C . Confocal images of control and NPC1I1061T patient fibroblasts expressing ARF1 - GFP . Insets show enlarged views of ARF1 in the Golgi area . Tubular structures were observed in NPC1I1061T patient fibroblasts as indicated by the black arrows . D . Quantification of ΑRF - 1 fluorescence intensity in Golgi normalized to the cytoplasm in control and NPC1I1061T patient fibroblasts . Control : n = 10 ; NPC1I1061T : n = 11 .

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C. Confocal images of control and NPC1I1061T patient fibroblasts expressing ARF1-GFP. Insets show enlarged views of ARF1 in the Golgi area. Tubular structures were observed in NPC1I1061T patient fibroblasts as indicated by the black arrows. D. Quantification of ΑRF-1 fluorescence intensity in Golgi normalized to the cytoplasm in control and NPC1I1061T patient fibroblasts. Control: n=10; NPC1I1061T: n=11.


Human tissue sections were probed with Siglec ‐ 5 Fc and Siglec ‐ 5 R119A Fc to determine the presence of potential sialic acid ‐ independent ligands . Positive signals ( in blue ) for such ligands were identified in the lumen of the lung bronchioles and prostate ducts , as well as in pancreatic tissues .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Human tissue sections were probed with Siglec‐5 Fc and Siglec‐5 R119A Fc to determine the presence of potential sialic acid‐independent ligands. Positive signals (in blue) for such ligands were identified in the lumen of the lung bronchioles and prostate ducts, as well as in pancreatic tissues.


Pancreatic tissue lysate or lung BAL were incubated with Siglec ‐ 5 R119A Fc ( Sig5 ‐ RA Fc ) or Siglec ‐ 7 Fc ( Sig7Fc ) immobilized onto Protein G beads . The proteins captured by these complexes were identified by mass spectrometric analysis . Hsp70 was the only protein that was captured by the Sig5 ‐ RA Fc bead complex from pancreas lysate and bronchoalveolar lavage , but not captured by Sig7Fc bead complex .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Pancreatic tissue lysate or lung BAL were incubated with Siglec‐5 R119A Fc (Sig5‐RA Fc) or Siglec‐7 Fc (Sig7Fc) immobilized onto Protein G beads. The proteins captured by these complexes were identified by mass spectrometric analysis. Hsp70 was the only protein that was captured by the Sig5‐RA Fc bead complex from pancreas lysate and bronchoalveolar lavage, but not captured by Sig7Fc bead complex.


Co ‐ localization of Hsp70 with Siglec ‐ 5 and Siglec ‐ 14 ligands in lung bronchiolesImmunofluorescence of human lung and prostate sections probed with Siglec ‐ 5 R119A Fc or Siglec ‐ 14 R119A Fc ( red ) with an anti ‐ Hsp70 antibody ( green ) . The co ‐ localized signals for the Siglec ‐ Fc probes with an anti ‐ Hsp70 antibody suggest that Hsp70 is one of the possible sialic acid ‐ independent ligands of Siglec ‐ 5 and Siglec ‐ 14 in the lung bronchiole . White dashes mark the lung bronchiole epithelium . All images are representative of at least 2 - 3 replicates .

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Co‐localization of Hsp70 with Siglec‐5 and Siglec‐14 ligands in lung bronchiolesImmunofluorescence of human lung and prostate sections probed with Siglec‐5 R119A Fc or Siglec‐14 R119A Fc (red) with an anti‐Hsp70 antibody (green). The co‐localized signals for the Siglec‐Fc probes with an anti‐Hsp70 antibody suggest that Hsp70 is one of the possible sialic acid‐independent ligands of Siglec‐5 and Siglec‐14 in the lung bronchiole. White dashes mark the lung bronchiole epithelium. All images are representative of at least 2-3 replicates.


Immunoblot probed with Siglec ‐ 5 Fc and Siglec ‐ 14 Fc to detect direct binding to Hsp70 . B6N domain of GBS β ‐ protein was used as a positive control , and clusterin was used as the negative control .

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Immunoblot probed with Siglec‐5 Fc and Siglec‐14 Fc to detect direct binding to Hsp70. B6N domain of GBS β‐protein was used as a positive control, and clusterin was used as the negative control.


ELISA demonstrates direct binding of Siglec ‐ 5 Fc and Siglec ‐ 14 Fc , but not Siglec ‐ 7 Fc , to Hsp70 . Bovine serum albumin ( BSA ) was used as the negative control .

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ELISA demonstrates direct binding of Siglec‐5 Fc and Siglec‐14 Fc, but not Siglec‐7 Fc, to Hsp70. Bovine serum albumin (BSA) was used as the negative control.


Siglec ‐ 5 or Siglec ‐ 14 Fc immobilized on magnetic beads was incubated with heat ‐ shocked HEK293A cell lysates . A Western blot of the captured proteins using an anti ‐ Hsp70 antibody demonstrates Hsp70 endogenously produced by HEK293A cells interacts with Siglec ‐ 5 and Siglec ‐ 14 .

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Siglec‐5 or Siglec‐14 Fc immobilized on magnetic beads was incubated with heat‐shocked HEK293A cell lysates. A Western blot of the captured proteins using an anti‐Hsp70 antibody demonstrates Hsp70 endogenously produced by HEK293A cells interacts with Siglec‐5 and Siglec‐14.


HEK293A cells were transfected with expression plasmids for Siglec ‐ 7 , Siglec ‐ 5 or Siglec ‐ 14 , and then subsequently incubated with ( red ) or without ( gray ) recombinant Hsp70 ( bottom panels ) . Flow cytometry analysis reveals Hsp70 binding to cell surfaces of HEK293A cells expressing Siglec ‐ 5 and Siglec ‐ 14 , but not Siglec ‐ 7 . Histogram of Siglec expression after transfection is shown as blue lines ( top panels ) .

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HEK293A cells were transfected with expression plasmids for Siglec‐7, Siglec‐5 or Siglec‐14, and then subsequently incubated with (red) or without (gray) recombinant Hsp70 (bottom panels). Flow cytometry analysis reveals Hsp70 binding to cell surfaces of HEK293A cells expressing Siglec‐5 and Siglec‐14, but not Siglec‐7. Histogram of Siglec expression after transfection is shown as blue lines (top panels).


Using flow cytometry , undifferentiated or PMA ‐ differentiated THP1 cells with empty vector ( EV ) , Siglec ‐ 5 ( Sig5 ) , or Siglec ‐ 14 ( Sig14 ) overexpression were assessed for the presence of endogenously secreted Hsp70 bound on the cell surface . The mean fluorescence intensity ( MFI ) of signals from an anti ‐ Hsp70 antibody on unpermeabilized THP1 cells is shown .

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Using flow cytometry, undifferentiated or PMA‐differentiated THP1 cells with empty vector (EV), Siglec‐5 (Sig5), or Siglec‐14 (Sig14) overexpression were assessed for the presence of endogenously secreted Hsp70 bound on the cell surface. The mean fluorescence intensity (MFI) of signals from an anti‐Hsp70 antibody on unpermeabilized THP1 cells is shown.


A comparison of the relative binding affinities between Siglec ‐ 5 WT versus R119A Fc , and Siglec ‐ 14 WT versus R119A Fc to Hsp70 by ELISA . The arginine ‐ mutated Siglecs display lower affinity for Hsp70 in comparison with their wild ‐ type sequence .

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A comparison of the relative binding affinities between Siglec‐5 WT versus R119A Fc, and Siglec‐14 WT versus R119A Fc to Hsp70 by ELISA. The arginine‐mutated Siglecs display lower affinity for Hsp70 in comparison with their wild‐type sequence.


A lysine ‐ to ‐ alanine mutation in residue 134 in Siglec ‐ 14 Fc ( K134A ) also abrogates its affinity for Hsp70 , as determined by ELISA . Lysine 134 was identified as a possible residue that changes binding affinity by aligning the sequences of Siglec ‐ 5 and Siglec ‐ 14 with Siglec ‐ 7 . The Siglec ‐ 14 R119A and K134A double mutant ( DM ) Fc reduces the interaction with Hsp70 to background levels .

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A lysine‐to‐alanine mutation in residue 134 in Siglec‐14 Fc (K134A) also abrogates its affinity for Hsp70, as determined by ELISA. Lysine 134 was identified as a possible residue that changes binding affinity by aligning the sequences of Siglec‐5 and Siglec‐14 with Siglec‐7. The Siglec‐14 R119A and K134A double mutant (DM) Fc reduces the interaction with Hsp70 to background levels.


Antibodies against Siglec ‐ 5 and Siglec ‐ 14 neutralized the interaction between Siglec ‐ 14 Fc to Hsp70 , as determined by ELISA .

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Antibodies against Siglec‐5 and Siglec‐14 neutralized the interaction between Siglec‐14 Fc to Hsp70, as determined by ELISA.


Co ‐ incubation of B6N peptide with Siglec ‐ 5 and Siglec ‐ 14 Fc decreased binding of each Siglec to Hsp70 in an ELISA .

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Co‐incubation of B6N peptide with Siglec‐5 and Siglec‐14 Fc decreased binding of each Siglec to Hsp70 in an ELISA.


Secretion of pro ‐ inflammatory cytokine TNFα by THP1 cells transfected with an empty vector ( EV ) or Siglec ‐ 5 ( Sig5 ) expression plasmid was evaluated by ELISA after concurrent exposure with LPS and 10 μg / ml Hsp70 or LPS alone . Secretion of TNFα was reduced only in THP1 Sig5 cells exposed to both LPS and Hsp70 but not LPS alone .

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Secretion of pro‐inflammatory cytokine TNFα by THP1 cells transfected with an empty vector (EV) or Siglec‐5 (Sig5) expression plasmid was evaluated by ELISA after concurrent exposure with LPS and 10 μg/ml Hsp70 or LPS alone. Secretion of TNFα was reduced only in THP1 Sig5 cells exposed to both LPS and Hsp70 but not LPS alone.


Similarly , IL ‐ 8 secretion from THP1 EV and Sig5 cells was evaluated by ELISA after concurrent exposure with LPS and 10 μg / ml Hsp70 . Secretion of IL ‐ 8 was also reduced in only THP1 Sig5cells exposed to both LPS and Hsp70 but not LPS alone .

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Similarly, IL‐8 secretion from THP1 EV and Sig5 cells was evaluated by ELISA after concurrent exposure with LPS and 10 μg/ml Hsp70. Secretion of IL‐8 was also reduced in only THP1 Sig5cells exposed to both LPS and Hsp70 but not LPS alone.


THP1 Sig5 cells were incubated with Hsp70 , and the levels of SHP ‐ 1 recruitment to Siglec ‐ 5 were evaluated by immunoprecipitation of Siglec ‐ 5 and Western blot for SHP ‐ 1 . Higher levels of SHP ‐ 1 were co ‐ immunoprecipitated with Siglec ‐ 5 when the cells were incubated with Hsp70 .

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THP1 Sig5 cells were incubated with Hsp70, and the levels of SHP‐1 recruitment to Siglec‐5 were evaluated by immunoprecipitation of Siglec‐5 and Western blot for SHP‐1. Higher levels of SHP‐1 were co‐immunoprecipitated with Siglec‐5 when the cells were incubated with Hsp70.


Western blots demonstrate phosphorylated p38 ( p ‐ p38 ) was reduced in THP1Siglec ‐ 5cells when exposed to both LPS and Hsp70 in comparison with LPS alone . This reduction was also greater in THP1Siglec ‐ 5cells in comparison with the control THP1cells . Numbers below immunoblots indicate densitometric analysis of each band normalized to the respective cell line ' s unstimulated control group , divided by the respective loading control ( total p38 ) .

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Western blots demonstrate phosphorylated p38 (p‐p38) was reduced in THP1Siglec‐5cells when exposed to both LPS and Hsp70 in comparison with LPS alone. This reduction was also greater in THP1Siglec‐5cells in comparison with the control THP1cells. Numbers below immunoblots indicate densitometric analysis of each band normalized to the respective cell line's unstimulated control group, divided by the respective loading control (total p38).


Secretion of pro ‐ inflammatory cytokine IL ‐ 8 by PMA ‐ differentiated THP1 EV or THP Sig5 cells was evaluated by ELISA after concurrent exposure with 10 ng / ml LPS with an anti ‐ Hsp70 antibody or isotype ‐ matched antibody . Secretion of IL ‐ 8 was increased in only THP1 Sig5 cells exposed to LPS and an anti ‐ Hsp70 antibody in comparison with the isotype control antibody , but this increase was not replicated in THP1 EV cells .

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Secretion of pro‐inflammatory cytokine IL‐8 by PMA‐differentiated THP1 EV or THP Sig5 cells was evaluated by ELISA after concurrent exposure with 10 ng/ml LPS with an anti‐Hsp70 antibody or isotype‐matched antibody. Secretion of IL‐8 was increased in only THP1 Sig5 cells exposed to LPS and an anti‐Hsp70 antibody in comparison with the isotype control antibody, but this increase was not replicated in THP1 EV cells.


Secretion of TNFα by THP1 cells transfected with a Siglec ‐ 5 or Siglec ‐ 14 expression vector was evaluated by ELISA after exposure to Hsp70 at the various concentrations in the presence of endotoxin ‐ neutralizing agent polymyxin B . Only THP1 Siglec ‐ 14 cells exhibited significant secretion of TNFα in an Hsp70 dose ‐ dependent manner .

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Secretion of TNFα by THP1 cells transfected with a Siglec‐5 or Siglec‐14 expression vector was evaluated by ELISA after exposure to Hsp70 at the various concentrations in the presence of endotoxin‐neutralizing agent polymyxin B. Only THP1 Siglec‐14 cells exhibited significant secretion of TNFα in an Hsp70 dose‐dependent manner.


IL ‐ 8 secretion was also evaluated after THP1Siglec ‐ 5 and Siglec ‐ 14cells were exposed to Hsp70 . Similarly , only THP1Siglec ‐ 14cells exhibited significant secretion of IL ‐ 8 in an Hsp70 dose ‐ dependent manner .

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IL‐8 secretion was also evaluated after THP1Siglec‐5 and Siglec‐14cells were exposed to Hsp70. Similarly, only THP1Siglec‐14cells exhibited significant secretion of IL‐8 in an Hsp70 dose‐dependent manner.


Western blot analysis demonstrates a greater increase in phosphorylated p38 ( p ‐ p38 ) from THP1 Siglec ‐ 14 cells after exposure to Hsp70 in comparison with control THP1 EV cells in a time ‐ dependent manner . Graph below immunoblots indicates densitometric analysis of each band normalized to the respective cell line ' s unstimulated control group , divided by the respective loading control .

Western blot analysis demonstrates a greater increase in phosphorylated p38 (p‐p38) from THP1 Siglec‐14 cells after exposure to Hsp70 in comparison with control THP1 EV cells in a time‐dependent manner. Graph below immunoblots indicates densitometric analysis of each band normalized to the respective cell line's unstimulated control group, divided by the respective loading control.


Exposure to Hsp70 also yielded greater degradation of the NF ‐ κB inhibitor , IκBα , in THP1 Siglec ‐ 14 cells in comparison with control THP1 cells , augmentation of the pro ‐ inflammatory response . Graph below immunoblots indicates densitometric analysis of each band normalized to the respective cell line ' s unstimulated control group , divided by the respective loading control .

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Exposure to Hsp70 also yielded greater degradation of the NF‐κB inhibitor, IκBα, in THP1 Siglec‐14 cells in comparison with control THP1 cells, augmentation of the pro‐inflammatory response. Graph below immunoblots indicates densitometric analysis of each band normalized to the respective cell line's unstimulated control group, divided by the respective loading control.


Secretion of pro ‐ inflammatory cytokine IL ‐ 8 by PMA ‐ differentiated THP1 EV or THP Sig 14 cells was evaluated by ELISA after concurrent exposure with an anti ‐ Hsp70 antibody or isotype ‐ matched antibody . Secretion of IL ‐ 8 was decreased in only THP1 Sig 14 cells exposed to LPS and an anti ‐ Hsp70 antibody in comparison with the isotype control antibody , but this change was not observed in THP1 EV cells .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Secretion of pro‐inflammatory cytokine IL‐8 by PMA‐differentiated THP1 EV or THP Sig 14 cells was evaluated by ELISA after concurrent exposure with an anti‐Hsp70 antibody or isotype‐matched antibody. Secretion of IL‐8 was decreased in only THP1 Sig 14 cells exposed to LPS and an anti‐Hsp70 antibody in comparison with the isotype control antibody, but this change was not observed in THP1 EV cells.


TNFα secretion from SIGLEC14 + / + monocytes was measured after exposure to LPS or Hsp70 in the presence or absence of endotoxin ‐ neutralizing compound polymyxin B . Hsp70 was able to induce secretion of TNFα even in the presence of polymyxin B , suggesting a mechanism independent of endotoxin contaminants . DnaK , the E . coli homolog of human Hsp70 , did not induce significant secretion of TNFα above background levels .

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TNFα secretion from SIGLEC14+/+ monocytes was measured after exposure to LPS or Hsp70 in the presence or absence of endotoxin‐neutralizing compound polymyxin B. Hsp70 was able to induce secretion of TNFα even in the presence of polymyxin B, suggesting a mechanism independent of endotoxin contaminants. DnaK, the E. coli homolog of human Hsp70, did not induce significant secretion of TNFα above background levels.


TNFα secretion from SIGLEC14 + / + monocytes by Hsp70 was reduced when the cells were exposed to an anti ‐ Siglec ‐ 5 / Siglec ‐ 14 blocking antibody .

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TNFα secretion from SIGLEC14+/+ monocytes by Hsp70 was reduced when the cells were exposed to an anti‐Siglec‐5/Siglec‐14 blocking antibody.


Human monocytes isolated from SIGLEC14 + / + , SIGLEC14 + / − , and SIGLEC14 − / − individuals were exposed to varying concentrations of Hsp70 , and TNFα secretion was evaluated . The amount of TNFα secreted was normalized to the respective cells ' response to 1 ng / ml LPS to minimize the impact of other possible polymorphisms and factors that contribute to variation between individuals .

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Human monocytes isolated from SIGLEC14+/+, SIGLEC14+/−, and SIGLEC14−/− individuals were exposed to varying concentrations of Hsp70, and TNFα secretion was evaluated. The amount of TNFα secreted was normalized to the respective cells' response to 1 ng/ml LPS to minimize the impact of other possible polymorphisms and factors that contribute to variation between individuals.


TNFα secretion from SIGLEC14 − / − monocytes stimulated with 15 ng / ml LPS and various concentrations of Hsp70 . TNFα secretion was reduced with increasing concentrations of Hsp70 .

TNFα secretion from SIGLEC14−/− monocytes stimulated with 15 ng/ml LPS and various concentrations of Hsp70. TNFα secretion was reduced with increasing concentrations of Hsp70.


A Representative immunoblots of the indicated proteins in lysates from HeLa cells treated with indicated concentrations of halofuginone ( HF ) for 5 h . B Quantification of ATF4 and P - eIF2α from experiments as in ( A ) . Data are mean ± SD ( n = 3 biological replicates ) . * p ≤ 0 . 0414 , * * p ≤ 0 . 0019 , * * * * p < 0 . 0001 , as determined by one - way ANOVA with Dunnett ' s multiple comparison test . Data information : Representative results of at least three independent experiments are shown in each panel .

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A Representative immunoblots of the indicated proteins in lysates from HeLa cells treated with indicated concentrations of halofuginone (HF) for 5 h. B Quantification of ATF4 and P-eIF2α from experiments as in (A). Data are mean ± SD (n = 3 biological replicates). *p ≤ 0.0414, **p ≤ 0.0019, ****p < 0.0001, as determined by one-way ANOVA with Dunnett's multiple comparison test. Data information: Representative results of at least three independent experiments are shown in each panel.


C Similar to ( A ) , but with HeLa cells treated with indicated concentrations of tunicamycin for 5 h . Data information : Representative results of at least three independent experiments are shown in each panel .

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C Similar to (A), but with HeLa cells treated with indicated concentrations of tunicamycin for 5 h. Data information: Representative results of at least three independent experiments are shown in each panel.


D Similar to ( A ) , but using lysates from HeLa cells treated with 62 . 5 nM and 312 . 5 nM HF for indicated times . Data information : Representative results of at least three independent experiments are shown in each panel .

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D Similar to (A), but using lysates from HeLa cells treated with 62.5 nM and 312.5 nM HF for indicated times. Data information: Representative results of at least three independent experiments are shown in each panel.


A Representative immunoblots of indicated proteins in lysates from mouse embryonic fibroblasts ( MEFs ) of indicated genotype after treatment with 12 . 5 nM or 200 nM HF for 5 h . Data information : Representative results of at least three independent experiments are shown in each panel .

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A Representative immunoblots of indicated proteins in lysates from mouse embryonic fibroblasts (MEFs) of indicated genotype after treatment with 12.5 nM or 200 nM HF for 5 h. Data information: Representative results of at least three independent experiments are shown in each panel.


B Similar to ( A ) , but using HeLa cells untreated or treated with GCN2 siRNA 48 h before treatment with indicated concentrations of HF for 5 h . C Similar to ( A ) , but with high HF concentrations . Data information : Representative results of at least three independent experiments are shown in each panel .

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B Similar to (A), but using HeLa cells untreated or treated with GCN2 siRNA 48 h before treatment with indicated concentrations of HF for 5 h. C Similar to (A), but with high HF concentrations. Data information: Representative results of at least three independent experiments are shown in each panel.


B Representative immunoblots of indicated proteins in lysates from HeLa cells after 5 h treatment with indicated concentrations of HF . C Similar to ( B ) , but using HeLa cells untreated or treated with GCN2 siRNA 48 h before treatment with indicated concentrations of HF . Data information : Representative results of at least three independent experiments are shown in each panel .

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B Representative immunoblots of indicated proteins in lysates from HeLa cells after 5 h treatment with indicated concentrations of HF. C Similar to (B), but using HeLa cells untreated or treated with GCN2 siRNA 48 h before treatment with indicated concentrations of HF. Data information: Representative results of at least three independent experiments are shown in each panel.


D Representative immunoblots of lysates from HeLa cells treated with indicated concentrations of HF for 5 h with or without the Nedd8 - activating enzyme inhibitor MLN4924 ( 1 μM ) . Data information : Representative results of at least three independent experiments are shown in each panel .

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D Representative immunoblots of lysates from HeLa cells treated with indicated concentrations of HF for 5 h with or without the Nedd8-activating enzyme inhibitor MLN4924 (1 μM). Data information: Representative results of at least three independent experiments are shown in each panel.


E . Representative immunoblots of lysates from HeLa cells treated with indicated concentrations of HF for 5 h with or without proteasome inhibitor MG - 132 ( 10 μM ) . Data information : Representative results of at least three independent experiments are shown in each panel .

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E. Representative immunoblots of lysates from HeLa cells treated with indicated concentrations of HF for 5 h with or without proteasome inhibitor MG-132 (10 μM). Data information: Representative results of at least three independent experiments are shown in each panel.


A Newly synthesized proteins pulse - labeled with 35S - methionine for 10 min in HeLa cells pre - treated with indicated compounds for 2 . 5h , except ( E ) ( Bottom ) Coomassie - stained gel .

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A Newly synthesized proteins pulse-labeled with 35S-methionine for 10 min in HeLa cells pre-treated with indicated compounds for 2.5h, except (E) (Bottom) Coomassie-stained gel.


B Newly synthesized proteins in HeLa cells treated with 2 . 5 μg / ml Tm or 50 μg / ml cycloheximide ( CHX ) .

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B Newly synthesized proteins in HeLa cells treated with 2.5 μg/ml Tm or 50 μg/ml cycloheximide (CHX).


C Newly synthesized proteins in 4KO or 4KO + GCN2 MEF cells treated with 12 . 5 or 200 nM HF .

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C Newly synthesized proteins in 4KO or 4KO+GCN2 MEF cells treated with 12.5 or 200 nM HF.


D Newly synthesized proteins in eIF2αS / S and eIF2αA / A MEF cells treated with 12 . 5 or 200 nM HF .

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D Newly synthesized proteins in eIF2αS/S and eIF2αA/A MEF cells treated with 12.5 or 200 nM HF.


E Newly synthesized proteins in eIF2αS / S and eIF2αA / A MEF cells treated with indicated concentrations of Tm for 2 . 5 or 5 h .

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

E Newly synthesized proteins in eIF2αS/S and eIF2αA/A MEF cells treated with indicated concentrations of Tm for 2.5 or 5 h.


F Newly synthesized proteins in HeLa cells untreated or treated with GCN2 siRNA 48 h before treatment with indicated concentrations of HF .

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

F Newly synthesized proteins in HeLa cells untreated or treated with GCN2 siRNA 48 h before treatment with indicated concentrations of HF.


G Newly synthesized proteins in HeLa cells treated with indicated concentrations of HF with or without proline supplementation ( 10 mM ) .

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

G Newly synthesized proteins in HeLa cells treated with indicated concentrations of HF with or without proline supplementation (10 mM).


H Representative immunoblots of indicated proteins in lysates from HeLa cells after treatment with indicated concentrations of HF with or without proline supplementation ( 10 mM ) .

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

H Representative immunoblots of indicated proteins in lysates from HeLa cells after treatment with indicated concentrations of HF with or without proline supplementation (10 mM).


I Newly synthesized proteins in HeLa cells untreated or treated with GCN2 siRNA 48 h before treatment with indicated concentrations of histidinol ( His ) or borrelidin ( Bor ) .

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

I Newly synthesized proteins in HeLa cells untreated or treated with GCN2 siRNA 48 h before treatment with indicated concentrations of histidinol (His) or borrelidin (Bor).


G , H Analysis of IC50 values of HF on confluency at 72h endpoint of H441 ( G ) and H23 ( H ) cells normalised to control conditions and determined by nonlinear regression curve fit for inhibitory dose - response ( IC50 ) . Each point represents mean ( n = 3 biological replicates ) ± SD .

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

G, H Analysis of IC50 values of HF on confluency at 72h endpoint of H441 (G) and H23 (H) cells normalised to control conditions and determined by nonlinear regression curve fit for inhibitory dose-response (IC50). Each point represents mean (n = 3 biological replicates) ± SD.


( D ) U2OS ( EGFP - BIR - 5085 ) cells expressing shRNAs for RAD51 , POLD3 or shRNA vector ( Ctrl ) were infected by lentiviruses encoding endonuclease I - SceI . The percentage of EGFP positive cells was assayed by FACS analysis 4 days later ( left ) . Expression of RAD51 and POLD3 is shown by Western blot analysis ( right ) . Data information : Error bars represent the standard deviation ( SD ) of at least three independent experiments . Signiﬁcance of the differences was assayed by Two - tailed non - paired parameters were applied in Student ' s t - test . The P value is indicated as * * P < 0 . 01 , * * * * P < 0 . 0001 and n . s . ( not significant ) P > 0 . 05 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) U2OS (EGFP-BIR-5085) cells expressing shRNAs for RAD51, POLD3 or shRNA vector (Ctrl) were infected by lentiviruses encoding endonuclease I-SceI. The percentage of EGFP positive cells was assayed by FACS analysis 4 days later (left). Expression of RAD51 and POLD3 is shown by Western blot analysis (right). Data information: Error bars represent the standard deviation (SD) of at least three independent experiments. Signiﬁcance of the differences was assayed by Two-tailed non-paired parameters were applied in Student's t-test. The P value is indicated as **P < 0.01, ****P < 0.0001 and n.s. (not significant) P>0.05.


( B ) U2OS WT or PIF1 - KO cells were labeled with CldU for 30 min followed by incubation with 2 mM HU for 2 hr and then IdU for another 30 min . Labeled cells were processed for DNA fiber analysis . Representative images of stalled or restarted forks and forks with new origin firing were shown ( left ) . The percentage of restarted forks was quantified by analyzing of 110 - 130 fibers for each experiment ( right ) . Experiments were repeated four times for each sample . Data information : Error bars represent the standard deviation ( SD ) of at least three independent experiments . Signiﬁcance of the differences was assayed by Two - tailed non - paired parameters were applied in Student ' s t - test . The P value is indicated as * * P < 0 . 01 , * * * P < 0 . 001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) U2OS WT or PIF1-KO cells were labeled with CldU for 30 min followed by incubation with 2 mM HU for 2 hr and then IdU for another 30 min. Labeled cells were processed for DNA fiber analysis. Representative images of stalled or restarted forks and forks with new origin firing were shown (left). The percentage of restarted forks was quantified by analyzing of 110-130 fibers for each experiment (right). Experiments were repeated four times for each sample. Data information: Error bars represent the standard deviation (SD) of at least three independent experiments. Signiﬁcance of the differences was assayed by Two-tailed non-paired parameters were applied in Student's t-test. The P value is indicated as **P < 0.01, ***P < 0.001.


( D ) U2OS ( EGFP - BIR - 5085 ) cell lines carrying Dox - inducible Cas9 / sgRNA - 5085 ( Dox - Cas9 ) or Cas9n / sgRNA - 5085 ( Dox - Cas9n ) were incubated with or without Dox ( 5 µg / ml ) and assayed by FACS analysis 2 days later ( left ) . U2OS ( EGFP - BIR - 5085 , Dox - Cas9 or Dox - Cas9n ) cells expressing shRNAs RAD51 , POLD3 and PIF1 or shRNA vector ( Ctrl ) were incubated with 5 µg / ml Dox , and FACS analysis was performed after 2 days ( right ) . Data information : Error bars represent the standard deviation ( SD ) of at least three independent experiments . Signiﬁcance of the differences was assayed by Two - tailed non - paired parameters were applied in Student ' s t - test . The P value is indicated as * * P < 0 . 01 , * * * P < 0 . 001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) U2OS (EGFP-BIR-5085) cell lines carrying Dox-inducible Cas9/sgRNA-5085 (Dox-Cas9) or Cas9n/sgRNA-5085 (Dox-Cas9n) were incubated with or without Dox (5 µg/ml) and assayed by FACS analysis 2 days later (left). U2OS (EGFP-BIR-5085, Dox-Cas9 or Dox-Cas9n) cells expressing shRNAs RAD51, POLD3 and PIF1 or shRNA vector (Ctrl) were incubated with 5 µg/ml Dox, and FACS analysis was performed after 2 days (right). Data information: Error bars represent the standard deviation (SD) of at least three independent experiments. Signiﬁcance of the differences was assayed by Two-tailed non-paired parameters were applied in Student's t-test. The P value is indicated as **P < 0.01, ***P < 0.001.


( E ) U2OS ( EGFP - Flex1 - BIR - 5086 ) cells expressing shRNAs for RAD51 , POLD3 and PIF1 or shRNA vector ( Ctrl ) were infected by retroviruses expressing RAS , and the percentage of EGFP positive cells was quantified by FACS analysis 4 days after retrovirus infection ( top ) . Expression level of RAD51 and POLD3 is shown by Western blot analysis ( bottom ) . Data information : Error bars represent the standard deviation ( SD ) of at least three independent experiments . Signiﬁcance of the differences was assayed by Two - tailed non - paired parameters were applied in Student ' s t - test . The P value is indicated as * * P < 0 . 01 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) U2OS (EGFP-Flex1-BIR-5086) cells expressing shRNAs for RAD51, POLD3 and PIF1 or shRNA vector (Ctrl) were infected by retroviruses expressing RAS, and the percentage of EGFP positive cells was quantified by FACS analysis 4 days after retrovirus infection (top). Expression level of RAD51 and POLD3 is shown by Western blot analysis (bottom). Data information: Error bars represent the standard deviation (SD) of at least three independent experiments. Signiﬁcance of the differences was assayed by Two-tailed non-paired parameters were applied in Student's t-test. The P value is indicated as **P < 0.01.


( F ) U2OS WT or PIF1 - KO cells were infected by retrovirus expressing RAS or empty vector . Cell profiling was plotted by counting cell numbers every 24 hr , and normalized to the cell number on the first day . Data information : Error bars represent the standard deviation ( SD ) of at least three independent experiments . Signiﬁcance of the differences was assayed by Two - tailed non - paired parameters were applied in Student ' s t - test . The P value is indicated as * * P < 0 . 01 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) U2OS WT or PIF1-KO cells were infected by retrovirus expressing RAS or empty vector. Cell profiling was plotted by counting cell numbers every 24 hr, and normalized to the cell number on the first day. Data information: Error bars represent the standard deviation (SD) of at least three independent experiments. Signiﬁcance of the differences was assayed by Two-tailed non-paired parameters were applied in Student's t-test. The P value is indicated as **P < 0.01.


( C ) U2OS ( EGFP - STGC - 1731 ) cell lines carrying Dox - inducible Cas9 / sgRNA - 1731 ( Dox - Cas9 ) or Cas9n / sgRNA ( Dox - Cas9n ) were incubated with or without Dox ( 5 µg / ml ) , and the percentage of EGFP positive cells was quantified by FACS analysis 2 days later ( top ) . U2OS ( EGFP - STGC - 1731 , Dox - Cas9 or Dox - Cas9n ) cells expressing shRNAs for RAD51 , POLD3 and PIF1 or shRNA control ( Ctrl ) were incubated with Dox ( 5 µg / ml ) , and the percentage of EGFP positive cells was quantified by FACS analysis after 2 days ( bottom ) . Data information : Error bars represent the standard deviation ( SD ) of at least three independent experiments . Signiﬁcance of the differences was assayed by Two - tailed non - paired parameters were applied in Student ' s t - test . The P value is indicated as * * P < 0 . 01 and n . s . ( not significant ) P > 0 . 05 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) U2OS (EGFP-STGC-1731) cell lines carrying Dox-inducible Cas9/sgRNA-1731 (Dox-Cas9) or Cas9n/sgRNA (Dox-Cas9n) were incubated with or without Dox (5 µg/ml), and the percentage of EGFP positive cells was quantified by FACS analysis 2 days later (top). U2OS (EGFP-STGC-1731, Dox-Cas9 or Dox-Cas9n) cells expressing shRNAs for RAD51, POLD3 and PIF1 or shRNA control (Ctrl) were incubated with Dox (5 µg/ml), and the percentage of EGFP positive cells was quantified by FACS analysis after 2 days (bottom). Data information: Error bars represent the standard deviation (SD) of at least three independent experiments. Signiﬁcance of the differences was assayed by Two-tailed non-paired parameters were applied in Student's t-test. The P value is indicated as **P < 0.01 and n.s. (not significant) P>0.05.


( E ) U2OS ( EGFP - Flex1 - STGC - 1541 ) cells were treated with or without 2 mM HU for 24 hr , and the percentage of EGFP positive cells by HU induction was quantified by FACS analysis 4 days after removal of HU ( left ) . U2OS ( EGFP - Flex1 - STGC - 1541 ) cells expressing shRNAs for RAD51 , POLD3 and PIF1 or shRNA vector ( Ctrl ) were treated with 2 mM HU for 24 hr , and the percentage of EGFP positive cells was quantified by FACS analysis 4 days later ( right ) . Data information : Error bars represent the standard deviation ( SD ) of at least three independent experiments . Signiﬁcance of the differences was assayed by Two - tailed non - paired parameters were applied in Student ' s t - test . The P value is indicated as * * P < 0 . 01 and n . s . ( not significant ) P > 0 . 05 .

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

(E) U2OS (EGFP-Flex1-STGC-1541) cells were treated with or without 2 mM HU for 24 hr, and the percentage of EGFP positive cells by HU induction was quantified by FACS analysis 4 days after removal of HU (left). U2OS (EGFP-Flex1-STGC-1541) cells expressing shRNAs for RAD51, POLD3 and PIF1 or shRNA vector (Ctrl) were treated with 2 mM HU for 24 hr, and the percentage of EGFP positive cells was quantified by FACS analysis 4 days later (right). Data information: Error bars represent the standard deviation (SD) of at least three independent experiments. Signiﬁcance of the differences was assayed by Two-tailed non-paired parameters were applied in Student's t-test. The P value is indicated as **P < 0.01 and n.s. (not significant) P>0.05.


( F ) U2OS ( EGFP - Flex1 - STGC - 1541 ) cells were infected by retroviruses expressing RAS or empty vector , and the percentage of EGFP positive cells was assayed by FACS 5 days following infection ( left ) . U2OS ( EGFP - Flex1 - STGC - 1541 ) cells expressing shRNAs for RAD51 , POLD3 and PIF1 shRNAs or shRNA vector were infected by retroviruses expressing RAS , and the percentage of EGFP positive cells was assayed by FACS 5 days after infection ( middle ) . Expression level of RAD51 and POLD3 is shown by Western blot analysis ( right ) . Data information : Error bars represent the standard deviation ( SD ) of at least three independent experiments . Signiﬁcance of the differences was assayed by Two - tailed non - paired parameters were applied in Student ' s t - test . The P value is indicated as * * P < 0 . 01 and n . s . ( not significant ) P > 0 . 05 .

(F) U2OS (EGFP-Flex1-STGC-1541) cells were infected by retroviruses expressing RAS or empty vector, and the percentage of EGFP positive cells was assayed by FACS 5 days following infection (left). U2OS (EGFP-Flex1-STGC-1541) cells expressing shRNAs for RAD51, POLD3 and PIF1 shRNAs or shRNA vector were infected by retroviruses expressing RAS, and the percentage of EGFP positive cells was assayed by FACS 5 days after infection (middle). Expression level of RAD51 and POLD3 is shown by Western blot analysis (right). Data information: Error bars represent the standard deviation (SD) of at least three independent experiments. Signiﬁcance of the differences was assayed by Two-tailed non-paired parameters were applied in Student's t-test. The P value is indicated as **P < 0.01 and n.s. (not significant) P>0.05.


( D ) U2OS ( EGFP / STGC - mCherry / LTGC - 5034 , Dox - Cas9 ( left ) or Dox - Cas9n ( right ) ) cells expressing shRNAs for POLD3 and PIF1 or shRNA vector ( Ctrl ) were incubated with Dox ( 5 µg / ml ) . The percentage of EGFP or mCherry positive cells after induction was quantified by FACS analysis 2 days later to determine the percentage of EGFP or mCherry positive cells . Data information : Error bars represent the standard deviation ( SD ) of at least three independent experiments . Signiﬁcance of the differences was assayed by Two - tailed non - paired parameters were applied in Student ' s t - test . The P value is indicated as * * P < 0 . 01 and n . s . ( not significant ) P > 0 . 05 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) U2OS (EGFP/STGC-mCherry/LTGC-5034, Dox-Cas9 (left) or Dox-Cas9n (right)) cells expressing shRNAs for POLD3 and PIF1 or shRNA vector (Ctrl) were incubated with Dox (5 µg/ml). The percentage of EGFP or mCherry positive cells after induction was quantified by FACS analysis 2 days later to determine the percentage of EGFP or mCherry positive cells. Data information: Error bars represent the standard deviation (SD) of at least three independent experiments. Signiﬁcance of the differences was assayed by Two-tailed non-paired parameters were applied in Student's t-test. The P value is indicated as **P < 0.01 and n.s. (not significant) P>0.05.


( E ) U2OS ( EGFP - Flex1 - STGC - 1541 ) cells expressing shRNAs for RFC1 and PCNA or vector ( Ctrl ) were treated with 2 mM HU for 24 hr . The percentage of EGFP positive cells after HU treatment was quantified by FACS analysis 3 days after HU removal ( left ) . U2OS ( EGFP - STGC - 1731 ) cells expressing shRNAs for RFC1 and PCNA or vector ( Ctrl ) were infected by lentivirus expressing I - SceI . The percentage of EGFP positive cells by I - SceI induction was quantified by FACS analysis 4 days later ( right ) . Data information : Error bars represent the standard deviation ( SD ) of at least three independent experiments . Signiﬁcance of the differences was assayed by Two - tailed non - paired parameters were applied in Student ' s t - test . The P value is indicated as * * P < 0 . 01 and n . s . ( not significant ) P > 0 . 05 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) U2OS (EGFP-Flex1-STGC-1541) cells expressing shRNAs for RFC1 and PCNA or vector (Ctrl) were treated with 2 mM HU for 24 hr. The percentage of EGFP positive cells after HU treatment was quantified by FACS analysis 3 days after HU removal (left). U2OS (EGFP-STGC-1731) cells expressing shRNAs for RFC1 and PCNA or vector (Ctrl) were infected by lentivirus expressing I-SceI. The percentage of EGFP positive cells by I-SceI induction was quantified by FACS analysis 4 days later (right). Data information: Error bars represent the standard deviation (SD) of at least three independent experiments. Signiﬁcance of the differences was assayed by Two-tailed non-paired parameters were applied in Student's t-test. The P value is indicated as **P < 0.01 and n.s. (not significant) P>0.05.


( F ) U2OS ( EGFP / STGC - mCherry / LTGC - 5034 , Dox - Cas9 ( left ) or Dox - Cas9n ( right ) ) cells expressing shRNAs for RFC1 and PCNA or vector ( Ctrl ) were incubated with Dox ( 5 µg / ml ) . The percentage of EGFP or mCherry positive cells after induction was quantified by FACS analysis 2 days later . Data information : Error bars represent the standard deviation ( SD ) of at least three independent experiments . Signiﬁcance of the differences was assayed by Two - tailed non - paired parameters were applied in Student ' s t - test . The P value is indicated as * * P < 0 . 01 and n . s . ( not significant ) P > 0 . 05 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) U2OS (EGFP/STGC-mCherry/LTGC-5034, Dox-Cas9 (left) or Dox-Cas9n (right)) cells expressing shRNAs for RFC1 and PCNA or vector (Ctrl) were incubated with Dox (5 µg/ml). The percentage of EGFP or mCherry positive cells after induction was quantified by FACS analysis 2 days later. Data information: Error bars represent the standard deviation (SD) of at least three independent experiments. Signiﬁcance of the differences was assayed by Two-tailed non-paired parameters were applied in Student's t-test. The P value is indicated as **P < 0.01 and n.s. (not significant) P>0.05.


( G ) FLAG - PIF1 was stably expressed in U2OS ( EGFP - Flex1 - STGC - 1541 ) cells by lentiviral infection . Enrichment of FLAG - PIF1 at Flex1 site or GAPDH site with or without HU ( 2 mM , 10 hr ) treatment was calculated by anti - FLAG ChIP ( left ) . When PCNA was depleted by shRNA using vector as a control ( Ctrl ) , enrichment of FLAG - PIF1 at Flex1 site was calculated by anti - FLAG ChIP ( right ) . ChIP value in cells without HU treatment is set up as 1 for normalization . Data information : Error bars represent the standard deviation ( SD ) of at least three independent experiments . Signiﬁcance of the differences was assayed by Two - tailed non - paired parameters were applied in Student ' s t - test . The P value is indicated as * * P < 0 . 01 and n . s . ( not significant ) P > 0 . 05 .

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

(G) FLAG-PIF1 was stably expressed in U2OS (EGFP-Flex1-STGC-1541) cells by lentiviral infection. Enrichment of FLAG-PIF1 at Flex1 site or GAPDH site with or without HU (2 mM, 10 hr) treatment was calculated by anti-FLAG ChIP (left). When PCNA was depleted by shRNA using vector as a control (Ctrl), enrichment of FLAG-PIF1 at Flex1 site was calculated by anti-FLAG ChIP (right). ChIP value in cells without HU treatment is set up as 1 for normalization. Data information: Error bars represent the standard deviation (SD) of at least three independent experiments. Signiﬁcance of the differences was assayed by Two-tailed non-paired parameters were applied in Student's t-test. The P value is indicated as **P < 0.01 and n.s. (not significant) P>0.05.


( D ) Growth curves of WT or FANCM - KO HCT116 cells were plotted after expressing POLD3 ( left ) or PIF1 ( right ) shRNA or shRNA vector ( Ctrl ) . Cell number was normalized to that on day one . Data information : Error bars represent the standard deviation ( SD ) of at least three independent experiments . Signiﬁcance of the differences was assayed by Two - tailed non - paired parameters were applied in Student ' s t - test . The P value is indicated as * * P < 0 . 01 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Growth curves of WT or FANCM-KO HCT116 cells were plotted after expressing POLD3 (left) or PIF1 (right) shRNA or shRNA vector (Ctrl). Cell number was normalized to that on day one. Data information: Error bars represent the standard deviation (SD) of at least three independent experiments. Signiﬁcance of the differences was assayed by Two-tailed non-paired parameters were applied in Student's t-test. The P value is indicated as **P < 0.01.


( C ) Anti - γH2AX ChIP analysis at FRA3B , FRA16D and GAPDH genomic loci was performed in U2OS WT and PIF1 - KO cells before and after APH treatment ( 0 . 4 μM , 24 hr ) . Enrichment of γH2AX at FRA3B , FRA16D and GAPDH loci in WT and PIF1 KO cells was calculated using ChIP value in WT cells as 1 for normalization . Data information : Error bars represent the standard deviation ( SD ) of at least three independent experiments . Signiﬁcance of the differences was assayed by Two - tailed non - paired parameters were applied in Student ' s t - test . The P value is indicated as * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 and n . s . ( not significant ) P > 0 . 05 .

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

(C) Anti-γH2AX ChIP analysis at FRA3B, FRA16D and GAPDH genomic loci was performed in U2OS WT and PIF1-KO cells before and after APH treatment (0.4 μM, 24 hr). Enrichment of γH2AX at FRA3B, FRA16D and GAPDH loci in WT and PIF1 KO cells was calculated using ChIP value in WT cells as 1 for normalization. Data information: Error bars represent the standard deviation (SD) of at least three independent experiments. Signiﬁcance of the differences was assayed by Two-tailed non-paired parameters were applied in Student's t-test. The P value is indicated as *P < 0.05, **P < 0.01, ***P < 0.001 and n.s. (not significant) P>0.05.


( E ) U2OS ( EGFP - BIR - 5085 ) cells expressing PIF1 WT or L319P mutant with endogenous PIF1 silenced by shRNA were infected by lentiviruses to express I - SceI . The percentage of EGFP positive cells was quantified by FACS analysis 5 days after I - SceI infection . Data information : Error bars represent the standard deviation ( SD ) of at least three independent experiments . Signiﬁcance of the differences was assayed by Two - tailed non - paired parameters were applied in Student ' s t - test . The P value is indicated as * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 and n . s . ( not significant ) P > 0 . 05 .

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(E) U2OS (EGFP-BIR-5085) cells expressing PIF1 WT or L319P mutant with endogenous PIF1 silenced by shRNA were infected by lentiviruses to express I-SceI. The percentage of EGFP positive cells was quantified by FACS analysis 5 days after I-SceI infection. Data information: Error bars represent the standard deviation (SD) of at least three independent experiments. Signiﬁcance of the differences was assayed by Two-tailed non-paired parameters were applied in Student's t-test. The P value is indicated as *P < 0.05, **P < 0.01, ***P < 0.001 and n.s. (not significant) P>0.05.


( I ) Anti - γH2AX ChIP analysis at FRA3B , FRA16D and GAPDH genomic loci was performed in U2OS WT and PIF1 - KO cells , or PIF1 - KO cells expressing PIF1 - WT , and E307Q and L319P mutants before and after APH treatment ( 0 . 4 μM , 24 hr ) . Enrichment of γH2AX at FRA3B , FRA16D and GAPDH loci was calculated using ChIP value in WT cells as 1 for normalization . Data information : Error bars represent the standard deviation ( SD ) of at least three independent experiments . Signiﬁcance of the differences was assayed by Two - tailed non - paired parameters were applied in Student ' s t - test . The P value is indicated as * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 and n . s . ( not significant ) P > 0 . 05 .

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(I) Anti-γH2AX ChIP analysis at FRA3B, FRA16D and GAPDH genomic loci was performed in U2OS WT and PIF1-KO cells, or PIF1-KO cells expressing PIF1-WT, and E307Q and L319P mutants before and after APH treatment (0.4 μM, 24 hr). Enrichment of γH2AX at FRA3B, FRA16D and GAPDH loci was calculated using ChIP value in WT cells as 1 for normalization. Data information: Error bars represent the standard deviation (SD) of at least three independent experiments. Signiﬁcance of the differences was assayed by Two-tailed non-paired parameters were applied in Student's t-test. The P value is indicated as *P < 0.05, **P < 0.01, ***P < 0.001 and n.s. (not significant) P>0.05.


Figure 1 . Identification of non - canonical p53 binding sites in human subtelomeres . ( A and B ) ChIP - seq tracks for p53 in camptothecin ( CPT ) treated or untreated HCT116 cells were aligned with RNAPII , CTCF , RAD21 , and SMC1 for human chromosome18q ( A ) or 13q ( B ) subtelomeres . Position relative to the centromeric end of the terminal TTAGGG repeat tract are indicated in bp at the top . Subtelomeric DNA paralogy segment tracks , internal telomere - like sequence tracks ( red CCCTAA ) , and annotated RefSeq gene tracks are indicated above the ChIP - seq tracks . ( C ) Alignment of human subtelomeric elements containing non - canonical p53 recognition sequences . The position of the RepeatMasker - defined LTR10c element is shown ; also note the ( TG ) n microsatellite repeat adjacent to the p53 binding site . ( D ) Schematic organization of non - canonical p53 binding sites found in human subtelomeres .

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Figure 1. Identification of non-canonical p53 binding sites in human subtelomeres. (A and B) ChIP-seq tracks for p53 in camptothecin (CPT) treated or untreated HCT116 cells were aligned with RNAPII, CTCF, RAD21, and SMC1 for human chromosome18q (A) or 13q (B) subtelomeres. Position relative to the centromeric end of the terminal TTAGGG repeat tract are indicated in bp at the top. Subtelomeric DNA paralogy segment tracks, internal telomere-like sequence tracks (red CCCTAA), and annotated RefSeq gene tracks are indicated above the ChIP-seq tracks. (C) Alignment of human subtelomeric elements containing non-canonical p53 recognition sequences. The position of the RepeatMasker-defined LTR10c element is shown; also note the (TG)n microsatellite repeat adjacent to the p53 binding site. (D) Schematic organization of non-canonical p53 binding sites found in human subtelomeres.


Stress - induced p53 binding at human subtelomeres . ( A ) Western blot of HCT116 p53 - / - and p53 + / + cells treated with DMSO ( D ) or 50 µM etoposide ( E ) for 24 hrs . Cell lysates were assayed for total p53 , phospho - Ser15 p53 ( p - p53 ) , γH2AX , or GAPDH .

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Stress-induced p53 binding at human subtelomeres. (A) Western blot of HCT116 p53-/- and p53+/+ cells treated with DMSO (D) or 50 µM etoposide (E) for 24 hrs. Cell lysates were assayed for total p53, phospho-Ser15 p53 (p-p53), γH2AX, or GAPDH.


( B ) ChIP - qPCR was assayed in HCT116 p53 - / - or p53 + / + cells treated with DMSO or etoposide as in panel A , and assayed by ChIP with antibodies to p53 ( Ab6 ) or control IgG , followed by qPCR for p53 binding site in subtelomere 18q ( top panel ) or 13q ( lower panel ) .

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(B) ChIP-qPCR was assayed in HCT116 p53-/- or p53 +/+ cells treated with DMSO or etoposide as in panel A, and assayed by ChIP with antibodies to p53 (Ab6) or control IgG, followed by qPCR for p53 binding site in subtelomere 18q (top panel) or 13q (lower panel).


( C ) HCT116 p53 - / - and p53 + / + cells were cultured in DMEM with 10 % FBS or Hank ' s buffered saline solution ( HBSS ) lacking FBS and then assayed by ChIP with either IgG control ( grey ) or two different p53 - specific antibodies Ab6 ( red ) or FL ( black ) . ChIP was assayed by qPCR with primers proximal to p53 binding sites at 18q , 13q , or p21 promoter .

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(C) HCT116 p53-/- and p53+/+ cells were cultured in DMEM with 10% FBS or Hank's buffered saline solution (HBSS) lacking FBS and then assayed by ChIP with either IgG control (grey) or two different p53-specific antibodies Ab6 (red) or FL(black). ChIP was assayed by qPCR with primers proximal to p53 binding sites at 18q, 13q, or p21 promoter.


. ( D ) Doxycyclin ( Dox ) - inducible p53 H1299 cells were treated without ( - ) or with ( + ) Dox in media containing 10 % serum or in serum free HBSS , and then subject to ChIP assay with control IgG or p53 - specific Ab6 antibody . ChIP DNA was assayed by qPCR using primers specific for p53 binding site at p21 ( top panel ) , p53 - negative control IGX1A ( middle panel ) , or p21 positive control ( lower panel ) . Error bars represent SD , and * indicates p value < . 05 . p - values were determined by Student t - test .

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. (D) Doxycyclin (Dox)-inducible p53 H1299 cells were treated without (-) or with (+) Dox in media containing 10% serum or in serum free HBSS, and then subject to ChIP assay with control IgG or p53-specific Ab6 antibody. ChIP DNA was assayed by qPCR using primers specific for p53 binding site at p21 (top panel), p53-negative control IGX1A (middle panel), or p21 positive control (lower panel). Error bars represent SD, and * indicates p value < .05. p-values were determined by Student t-test.


. Transcriptional activity of subtelomeric p53 - response elements . ( A ) Luciferase test constructs containing either empty test vector ( top ) , p53 - response elements from 18q subtelomere ( middle ) or MDM2 promoter ( lower ) were assayed in HCT p53 - / - cells transfected with two independent preparations of empty vector or p53 WT expression vector .

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. Transcriptional activity of subtelomeric p53-response elements. (A) Luciferase test constructs containing either empty test vector (top), p53-response elements from 18q subtelomere (middle) or MDM2 promoter (lower) were assayed in HCT p53-/- cells transfected with two independent preparations of empty vector or p53 WT expression vector.


( B ) RT - qPCR measure of p21 ( top panel ) or PARD6G ( lower panel ) in HCT116 p53 - / - or p53 + / + cells after treatment with DMSO or 50 µM etoposide .

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(B) RT-qPCR measure of p21 (top panel) or PARD6G (lower panel) in HCT116 p53-/- or p53+/+ cells after treatment with DMSO or 50 µM etoposide.


( C ) RT - qPCR analysis of TERRA transcripts from subtelomere 13q ( upper ) or 2q ( lower ) .

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(C) RT-qPCR analysis of TERRA transcripts from subtelomere 13q (upper) or 2q (lower).


( D ) RT - qPCR analysis of TERRA transcripts from 18q using two independent primer sets pr1 ( top panel ) or pr2 ( lower panel ) from HCT116 p53 + / + cells cultured with serum ( grey bars ) or in HBSS lacking serum ( black bars ) . Error bars represent SD and * indicates p value < . 05 using student t - test comparing treated and untreated samples .

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(D) RT-qPCR analysis of TERRA transcripts from 18q using two independent primer sets pr1 (top panel) or pr2 (lower panel) from HCT116 p53+/+ cells cultured with serum (grey bars) or in HBSS lacking serum (black bars). Error bars represent SD and * indicates p value <.05 using student t-test comparing treated and untreated samples.


. p53 - dependent cell viability and telomere protection in response to DNA damage . ( A ) Western blot of total p53 , γH2AX , and GAPDH in HCT116 p53 - / - or p53 + / + cells treated with 1 µM or 50 µM etoposide for 24 hrs .

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. p53-dependent cell viability and telomere protection in response to DNA damage. (A) Western blot of total p53, γH2AX, and GAPDH in HCT116 p53-/- or p53+/+ cells treated with 1 µM or 50 µM etoposide for 24 hrs.


s . ( B ) Cell cycle analysis of HCT116 p53 - / - or p53 + / + cells untreated or treated with 50 µM etoposide for 24 hrs . Cell cycle was determined by FACS analysis of propidium iodide stained cells .

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s. (B) Cell cycle analysis of HCT116 p53-/- or p53+/+ cells untreated or treated with 50 µM etoposide for 24 hrs. Cell cycle was determined by FACS analysis of propidium iodide stained cells.


( C ) Cell clonal survival assay for HCT116 p53 - / - or p53 + / + cells treated with DMSO or 1 , 1 . 5 or 2 µM etoposide for 24 hr prior to replating .

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(C) Cell clonal survival assay for HCT116 p53-/- or p53+/+ cells treated with DMSO or 1, 1.5 or 2 µM etoposide for 24 hr prior to replating.


. ( D ) Quantification of clonal survival assay relative total cell numbers ( top panel ) or normalized to DMSO controls ( lower panel ) .

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. (D) Quantification of clonal survival assay relative total cell numbers (top panel) or normalized to DMSO controls (lower panel).


E ) TIF assay for HCT116 p53 + / + or p53 - / - cells treated with 1 µM etoposide for 3 hrs . Telomere DNA FISH with TelC probe ( red ) , γH2AX ( green ) , Dapi ( blue ) .

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E) TIF assay for HCT116 p53+/+ or p53-/- cells treated with 1 µM etoposide for 3 hrs. Telomere DNA FISH with TelC probe (red), γH2AX (green), Dapi (blue).


( F ) Quantification of TIF assay for % cells with > 4 TIFs per cell for p53 + / + ( black ) or p53 - / - ( red ) cell .

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(F) Quantification of TIF assay for % cells with > 4 TIFs per cell for p53+/+ (black) or p53-/- (red) cell.


G ) Telomere length assay showing p53 - / - or p53 + / + cells treated with either DMS0 , 1 or 50 µM etoposide . Ethidium bromide ( EtBr ) stained agarose gels ( left panel ) , Southern blot hybridized with Tel C probe ( middle panel ) or with α Satellite DNA promote ( right panel ) . Quantification of telomereDNA signal relative to invariant bands in α Sat . Error bars represent SD and * indicates p values < . 05 determined by student t - test ( TLA ) , or chi - square ( TIF assay ) .

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G) Telomere length assay showing p53-/- or p53+/+ cells treated with either DMS0, 1 or 50 µM etoposide. Ethidium bromide (EtBr) stained agarose gels (left panel), Southern blot hybridized with Tel C probe (middle panel) or with α Satellite DNA promote (right panel). Quantification of telomereDNA signal relative to invariant bands in α Sat. Error bars represent SD and * indicates p values <.05 determined by student t-test (TLA), or chi-square (TIF assay).


( H ) Two - dimensional agarose gel analysis of telomeric DNA from HCT p53 + / + or or p53 - / - treated with 50 µM etoposide for 24 hrs . Ethidium gel ( top ) and Southern blot probed with TelG probe ( lower panel ) . Single stranded telomere DNA ( red arrow ) and recombination / replication - associated structures ( blue arrow ) are indicated .

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(H) Two-dimensional agarose gel analysis of telomeric DNA from HCT p53+/+ or or p53-/- treated with 50 µM etoposide for 24 hrs. Ethidium gel (top) and Southern blot probed with TelG probe (lower panel). Single stranded telomere DNA (red arrow) and recombination/ replication-associated structures (blue arrow) are indicated.


Figure 5 . p53 - dependent histone modification changes in response to DNA damage stress . HCT116p53 - / - ( left panels ) or p53 + / + ( right panels ) were treated with DMSO ( black ) or 50 µM etoposide ( red ) for 24 hrs and assayed by ChIP - qPCR at various locations relative to the TTAGGG repeats across the chromosome18qsubtelomere ( panel A ) or 13qsubtelomere ( panel B ) or for control regions ( p21 promoter , GKN1 / 2 , PARKIN ) as indicated below . ChIP - for p53 , H3K9Ac , H3K27Ac , γH2AX , or IgG control were quantified as % input . Error bars indicates SD and * indicates p value < . 05 using student t - test comparing ChIP values in p53 + / + relative to p53 - / - cells for specific primer pairs .

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Figure 5. p53-dependent histone modification changes in response to DNA damage stress. HCT116p53-/- (left panels) or p53 +/+ (right panels) were treated with DMSO (black) or 50 µM etoposide (red) for 24 hrs and assayed by ChIP-qPCR at various locations relative to the TTAGGG repeats across the chromosome18qsubtelomere (panel A) or 13qsubtelomere (panel B) or for control regions (p21 promoter, GKN1/2, PARKIN) as indicated below. ChIP- for p53, H3K9Ac, H3K27Ac, γH2AX, or IgG control were quantified as % input. Error bars indicates SD and * indicates p value<.05 using student t-test comparing ChIP values in p53+/+ relative to p53-/- cells for specific primer pairs.


( C ) Dot blot analysis of telomere repeat or Alu repeat DNA from p53 + / + or p53 - / - HCT116 cells treated with DMSO or etoposide ( ETP ) ( as described in panel A ) , that were subject to ChIP with antibodies to IgG , p53 , γH2AX , TRF2 , or TRF1 . Quantitation of three independent ChIP - assays for TRF1 and TRF2 is shown in the panel to the right . (

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(C) Dot blot analysis of telomere repeat or Alu repeat DNA from p53+/+ or p53-/- HCT116 cells treated with DMSO or etoposide (ETP) (as described in panel A), that were subject to ChIP with antibodies to IgG, p53, γH2AX, TRF2, or TRF1. Quantitation of three independent ChIP-assays for TRF1 and TRF2 is shown in the panel to the right. (


( E ) ChIP - qPCR for TRF2 is shown for cells treated as in panel A using primers across the subtelomeres of 18q ( left ) or 13q ( right ) .

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(E) ChIP-qPCR for TRF2 is shown for cells treated as in panel A using primers across the subtelomeres of 18q (left) or 13q (right).


( A ) HCT116 p53 + / + or p53 - / - cells treated with 50 µM etoposide were assayed by Western blot with antibodies for p53 , γH2AX , H3K9Ac , panH3 , and GAPDH at times 0 , 8 , 16 , or 24 hr post - treatment .

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(A) HCT116 p53+/+ or p53-/- cells treated with 50 µM etoposide were assayed by Western blot with antibodies for p53, γH2AX, H3K9Ac, panH3, and GAPDH at times 0, 8, 16, or 24 hr post-treatment.


( B ) ChIP - qPCR for γH2AX ( upper panels ) or IgG control ( lower panels ) for cells treated as indicated in panel A . ( C - D ) RT - qPCR analysis for cells treated as shown in panel A .

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(B) ChIP-qPCR for γH2AX (upper panels) or IgG control (lower panels) for cells treated as indicated in panel A. (C-D) RT-qPCR analysis for cells treated as shown in panel A.


C - D ) RT - qPCR analysis for cells treated as shown in panel A . ( C ) RT - PCR for the p21 ( top ) , or PARD6G mRNA ( lower ) . ( D ) RT - PCR for primer pairs spanning subtelomere18q as indicated below . Lower panel shows the No - RT control for 18qsubtelomere primers . RT - qPCR was quantified as relative to Actin with error bars indicating SD and * indicating p value < . 05 using student t - test comparing etoposide ( 24 hr ) relative to untreated for specific primer pairs .

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C-D) RT-qPCR analysis for cells treated as shown in panel A. (C) RT-PCR for the p21 (top), or PARD6G mRNA (lower). (D) RT-PCR for primer pairs spanning subtelomere18q as indicated below. Lower panel shows the No-RT control for 18qsubtelomere primers. RT-qPCR was quantified as relative to Actin with error bars indicating SD and * indicating p value <.05 using student t-test comparing etoposide (24 hr) relative to untreated for specific primer pairs.


( E ) Meta - data analysis of published GRO - Seq and ChIP Seq data sets . Gro - Seq Minus ( red / maroon ) or Plus ( purple / pink ) RNA strand from Control or Nutlin - 3a treated MCF7 cells was mapped to human 18q subtelomere . Histone H3K27ac , H3K4me1 , and H3K4me3ChIP - Seq data sets from untreated MCF7 are also shown for these subtelomeric regions . Right panel shows a zoom in of approximately 1000bp ( light blue on left panel ) surrounding the p53 response element within the subtelomere .

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(E) Meta-data analysis of published GRO-Seq and ChIP Seq data sets. Gro-Seq Minus (red/maroon) or Plus (purple/pink) RNA strand from Control or Nutlin -3a treated MCF7 cells was mapped to human 18q subtelomere. Histone H3K27ac, H3K4me1, and H3K4me3ChIP-Seq data sets from untreated MCF7 are also shown for these subtelomeric regions. Right panel shows a zoom in of approximately 1000bp (light blue on left panel) surrounding the p53 response element within the subtelomere.


( F ) Summary of GRO - Seq and ChIP - Seq metadata analyses from human subtelomeres with p53 binding sites . Each p53 response element is located upstream of a region of bidirectional transcription which is modulated in a p53 dependent manner along with local bidirectional transcription at the p53 response element itself .

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(F) Summary of GRO-Seq and ChIP-Seq metadata analyses from human subtelomeres with p53 binding sites. Each p53 response element is located upstream of a region of bidirectional transcription which is modulated in a p53 dependent manner along with local bidirectional transcription at the p53 response element itself.


( B ) Western blot analysis of CRISPR deleted cell lines 18q WT and 18q Δp53 treated with DMSO ( D ) or 50 µM etoposide ( E ) for 24 hr . Blots were probed with pan - p53 , phospho - p53 , γH2AX , or GAPDH ( as loading control )

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(B) Western blot analysis of CRISPR deleted cell lines 18q WT and 18q Δp53 treated with DMSO (D) or 50 µM etoposide (E) for 24 hr. Blots were probed with pan-p53, phospho-p53, γH2AX, or GAPDH (as loading control)


( C - E ) RT - qPCR for p21 ( C ) , PARDG6 ( D ) , or 18q subtelomeric RNA ( E ) in 18q WT or18q Δp53 treated with DMSO ( black ) or 50 μM etoposide ( red ) for 24 hr . ( F ) ChIP - qPCR assay for γH2AX at 18q subtelomere in 18q WT or 18q Δp53 treated as in panels B - E .

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(C-E) RT-qPCR for p21 (C), PARDG6 (D), or 18q subtelomeric RNA (E) in 18q WT or18q Δp53 treated with DMSO (black) or 50 μM etoposide (red) for 24 hr. (F) ChIP-qPCR assay for γH2AX at 18q subtelomere in 18q WT or 18q Δp53 treated as in panels B-E.


( A ) Fractionation of nuclear and cytosolic compartments using a standard NP - 40 lysis protocol and immunoblotting with chaperone specific antibodies

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(A) Fractionation of nuclear and cytosolic compartments using a standard NP-40 lysis protocol and immunoblotting with chaperone specific antibodies


( B ) Immunofluorescence of histone chaperones in fixed cells using the same antibodies used in ( A ) . Cells are segregated into those undergoing replication and those that are not . Scale bar represents 5 μm

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(B) Immunofluorescence of histone chaperones in fixed cells using the same antibodies used in (A). Cells are segregated into those undergoing replication and those that are not. Scale bar represents 5 μm


( D ) Real - time imaging of cells undergoing biochemical fractionation . sNASP is tagged with mCherry , whilst LamnA / C is tagged with EGFP . The left column of panels represents a maximum intensity projection . The time course on the right represents a plane in the z dimension reconstructed from 20 z - stacks penetrating 20 μm into the medium . Scale bars represent 5

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Real-time imaging of cells undergoing biochemical fractionation. sNASP is tagged with mCherry, whilst LamnA/C is tagged with EGFP. The left column of panels represents a maximum intensity projection. The time course on the right represents a plane in the z dimension reconstructed from 20 z-stacks penetrating 20 μm into the medium. Scale bars represent 5 μm


( A ) HeLa cell lines stably expressing eGFP - sNASP , eGFP - ASF1 and eGFP - HAT1 . Scale bars represent 10 μm

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(A) HeLa cell lines stably expressing eGFP-sNASP, eGFP-ASF1 and eGFP-HAT1. Scale bars represent 10 μm


( B ) Histone chaperone immunoprecipitations separated by 15 % SDS - PAGE and stained with Coomassie . Major bands that could be confidently identified by mass spectrometry are indicated . Due to the high percentage of acrylamide used , many of the higher molecular weight bands are not resolved . Asterisks represent partial degradation . Background - corrected densitometry profiles are shown for the portions of the gels covering H3 and H4 . Gel filtration purified recombinant H3 - H4 dimer is shown on the

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(B) Histone chaperone immunoprecipitations separated by 15% SDS-PAGE and stained with Coomassie. Major bands that could be confidently identified by mass spectrometry are indicated. Due to the high percentage of acrylamide used, many of the higher molecular weight bands are not resolved. Asterisks represent partial degradation. Background-corrected densitometry profiles are shown for the portions of the gels covering H3 and H4. Gel filtration purified recombinant H3-H4 dimer is shown on the right


( A ) MOG1 interacts genetically with the H2B deubiquitination machinery . Ten - fold serial dilutions of the indicated strains were spotted on YPD and incubated for 2 days at the indicated

(A) MOG1 interacts genetically with the H2B deubiquitination machinery. Ten-fold serial dilutions of the indicated strains were spotted on YPD and incubated for 2 days at the indicated temperatures


( B ) Expression levels of total H2B and H2Bub1 were analyzed in wild - type ( WT ) , mog1Δ , ubp8Δ and ubp8Δmog1Δ whole cell lysates by western blotting using anti - total H2B and anti - H2Bub1 antibodies . Bar graphs of H2Bub1 levels after H2B normalization show the mean and standard deviations of at least three independent experiments . The P - value was calculated using Student ´ s t - test . Error bars represent SD . The P - value was calculated using Student ´ s t - test ( * , P = 0 . 01 - 0 . 05 ; * * , P = 0 . 001 - 0 . 01 ; * * * , P < 0 . 001 )

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(B) Expression levels of total H2B and H2Bub1 were analyzed in wild-type (WT), mog1Δ, ubp8Δ and ubp8Δmog1Δ whole cell lysates by western blotting using anti-total H2B and anti-H2Bub1 antibodies. Bar graphs of H2Bub1 levels after H2B normalization show the mean and standard deviations of at least three independent experiments. The P-value was calculated using Student´s t-test. Error bars represent SD. The P-value was calculated using Student´s t-test (*, P = 0.01-0.05; **, P = 0.001-0.01; ***, P < 0.001)


Ten - fold serial dilutions of the indicated strains were spotted on YPD and incubated for 2 days at the indicated temperatures ( C ) , ( D and were then spotted on YPD or YPGal and incubated for 2

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Ten-fold serial dilutions of the indicated strains were spotted on YPD and incubated for 2 days at the indicated temperatures (C), (D and were then spotted on YPD or YPGal and incubated for 2 days


Ten - fold serial dilutions of the indicated strains were spotted on YPD and incubated for 2 days at the indicated temperatures ( E and were then spotted on YP and incubated for 2

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Ten-fold serial dilutions of the indicated strains were spotted on YPD and incubated for 2 days at the indicated temperatures (E and were then spotted on YP and incubated for 2 days


( A ) The expression levels of H3K4me3 and H3 in total extracts of wild - type ( WT ) , mog1Δ , set1Δ and spp1Δ strains were analyzed by western blotting using the indicated antibodies . Bar graphs of H3K4me3 levels after H3 normalization show the mean and standard deviations of at least three independent experiments . The P - value was calculated using Student ´ s t - test ( * , P = 0 . 01 - 0 . 05 ; * * , P = 0 . 001 - 0 . 01 ; * * * , P < 0 . 001 )

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(A) The expression levels of H3K4me3 and H3 in total extracts of wild-type (WT), mog1Δ, set1Δ and spp1Δ strains were analyzed by western blotting using the indicated antibodies. Bar graphs of H3K4me3 levels after H3 normalization show the mean and standard deviations of at least three independent experiments. The P-value was calculated using Student´s t-test (*, P = 0.01-0.05; **, P = 0.001-0.01; ***, P < 0.001)


( B ) ChIP analysis of H3K4me3 presence at ADH1 , PMA1 and YEF3 promoter ( P ) and 5 ' ORF in wild - type ( WT ) or mog1Δ strains relative to total H3 . The bar graphs indicate the mean and standard deviation for at least three independent experiments . The significance of the differences was calculated using Student ´ s t - test ( * , P value = 0 . 01 - 0 . 05 ; * * , P value = 0 . 001 - 0 . 01 ; * * * , P < 0 . 001 )

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(B) ChIP analysis of H3K4me3 presence at ADH1, PMA1 and YEF3 promoter (P) and 5'ORF in wild-type (WT) or mog1Δ strains relative to total H3. The bar graphs indicate the mean and standard deviation for at least three independent experiments. The significance of the differences was calculated using Student´s t-test (*, P value = 0.01-0.05; **, P value = 0.001-0.01; ***, P < 0.001)


( C ) MOG1 interacts genetically with the H3 methylases Set1 and Set2 . Ten - fold serial dilutions of the indicated strains were spotted on YPD and incubated for 2 days at the indicated temperatures . ( D ) histone H3K4A and H3K4Amog1Δ strains were compared with their isogenic strains as in ( C )

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(C) MOG1 interacts genetically with the H3 methylases Set1 and Set2. Ten-fold serial dilutions of the indicated strains were spotted on YPD and incubated for 2 days at the indicated temperatures. (D) histone H3K4A and H3K4Amog1Δ strains were compared with their isogenic strains as in (C)


( A ) Ten - fold serial dilutions of the indicated strains were spotted on YPD containing the indicated concentrations of hydroxyurea ( HU ) and were incubated for 2 - 3 days .

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(A) Ten-fold serial dilutions of the indicated strains were spotted on YPD containing the indicated concentrations of hydroxyurea (HU) and were incubated for 2-3 days.


( B ) mRNA synthesis rates ( SR ) as determined in Genomic Run - On analysis and ( C ) mRNA abundance ( RA ) , in mog1 ∆ compared with wild type ( WT ) cells . Bar graphs showing the average level and standard deviation of three experiments ( n = 3 ) for SR and RA values obtained as the median of the whole gene dataset values in arbitrary units ( x 107 ) from image analysis quantification . Error bars represent SD . Significance of the differences was obtained using Student ´ s t - test and statistical difference is considered as * , P value = 0 . 01 - 0 . 05 ; * * , P value = 0 . 001 - 0 . 01 ; * * * , P value < 0 . 001 . The scatter plots ( right panels ) show the variation of the transcription ( B ) or mRNA ( C ) levels of individual genes . Pearson correlation values are : - 0 . 039 ( SR ) and - 0 . 042 ( RA ) . This low correlation and the flat shape of the clouds on scatter plots ( see red tendency line ) indicates that there is no bias of the transcriptional effect with regard to expression level . ( D ) Box plots of SR and RA median levels of mog1Δ cells compared with WT for all genes ( Total ) , SAGA and TFIID - dominated genes . These data were obtained from three independent replicates ( n = 3 ) of the RA and SR data that were averaged as explained in the main text . The median and standard deviation for the whole data set ( Total ) and for 351 ( for RA ) or 316 ( SR ) SAGA - dominated genes and for the 3903 ( RA ) or 3403 ( SR ) TFIID - dominated genes are shown . Significance of the differences was calculated using Student ´ s t - test . No significant difference between the different groups was found . The similar variations in SRs and RAs indicate that there are no changes in mRNA half - lives upon MOG1 deletion ( shown in Fig EV4 ) . The total number of genes with confident value in the analyses was 3953 for RA and 3989 for

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) mRNA synthesis rates (SR) as determined in Genomic Run-On analysis and (C) mRNA abundance (RA), in mog1∆ compared with wild type (WT) cells. Bar graphs showing the average level and standard deviation of three experiments (n=3) for SR and RA values obtained as the median of the whole gene dataset values in arbitrary units (x 107) from image analysis quantification. Error bars represent SD. Significance of the differences was obtained using Student´s t-test and statistical difference is considered as *, P value = 0.01-0.05; **, P value = 0.001-0.01; ***, P value < 0.001. The scatter plots (right panels) show the variation of the transcription (B) or mRNA (C) levels of individual genes. Pearson correlation values are: -0.039 (SR) and -0.042 (RA). This low correlation and the flat shape of the clouds on scatter plots (see red tendency line) indicates that there is no bias of the transcriptional effect with regard to expression level. (D) Box plots of SR and RA median levels of mog1Δ cells compared with WT for all genes (Total), SAGA and TFIID-dominated genes. These data were obtained from three independent replicates (n=3) of the RA and SR data that were averaged as explained in the main text. The median and standard deviation for the whole data set (Total) and for 351 (for RA) or 316 (SR) SAGA-dominated genes and for the 3903 (RA) or 3403 (SR) TFIID-dominated genes are shown. Significance of the differences was calculated using Student´s t-test. No significant difference between the different groups was found. The similar variations in SRs and RAs indicate that there are no changes in mRNA half-lives upon MOG1 deletion (shown in Fig EV4). The total number of genes with confident value in the analyses was 3953 for RA and 3989 for SR


( A ) ChIP analysis of TAP - tagged Rad6 presence at ADH1 , PMA1 and YEF3 coding regions ( 5 ' & 3 ' ) in WT or mog1Δ strains . Bar charts indicate the mean and standard deviation for at least three independent experiments . Significance of the differences was obtained using Student ´ s t - test ( * , P value = 0 . 01 - 0 . 05 ; * * , P value = 0 . 001 - 0 . 01 , * * * , P value = 0 . 0001 - 0 . 001 , * * * * , P value < 0 . 0001 ) . ( B ) , ( C ) and ( D ) Bre1 - TAP , Rtf1 - TAP and Rpb3 respectively , were analyzed as described in ( A ) . ( E ) Bar charts showing the relative chromatin occupancy to 5 ' ORFs ( left panel ) or 3 ' ORFs ( right panel ) of each factor relative to Rpb3 occupancy at these gene positions .

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(A) ChIP analysis of TAP-tagged Rad6 presence at ADH1, PMA1 and YEF3 coding regions (5' & 3') in WT or mog1Δ strains. Bar charts indicate the mean and standard deviation for at least three independent experiments. Significance of the differences was obtained using Student´s t-test (*, P value = 0.01-0.05; **, P value = 0.001-0.01, ***, P value = 0.0001-0.001, ****, P value <0.0001). (B), (C) and (D) Bre1-TAP, Rtf1-TAP and Rpb3 respectively, were analyzed as described in (A). (E) Bar charts showing the relative chromatin occupancy to 5'ORFs (left panel) or 3'ORFs (right panel) of each factor relative to Rpb3 occupancy at these gene positions.


( A ) Co - purification of Rtf1 - PK in cells expressing Mog1 - TAP ( + , + ) . A non - tagged strain ( - , - ) and cells expressing only Rtf1 - PK ( + , - ) were included as negative controls for co - purification . Inputs and IPs are depicted for each strain .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Co-purification of Rtf1-PK in cells expressing Mog1-TAP (+,+). A non-tagged strain (-,-) and cells expressing only Rtf1-PK (+,-) were included as negative controls for co-purification. Inputs and IPs are depicted for each strain.


( B ) ChIP analysis of the presence of HA - tagged Mog1 compared to a non - tagged strain ( WT ) at PMA1 and YEF3 promoter ( P ) and coding regions ( 5 ' ORF ) . The occupancy level was calculated as the signal ratio of the IP samples in relation to the Input signal normalized to an Intergenic region . Bar charts indicate the mean and standard deviation for at least three independent experiments . Significance of the differences was obtained using Student ´ s t - test and is presented as a P - value ( * , P < 0 . 05 ) .

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(B) ChIP analysis of the presence of HA-tagged Mog1 compared to a non-tagged strain (WT) at PMA1 and YEF3 promoter (P) and coding regions (5'ORF). The occupancy level was calculated as the signal ratio of the IP samples in relation to the Input signal normalized to an Intergenic region. Bar charts indicate the mean and standard deviation for at least three independent experiments. Significance of the differences was obtained using Student´s t-test and is presented as a P-value (*, P<0.05).


( D ) Mog1 - HA immunoprecipitation in cells expressing Bre1 - PK ( upper panel ) and Mog1 - TAP co - precipitation in cells expressing Ada2 - HA ( lower panel ) are indicate as in ( A ) Inputs and IPs are depicted for each strain .

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(D) Mog1-HA immunoprecipitation in cells expressing Bre1-PK (upper panel) and Mog1-TAP co-precipitation in cells expressing Ada2-HA (lower panel) are indicate as in (A) Inputs and IPs are depicted for each strain.


Representative images of the analysis of nuclear mRNA export are shown . The cellular localization of poly ( A ) + RNA was assessed by in situ hybridization using Cy3 - labeled oligo ( dT ) probes ( red ) in the indicated strains . In situ hybridization was monitored at 30 ºC and after shifting of the cells to 39 ºC for 3 hours . DNA was stained by DAPI ( blue ) . In all images the scale bar corresponds to 2 μM . Images are representative pictures from 2 independent experiments of approximately 600 cells . The percentages indicate the number of cells showing mRNA export defect from these experiments .

O O O O O O O B-MEASURED_VAR O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Representative images of the analysis of nuclear mRNA export are shown. The cellular localization of poly(A)+RNA was assessed by in situ hybridization using Cy3-labeled oligo(dT) probes (red) in the indicated strains. In situ hybridization was monitored at 30 ºC and after shifting of the cells to 39 ºC for 3 hours. DNA was stained by DAPI (blue). In all images the scale bar corresponds to 2 μM. Images are representative pictures from 2 independent experiments of approximately 600 cells. The percentages indicate the number of cells showing mRNA export defect from these experiments.


( A ) The Mog1E65K Ran - binding mutant complements the slow growth of mog1ΔsetΔ mutants . Ten - fold serial dilutions of wild - type ( WT ) , mog1Δ , set1Δ , and mog1ΔsetΔ strains transformed with wild - type MOG1 ( pMOG1 ) or with the Ran - binding mutant ( pMOG1E65K ) as indicated were spotted on SC - LEU and incubated for 2 days at the indicated temperatures .

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(A) The Mog1E65K Ran-binding mutant complements the slow growth of mog1ΔsetΔ mutants. Ten-fold serial dilutions of wild-type (WT), mog1Δ, set1Δ, and mog1ΔsetΔ strains transformed with wild-type MOG1 (pMOG1) or with the Ran-binding mutant (pMOG1E65K) as indicated were spotted on SC-LEU and incubated for 2 days at the indicated temperatures.


( B ) The Mog1E65K Ran - binding mutant complements the low H3K4me3 and H2Bub1 levels of mog1Δ . The expression levels of H3K4me3 , total H3 , H2Bub1 , total H2B and PGK1 in whole cell lysates from wild type ( WT ) or from mog1Δ cells transformed with an empty plasmid ( + pEmpty ) , wild type MOG1 ( pMOG1 ) or a Ran - binding mutant ( pMOG1E65K ) were analyzed by western blotting using the corresponding antibodies .

O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(B) The Mog1E65K Ran-binding mutant complements the low H3K4me3 and H2Bub1 levels of mog1Δ. The expression levels of H3K4me3, total H3, H2Bub1, total H2B and PGK1 in whole cell lysates from wild type (WT) or from mog1Δ cells transformed with an empty plasmid (+pEmpty), wild type MOG1 (pMOG1) or a Ran-binding mutant (pMOG1E65K) were analyzed by western blotting using the corresponding antibodies.