10 ) - H3 antibody , anti - TIAR antibody and DAPI . Yellow arrows mark focal accumulation of TIAR in GMGs .

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

A HeLa cells, untreated or treated for 24 hours with 0.4 µM APH, were fixed with methanol and analyzed by IF microscopy after staining with anti-p(S10)-H3 antibody, anti-TIAR antibody and DAPI. Yellow arrows mark focal accumulation of TIAR in GMGs.


B IF microscopy was performed as in ( A ) to determine the intensity of the TIAR signal within GMGs ( mean ± SD , n = 3 independent experiments , approximately 100 GMGs in 25 cells were analyzed per experiment and condition ) . C IF microscopy was performed as in ( A ) to determine the area of the TIAR signal within GMGs ( mean ± SD , n = 3 ) . Data information statistical significance was determined by unpaired Student ' s t - test ; * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 .

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B IF microscopy was performed as in (A) to determine the intensity of the TIAR signal within GMGs (mean ± SD, n = 3 independent experiments, approximately 100 GMGs in 25 cells were analyzed per experiment and condition). C IF microscopy was performed as in (A) to determine the area of the TIAR signal within GMGs (mean ± SD, n = 3). Data information statistical significance was determined by unpaired Student's t-test; *, p < 0.05; **, p < 0.01; ***, p < 0.001.


D IF microscopy was performed as in ( A ) to quantify the percentage of late G2 / prophase cells containing GMGs ( mean ± SD , n = 3 independent experiments , 40 cells were analyzed per experiment and condition ) . Data information statistical significance was determined by unpaired Student ' s t - test ; * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 .

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D IF microscopy was performed as in (A) to quantify the percentage of late G2/prophase cells containing GMGs (mean ± SD, n = 3 independent experiments, 40 cells were analyzed per experiment and condition). Data information statistical significance was determined by unpaired Student's t-test; *, p < 0.05; **, p < 0.01; ***, p < 0.001.


E HeLa cells were processed for IF microscopy as in ( A ) , images depict different cell cycle phases .

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E HeLa cells were processed for IF microscopy as in (A), images depict different cell cycle phases.


HeLa cell were treated for 16 hours with ( F ) the CDK1 inhibitor Ro3306 ( 9 µM ) prior to fixation with methanol and staining with anti - TIAR antibody and DAPI . The graphs show the percentage of late G2 / prophase cells containing GMGs ( mean ± SD , n = 3 independent experiments , 40 cells were analyzed per experiment and condition ) . Data information : statistical significance was determined by unpaired Student ' s t - test ; * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 .

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HeLa cell were treated for 16 hours with (F) the CDK1 inhibitor Ro3306 (9 µM) prior to fixation with methanol and staining with anti-TIAR antibody and DAPI. The graphs show the percentage of late G2/prophase cells containing GMGs (mean ± SD, n = 3 independent experiments, 40 cells were analyzed per experiment and condition). Data information: statistical significance was determined by unpaired Student's t-test; *, p < 0.05; **, p < 0.01; ***, p < 0.001.


HeLa cell were treated for 16 hours with ( G ) ICFR - 193 ( 1 μM ) prior to fixation with methanol and staining with anti - TIAR antibody and DAPI . The graphs show the percentage of late G2 / prophase cells containing GMGs ( mean ± SD , n = 3 independent experiments , 40 cells were analyzed per experiment and condition ) .

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HeLa cell were treated for 16 hours with (G) ICFR-193 (1 μM) prior to fixation with methanol and staining with anti-TIAR antibody and DAPI. The graphs show the percentage of late G2/prophase cells containing GMGs (mean ± SD, n = 3 independent experiments, 40 cells were analyzed per experiment and condition).


A - G HeLa cells were treated with 0 . 4 µM APH for 24 hours , fixed and processed for IF microscopy after staining with anti - TIAR antibody , DAPI in combination with ( A ) anti - PCNA - 1 antibody , ( B ) anti - FANCD2 antibody , ( C ) anti - BRCA1 antibody , ( D ) anti - PRP19 antibody , ( E ) anti - Sm ( Y12 ) antibody , ( F ) anti - HuR antibody and ( G ) pS2 - RPOL2 ( H5 ) antibody . Intensity profiles along the white line in the merged image are presented on the right side ; yellow and green arrows mark GMGs .

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A-G HeLa cells were treated with 0.4 µM APH for 24 hours, fixed and processed for IF microscopy after staining with anti-TIAR antibody, DAPI in combination with (A) anti-PCNA-1 antibody, (B) anti-FANCD2 antibody, (C) anti-BRCA1 antibody, (D) anti-PRP19 antibody, (E) anti-Sm(Y12) antibody, (F) anti-HuR antibody and (G) pS2-RPOL2 (H5) antibody. Intensity profiles along the white line in the merged image are presented on the right side; yellow and green arrows mark GMGs.


A HeLa cells were treated with 0 . 4 µM APH for 24 hours , fixed and processed for IF microscopy after staining with anti - TIAR and anti - CDK1 antibodies . Intensity profiles along the white line in the merged image are presented on the right side ; yellow arrows mark GMGs .

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A HeLa cells were treated with 0.4 µM APH for 24 hours, fixed and processed for IF microscopy after staining with anti-TIAR and anti-CDK1 antibodies. Intensity profiles along the white line in the merged image are presented on the right side; yellow arrows mark GMGs.


B IF microscopy analysis as in ( A ) using anti - Cyclin B antibody .

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B IF microscopy analysis as in (A) using anti-Cyclin B antibody.


C HeLa cells transfected with control or TIAR siRNAs were treated with 0 . 4 µM APH 24 hours prior to fixation . Cells were processed for IF microscopy after staining with anti - PRP19 and anti - CDK1 antibodies . Intensity profiles from staining of the same cells with anti - TIAR and anti - CDK1 antibodies are depicted on the right side .

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C HeLa cells transfected with control or TIAR siRNAs were treated with 0.4 µM APH 24 hours prior to fixation. Cells were processed for IF microscopy after staining with anti-PRP19 and anti-CDK1 antibodies. Intensity profiles from staining of the same cells with anti-TIAR and anti-CDK1 antibodies are depicted on the right side.


D CDK1 was immunoprecipitated from HeLa cells transfected with control or TIAR siRNAs , tested for in vitro kinase activity using recombinant histone H1 as substrate , and visualized by Western blot analysis and autoradiography . The mean CDK1 activity ± SE was quantified from n = 5 experiments . Data information statistical significance was determined by unpaired Student ' s t - test . ; * , p < 0 . 05 ; * * * , p < 0 . 001 .

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D CDK1 was immunoprecipitated from HeLa cells transfected with control or TIAR siRNAs, tested for in vitro kinase activity using recombinant histone H1 as substrate, and visualized by Western blot analysis and autoradiography. The mean CDK1 activity ± SE was quantified from n = 5 experiments. Data information statistical significance was determined by unpaired Student's t-test. ; *, p < 0.05; ***, p < 0.001.


E CDK2 activity was analyzed following immunoprecipitation as in ( D ) ( mean ± SE , n = 3 ) . Data information statistical significance was determined by unpaired Student ' s t - test . * , p < 0 . 05 ; * * * , p < 0 . 001 .

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E CDK2 activity was analyzed following immunoprecipitation as in (D) (mean ± SE, n = 3). Data information statistical significance was determined by unpaired Student's t-test. *, p < 0.05; ***, p < 0.001.


F HeLa cells were transfected with control or TIAR siRNAs for 72 hours before measuring expression levels of p ( S22 ) - Lamin A by Western blot analysis ( mean ± SE , n = 3 ) . Data information statistical significance was determined by unpaired Student ' s t - test . ; * , p < 0 . 05 ; * * * , p < 0 . 001 .

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F HeLa cells were transfected with control or TIAR siRNAs for 72 hours before measuring expression levels of p(S22)-Lamin A by Western blot analysis (mean ± SE, n = 3). Data information statistical significance was determined by unpaired Student's t-test. ; *, p < 0.05; ***, p < 0.001.


G p ( S22 ) - Lamin A levels were analyzed by HTM in si - RNA transfected HeLa cells ( n = 3 , 1000 cells examined per experiment and condition ) . Data information statistical significance was determined by Wilcoxon rank - sum test ; * , p < 0 . 05 ; * * * , p < 0 . 001 .

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G p(S22)-Lamin A levels were analyzed by HTM in si-RNA transfected HeLa cells (n = 3, 1000 cells examined per experiment and condition). Data information statistical significance was determined by Wilcoxon rank-sum test; *, p < 0.05; ***, p < 0.001.


( A ) BI ‐ 1 WT and KO MEFs cells were stably transduced with retroviruses expressing cytochrome b5 - GFP to visualize the ER ( green ) . Then cells were stained with lysotracker ( red ) and observed with a confocal microscope . Nucleus was stained with Hoechst ( blue ) . Scale bar : 30 μm .

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(A) BI‐1 WT and KO MEFs cells were stably transduced with retroviruses expressing cytochrome b5-GFP to visualize the ER (green). Then cells were stained with lysotracker (red) and observed with a confocal microscope. Nucleus was stained with Hoechst (blue). Scale bar: 30 μm.


( B ) BI ‐ 1 WT and KO MEFs cells stimulated with EBSS for 3 h to induce autophagy . Acidic vesicles were visualized with a confocal microscope after lysotracker staining . Data represent the results of three independent experiments . Scale bar : 50 μm .

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(B) BI‐1 WT and KO MEFs cells stimulated with EBSS for 3 h to induce autophagy. Acidic vesicles were visualized with a confocal microscope after lysotracker staining. Data represent the results of three independent experiments. Scale bar: 50 μm.


( C ) ( a ) Epifluorescence imaging of BI ‐ 1 KO cells stained with lysotracker ; ( b ) electron micrograph of the same field of cells shown in ( a ) ; ( c - e ) , magnification of lysotracker ‐ positive vesicles in BI ‐ 1 KO cells exposed to EBSS for 3 h and visualized with a fluorescent microscope ( c ) and the same field subsequently imaged by EM ( d ) . Overlapping images are presented ( e ) . Left panel : analysis of vesicular structures by EM with morphologies resembling early ( f ) , intermediate ( g ) and late ( h ) autophagy vesicles .

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(C) (a) Epifluorescence imaging of BI‐1 KO cells stained with lysotracker; (b) electron micrograph of the same field of cells shown in (a); (c-e), magnification of lysotracker‐positive vesicles in BI‐1 KO cells exposed to EBSS for 3 h and visualized with a fluorescent microscope (c) and the same field subsequently imaged by EM (d). Overlapping images are presented (e). Left panel: analysis of vesicular structures by EM with morphologies resembling early (f), intermediate (g) and late (h) autophagy vesicles.


( D ) Left panel : the distribution of endogenous LC3 was monitored by immunofluorescence and confocal microscopy in BI ‐ 1 WT and KO MEFs cells at basal conditions ( NT ) or after exposure to EBSS for 3 h . Scale bar : left 15 μm and right 10 μm . Right panel : quantification of the number cells containing three or more LC3 ‐ positive vesicles ( N = 160 cells ) . Mean and standard deviation are presented ( N = 4 ) . Student ' s t ‐ test was used to analyse statistical significance , * * P0 . 001 and * P0 . 01 .

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(D) Left panel: the distribution of endogenous LC3 was monitored by immunofluorescence and confocal microscopy in BI‐1 WT and KO MEFs cells at basal conditions (NT) or after exposure to EBSS for 3 h. Scale bar: left 15 μm and right 10 μm. Right panel: quantification of the number cells containing three or more LC3‐positive vesicles (N=160 cells). Mean and standard deviation are presented (N=4). Student's t‐test was used to analyse statistical significance, **P0.001 and *P0.01.


( E ) BI ‐ 1 WT and KO cells were transiently transfected with an expression vector for a monomeric ‐ tandem LC3 - RFP - GFP construct . After 24 h , cells were exposed to EBSS for 3 h . Autophagy fluxes were monitored in living cells by visualizing the distribution of LC3 ‐ positive dots in the red and green channels using a confocal microscope . Scale bar : 10 μm . Right panel : quantification of the ratio between red and yellow dots per cell is presented . Mean and standard error of the analysis of 15 cells are shown .

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(E) BI‐1 WT and KO cells were transiently transfected with an expression vector for a monomeric‐tandem LC3-RFP-GFP construct. After 24 h, cells were exposed to EBSS for 3 h. Autophagy fluxes were monitored in living cells by visualizing the distribution of LC3‐positive dots in the red and green channels using a confocal microscope. Scale bar: 10 μm. Right panel: quantification of the ratio between red and yellow dots per cell is presented. Mean and standard error of the analysis of 15 cells are shown.


( A ) BI ‐ 1 WT and KO MEFs were treated with EBSS ( left panel ) or glucose / serum ‐ free RPMI media ( right panel ) for the indicated time points . Then , levels of LC3 were determined by western blot analysis . LC3 ‐ I and LC3 ‐ II forms are indicated . Hsp90 levels were assessed as loading control .

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(A) BI‐1 WT and KO MEFs were treated with EBSS (left panel) or glucose/serum‐free RPMI media (right panel) for the indicated time points. Then, levels of LC3 were determined by western blot analysis. LC3‐I and LC3‐II forms are indicated. Hsp90 levels were assessed as loading control.


( B ) Quantification of LC3 ‐ II levels relative to Hsp90 expression was performed in several experiments performed as presented in ( A ) .

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(B) Quantification of LC3‐II levels relative to Hsp90 expression was performed in several experiments performed as presented in (A).


( C ) Cells were pre ‐ treated with a cocktail of lysosomal inhibitors ( 200 nM bafilomycin A1 , 10 μg / ml pepstatin , and E64d ; left panel ) or 10 mM 3 ‐ methyladenine ( 3 ‐ MA ; right panel ) for 12 h and then exposed to starvation . LC3 levels monitored by western blot ( D ) and quantification of LC3 ‐ II levels relative to Hsp90 were performed in the experimental conditions described in ( C ) .

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(C)Cells were pre‐treated with a cocktail of lysosomal inhibitors (200 nM bafilomycin A1, 10 μg/ml pepstatin, and E64d; left panel) or 10 mM 3‐methyladenine (3‐MA; right panel) for 12 h and then exposed to starvation. LC3 levels monitored by western blot (D) and quantification of LC3‐II levels relative to Hsp90 were performed in the experimental conditions described in (C).


( E ) Basal autophagy flux was monitored in cells treated with a cocktail of lysosomal inhibitors ( Lys . Inh . ) in the presence of normal cell culture media . Right panel : quantification of independent experiments is presented .

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(E) Basal autophagy flux was monitored in cells treated with a cocktail of lysosomal inhibitors (Lys. Inh.) in the presence of normal cell culture media. Right panel: quantification of independent experiments is presented.


( F ) BI ‐ 1 KO cells were stably transduced with retroviruses expressing BI ‐ 1 - GFP or empty vector , and then levels of LC3 ‐ II were assessed over time by western blot after exposure to EBSS . Right panel : as control , the levels of BI ‐ 1 - GFP were monitored by western blot . Hsp90 levels were used as loading control . In ( B , D and E ) mean and standard deviation are presented . Two ‐ way ANOVA was applied to analyse statistical significance . In parenthesis , the number of independent experiments for each time point is indicated . Student ' s t ‐ test was also used in ( E ) to analyse the statistical significance between each time point ( * P0 . 001 ) . In ( B , D and E ) , normalization was performed as a ratio with the LC3 ‐ II / Hsp90 normalized levels from non ‐ treated BI ‐ 1 WT cells .

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(F) BI‐1 KO cells were stably transduced with retroviruses expressing BI‐1-GFP or empty vector, and then levels of LC3‐II were assessed over time by western blot after exposure to EBSS. Right panel: as control, the levels of BI‐1-GFP were monitored by western blot. Hsp90 levels were used as loading control. In (B, D and E) mean and standard deviation are presented. Two‐way ANOVA was applied to analyse statistical significance. In parenthesis, the number of independent experiments for each time point is indicated. Student's t‐test was also used in (E) to analyse the statistical significance between each time point (*P0.001). In (B, D and E), normalization was performed as a ratio with the LC3‐II/Hsp90 normalized levels from non‐treated BI‐1 WT cells.


( A ) Left panel : BI ‐ 1 WT and KO MEFs cells were incubated in EBSS , and then cell viability was monitored using the MTS assay . Right panel : a similar experiment was performed after treating cells with the indicated concentration of tunicamycin for 24 h . Mean and standard deviation are presented of triplicates representative of three independent experiments .

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(A) Left panel: BI‐1 WT and KO MEFs cells were incubated in EBSS, and then cell viability was monitored using the MTS assay. Right panel: a similar experiment was performed after treating cells with the indicated concentration of tunicamycin for 24 h. Mean and standard deviation are presented of triplicates representative of three independent experiments.


( B ) BI ‐ 1 WT and KO cells were treated with three different starvation stimuli for 6 and 24 h . Cell death was determined after propidium iodide ( PI ) staining and FACS analysis . In addition , cells were treated with 100 ng / ml tunicamycin ( Tm ) for 24 h . Mean and standard deviation are presented of one experiment performed in triplicates .

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(B) BI‐1 WT and KO cells were treated with three different starvation stimuli for 6 and 24 h. Cell death was determined after propidium iodide (PI) staining and FACS analysis. In addition, cells were treated with 100 ng/ml tunicamycin (Tm) for 24 h. Mean and standard deviation are presented of one experiment performed in triplicates.


( C ) Cells were exposed to EBSS for 6 h or 1 μg / ml Tm for 2 h , and then replated in normal cell culture media . After 5 days , cell viability was monitored by staining with crystal violet . Data are representative of three independent experiments .

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(C) Cells were exposed to EBSS for 6 h or 1 μg/ml Tm for 2 h, and then replated in normal cell culture media. After 5 days, cell viability was monitored by staining with crystal violet. Data are representative of three independent experiments.


( D ) BI ‐ 1 WT MEFs were stably transduced with lentiviral expression vectors to deliver an shRNA against bi ‐ 1 mRNA or control mRNA ( luciferase shRNA ) . Cell survival was measured after treatment of cells as described in ( B ) . Mean and standard deviation of an experiment made by triplicate , representative of two independent experiments .

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(D) BI‐1 WT MEFs were stably transduced with lentiviral expression vectors to deliver an shRNA against bi‐1 mRNA or control mRNA (luciferase shRNA). Cell survival was measured after treatment of cells as described in (B). Mean and standard deviation of an experiment made by triplicate, representative of two independent experiments.


( E ) BI ‐ 1 KO cells were stably transduced with retroviruses expressing BI ‐ 1 - GFP or empty vector , and then exposed to EBSS for indicated time points . Cell viability was monitored after PI staining by FACS . Mean and standard deviation are presented of triplicates representative of two independent experiments .

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

(E) BI‐1 KO cells were stably transduced with retroviruses expressing BI‐1-GFP or empty vector, and then exposed to EBSS for indicated time points. Cell viability was monitored after PI staining by FACS. Mean and standard deviation are presented of triplicates representative of two independent experiments.


( A ) BI ‐ 1 WT and KO MEFs were treated with EBSS for 2 h , and the levels of phospho ‐ p70S6k were determined by western blot analysis . Total p70S6k is also shown .

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

(A) BI‐1 WT and KO MEFs were treated with EBSS for 2 h, and the levels of phospho‐p70S6k were determined by western blot analysis. Total p70S6k is also shown.