Journal: Nucleic Acids Research

Article Title: Novel role of zinc-finger protein 518 in heterochromatin formation on α-satellite DNA

doi: 10.1093/nar/gkae1162

Figure Lengend Snippet: ZNF518A and ZNF518B are required for the formation of pericentromeric heterochromatin. ( A and D ) Growth curves of WT cells and two lines (#1 and #2) of ZNF518 dKO cells before ( A ) and after ( D ) siRNA knockdown of SUV39H1. The siRNA transfection was performed at 0 h in ( D ). * P < 0.05, ** P < 0.01. ( B ) Immunofluorescence staining of histone H3K9me3 and CENP-B at 0, 72 or 144 h after SUV39H1 knockdown in WT (top), ZNF518s dKO cells #1 (middle), or #2 (bottom). Areas marked by dashed boxes in the left panels are magnified in the right panels. Scale bar, 5 μm. ( C ) The percentage of CENP-B foci categorized by overlapping H3K9me3 was quantified (See ). Statistical significance was assessed using a Chi-square test. * P < 0.05, *** P < 0.005. ( E ) Tethering of fusion proteins with TetR-YFP to the tetO repeats for 2 weeks results in the accumulation of histone H3K9me3. ( F ) Quantification of the percentage of H3K9 trimethylation at the ectopic site. tethering TetR-YFP, TetR:YFP:CENP-B 133–599 , TetR-YFP-G9A, TetR-YFP-ZNF518B 555-1020 , TetR-YFP-ZNF518B 878-1020 or TetR-YFP-ZNF518B 878-987 in CENP-B KO cells based on the images shown in panel E. Each point represents an independent experiment, calculated from at least 50 individual YFP foci at ectopic sites. Black bars indicate the median of independent experiments within each category. *** P < 0.005. ( G ) Violin plots show the quantification of the fluorescence area of TetR-YFP signals at individual ectopic sites in CENP-B KO cells, based on the images shown in panel E. P -values compared to WT were calculated using the two-sided Mann-Whitney U test, repeated 20 times by randomly selecting 10 data points from each category. The final P -values represent the average of these 20 calculations. * P < 0.05, *** P < 0.005. ( H ) Model depicting the central role of ZNF518A and ZNF518B in pericentromeric heterochromatin formation via their interactions with CENP-B, HP1 and G9A.

Article Snippet: Small interfering RNA (siRNA) oligos to knock down SUV39H1 used in this study were described in previous research (s13658, silencer select, Thermo Fisher Scientific; ( )).

Techniques: Knockdown, Transfection, Immunofluorescence, Staining, Fluorescence, MANN-WHITNEY

Journal: Nucleic Acids Research

Article Title: EGFR-mediated HSP70 phosphorylation facilitates PCNA association with chromatin and DNA replication

doi: 10.1093/nar/gkae938

Figure Lengend Snippet: EGFR interacts and phosphorylates HSP70. ( A ) and ( B ) HSP70–EGFR interaction in HCC827 cells. The HSP70–EGFR complex was immunoprecipitated from HCC827 cell lysates using antibodies against HSP70 (panel A) or EGFR (panel B), respectively. The immunoprecipitated HSP70–EGFR complex was analyzed by western blot. ( C ) In vitro EGFR kinase assay detecting Y41 phosphorylation of HSP70. Purified EGFR kinase domain protein was used as the enzyme and purified Flag-tagged HSP70 (WT or Y41F) was used as the protein substrate. HSP70 Y41 phosphorylation was evaluated by western blot using the antibody against Y41 phosphorylated HSP70. ( D ) Enhancement of HSP70 phosphorylation by EGF. HCC827 cells expressing Flag-tagged WT or Y41F HSP70 were untreated or treated with EGF (100 ng/ml, 1 h). The phosphorylation of Flag-tagged HSP70 (WT or Y41F) was analyzed by Co-IP using anti-Flag-tag antibody and western blot using the antibody against Y41 phosphorylated HSP70. ( E ) Inhibition of HSP70 phosphorylation by EGFR–TKIs. HCC827 cells were treated with EGFR–TKIs osimertinib, erlotinib and gefitinib. The status of EGFR phosphorylation, EGFR level, HSP70 phosphorylation and HSP70 level were analyzed by western blot. GADPH was used as the loading control for western blot.

Article Snippet: For knockdown of HSP70 or EGFR by human HSP70 small interfering RNA (siRNA) (ORIGENE, Catalog No.: SR320559) or human EGFR siRNA (ORIGENE, Catalog No.: SR320009) transfection, cells were seeded to 50–70% confluency at the time of transfection.

Techniques: Immunoprecipitation, Western Blot, In Vitro, Kinase Assay, Purification, Expressing, Co-Immunoprecipitation Assay, FLAG-tag, Inhibition, Control

Journal: Nucleic Acids Research

Article Title: EGFR-mediated HSP70 phosphorylation facilitates PCNA association with chromatin and DNA replication

doi: 10.1093/nar/gkae938

Figure Lengend Snippet: EGFR mediated Y41 phosphorylation of HSP70 promotes HSP70 nuclear translocation. (A–C) Stimulation of HSP70 nuclear localization by EGF. ( A ) HCC827 cells were treated with EGF (100 ng/ml) for 0, 30, 60 and 120 min. Cytoplasmic and nuclear HSP70 was analyzed by western blot. ( B ) and ( C ) HCC827 cells were untreated or treated with EGF (100 ng/ml) for 30 min. Immunofluorescence (IF) staining of HSP70 was carried out to evaluate HSP70 in the nucleus (DAPI stained). Panel (B) shows the representative IF images with or without EGF treatment. Panel (C) is the quantification of nuclear HSP70 by Image J. The P -value was calculated using Student’s t -test. Scale bar: 20 μm. ( D ) EGFR–TKIs block HSP70 nuclear localization. HCC827 cells were untreated or treated with erlotinib (erlo.) (1 μM) or osimertinib (osi.) (1 μM) for 24 h. Cytoplasmic and nuclear HSP70 was analyzed by western blot. ( E ) Impact of HSP70 phosphorylation on its nuclear localization. HCC827 cells expressing WT, phosphorylation mimicking Y41D and phosphorylation-deficient mimicking Flag-tagged HSP70 were treated with erlotinib (erlo.) (1 μM) or osimertinib (osi.) (1 μM) for 24 h. Cytoplasmic and nuclear Flag-tagged WT, Y41D or Y41F HSP70 was analyzed by western blot.

Article Snippet: For knockdown of HSP70 or EGFR by human HSP70 small interfering RNA (siRNA) (ORIGENE, Catalog No.: SR320559) or human EGFR siRNA (ORIGENE, Catalog No.: SR320009) transfection, cells were seeded to 50–70% confluency at the time of transfection.

Techniques: Translocation Assay, Western Blot, Immunofluorescence, Staining, Blocking Assay, Expressing

Journal: Nucleic Acids Research

Article Title: EGFR-mediated HSP70 phosphorylation facilitates PCNA association with chromatin and DNA replication

doi: 10.1093/nar/gkae938

Figure Lengend Snippet: EGFR-mediated HSP70 phosphorylation facilitates PCNA association with chromatin. ( A ) HSP70 and PCNA were co-stained using specific antibodies against these two proteins. Nuclei were stained with DAPI. Representative images of interphase (with few PCNA foci) and S phase (with clear PCNA foci) HCC827 cells were shown. ( B ) Quantification of HSP70 foci that were co-localized with PCNA foci in HCC827 cells untreated or treated with EGF (100 ng/ml, 30 min). ( C ) EGF stimulated chromatin association of HSP70 and PCNA. Nonchromatin and chromatin fractions were prepared from HCC827 cells treated with EGF (100 ng/ml) for 0, 30 and 60 min. HSP70 and PCNA in these fractions were analyzed by western blot. Tubulin and histone H3 were used as the loading controls for nonchromatin and chromatin fractions, respectively. ( D ) and ( E ) Impact of MG132 on the level of nuclear HSP70 and PCNA. Panel (D) shows representative co-IF staining images of HSP70 and PCNA in HCC827 cells without or with MG132 treatment (1 μM, 1 h) and panel (E) is the quantification of nuclear HSP70 and PCNA in the cells as shown in panel (D). ( F ) and ( G ) IF staining of PCNA in parental and HSP70 knockdown cells. HCC827 cells HSP70 knockdown was performed by siRNA transfection. Panel (F) shows the representative image and panel (G) is the quantification. ( H ) Impact of HSP70 knockdown on chromatin association of PCNA. PCNA expression from each cell fraction was examined by western blotting. Tubulin and histone H3 were used as the loading controls for nonchromatin and chromatin fractions, respectively. ( I ) Impact of HSP70 phosphorylation on chromatin association of PCNA. HCC827 cells expressing WT, phosphorylation mimicking Y41D and phosphorylation-deficient mimicking Y41 Flag-tagged HSP70 were untreated (ctr.) or treated with erlotinib (erlo.) (1 μM) or osimertinib (osi.) (1 μM) for 24 h. Nonchromatin and chromatin fractions were prepared from these cells. The level of nonchromatin PCNA and chromatin associated PCNA and HSP70 were analyzed by western blot. Tubulin and histone H3 were used as loading controls for nonchromatin and chromatin fraction, respectively. The P -values in panels (B), (E) and (G) were calculated using Student’s t -test.

Article Snippet: For knockdown of HSP70 or EGFR by human HSP70 small interfering RNA (siRNA) (ORIGENE, Catalog No.: SR320559) or human EGFR siRNA (ORIGENE, Catalog No.: SR320009) transfection, cells were seeded to 50–70% confluency at the time of transfection.

Techniques: Staining, Western Blot, Knockdown, Transfection, Expressing

Journal: Nucleic Acids Research

Article Title: EGFR-mediated HSP70 phosphorylation facilitates PCNA association with chromatin and DNA replication

doi: 10.1093/nar/gkae938

Figure Lengend Snippet: HSP70 deficiency or chemical inhibition impairs OFM and DNA replication. ( A – D ) NEs-based OFM assays. Panels (A) and (C) show RPR assays using the NEs isolated from parental or HSP70 knockdown HCC827 cells or cells treated with EGFR–TKI (erlotinib, 1 μM) for 24 h, and panels (B) and (D) show the Polα error editing (AEE) assays using NEs isolated from parental or HSP70 knockdown cells or cells treated with EGFR–TKI (erlotinib, 1 μM) for 24 h. The diagrams on the top of panels (A) and (B) are the oligo-based DNA substrates. The images in panels (A–D) are the representative denaturing PAGE image of the RPR and AEE reaction products. Reactions were carried out at 37°C for 0, 10, 20, 40, 60 and 80 min. The unligated products (Nonlig. prod.) and ligated (Lig. prod.) were indicated. ( E ) and ( F ) Analysis of DNA synthesis with the DNA fiber assays in HCC827 cells treated with DMSO (control, 1 or 4 h) or HSP70 inhibitor VER155008 (1 or 4 h). Panel (E) shows the diagram of IdU and CldU labeling and the drug treatment (upper portion) and representative fiber images in the control and VER155008-treated cells (bottom portion). Panel (F) shows the CldU track length in the IdU labeled tracks. The P -values in panels (F) were calculated using Student’s t -test.

Article Snippet: For knockdown of HSP70 or EGFR by human HSP70 small interfering RNA (siRNA) (ORIGENE, Catalog No.: SR320559) or human EGFR siRNA (ORIGENE, Catalog No.: SR320009) transfection, cells were seeded to 50–70% confluency at the time of transfection.

Techniques: Inhibition, Isolation, Knockdown, DNA Synthesis, Control, Labeling

Journal: Nucleic Acids Research

Article Title: EGFR-mediated HSP70 phosphorylation facilitates PCNA association with chromatin and DNA replication

doi: 10.1093/nar/gkae938

Figure Lengend Snippet: HSP70 inhibition causes replicative DNA damage and alters cell cycle progression. ( A ) and ( B ) γH2AX foci in replicating untreated cells or cells treated with HSP70 inhibitor VER155008 (5 μM) and osimertinib (5 μM) individually or in combination for 2 h. Panel (A) shows the representative image of γH2AX foci in EdU-positive cells. HCC827 cells were pre-treated with DMSO (untreated control) or VER155008 (5 μM) for 2 h. The cells were labeled with EdU (10 μM) for 30 min. The γH2AX-EdU co-staining was carried out and γH2AX foci in EdU positive cells were scored as described in Figure . Scale bar: 20 μm. Panel (B) shows quantification of γH2AX foci number in the EdU-positive cells that were pre-treated with DMSO (untreated control) or VER155008 and osimertinib individually or in combination for 2 h using Image J. ( C ) and ( D ) Genome wide DNA damage analysis with the comet assays in HCC827 cells treated with DMSO (control) or VER155008 (10 μM) and osimertinib (5 μM) individually or in combination for 24 h. Panel (C) shows the representative images of nuclei in the control and treatment groups and panel (D) is the quantification of comet tails in the control or VER155008 and/or osimertinib-treated cells. ( E ) and ( F ) Flow cytometry-based cell cycle analysis of HCC827 cells treated with DMSO (untreated control) or VER155008 (10 μM) and osimertinib (5 μM) individually or in combination for 24 h. Panel (E) shows the PI-stained cells in each group and panel (F) indicated the percentages of each cell cycle phase in different groups of HCC827 cells [(i) DMSO, (ii) osimertinib (5 μM), (iii) VER155008 (10 μM) and (iv) combination of osimertinib and VER155008) for 24 h. The P -values in panels (B) and (D) were calculated using Student’s t -test.

Article Snippet: For knockdown of HSP70 or EGFR by human HSP70 small interfering RNA (siRNA) (ORIGENE, Catalog No.: SR320559) or human EGFR siRNA (ORIGENE, Catalog No.: SR320009) transfection, cells were seeded to 50–70% confluency at the time of transfection.

Techniques: Inhibition, Control, Labeling, Staining, Genome Wide, Flow Cytometry, Cell Cycle Assay

Journal: Nucleic Acids Research

Article Title: EGFR-mediated HSP70 phosphorylation facilitates PCNA association with chromatin and DNA replication

doi: 10.1093/nar/gkae938

Figure Lengend Snippet: HSP70 inhibitor VER155008 and EGFR–TKI osimertinib synergistically kill HCC827 cells in vitro and in vivo . ( A ) and ( B ) Flow cytometry-based apoptosis analysis of HCC827 cells treated with DMSO (untreated control) or VER155008 (10 μM) and osimertinib (5 μM) individually or in combination for 24 h. Panel (A) shows the Annexin V-FITC and PI co-stained cells in each group and panel (B) indicated the percentage of apoptotic cells (Q2 in panel A) in different groups of HCC827 cells. ( C ) and ( D ) The synergy between the HSP70 inhibitor VER155008 and the EGFR–TKI inhibitor osimertinib was assayed by clonogenic assay. The values are means ± SEM of three independent clonogenic assays. Panel (C) is the inhibition curve of varying concentrations of VER155008 from 0 to 2 μM in combination with osimertinib (0 or 2 nM). Panel (D) is the inhibition curve of varying concentrations of osimertinib from 0 to 4 nM in combination with VER155008 (0 or 1 μM). ( E ) and ( F ) VER155008 potentiates osimertinib-induced HCC827 tumor cell killing in xenografting mice. Panel (E) shows the tumor growth rate in mice treated with DMSO (untreated control, n = 10) or VER155008 ( n = 13) and osimertinib ( n = 13) individually or in combination ( n = 14). P -values were calculated using the Student's t -test. Panel (F) is the survival curve of each group.

Article Snippet: For knockdown of HSP70 or EGFR by human HSP70 small interfering RNA (siRNA) (ORIGENE, Catalog No.: SR320559) or human EGFR siRNA (ORIGENE, Catalog No.: SR320009) transfection, cells were seeded to 50–70% confluency at the time of transfection.

Techniques: In Vitro, In Vivo, Flow Cytometry, Control, Staining, Clonogenic Assay, Inhibition

Journal: Cell Death & Disease

Article Title: Reg4 deficiency aggravates pancreatitis by increasing mitochondrial cell death and fibrosis

doi: 10.1038/s41419-024-06738-y

Figure Lengend Snippet: A Co-localization of REG4 (green) with EXTL3 (red) in mice pancreas sections using immunofluorescence microscopy ( n = 3). B Co-immunoprecipitation of REG4 and EXTL3 in mice pancreas ( n = 2). C Knockdown of Extl3 by siRNA (20 nM) in the context of the MIN6 cell line and treated with cerulean (200 μg/ml) for 6 h. Western-blot analysis for PUMA, Cleaved-Caspase 3, Caspase 3, LC3, and β-actin ( n ≥ 3 for each treatment). D Quantification of ( C ). β-actin was used as the internal reference. Randomized one-way ANOVA for ( C ) (ns not significant; ** P < 0.01).

Article Snippet: Small interfering RNA (siRNA) oligonucleotides targeting Extl3 (#SR421199) were purchased from the Origene.

Techniques: Immunofluorescence, Microscopy, Immunoprecipitation, Knockdown, Western Blot