Journal: Science Advances

Article Title: HMCES corrupts replication fork stability during base excision repair in homologous recombination–deficient cells

doi: 10.1126/sciadv.ads3227

Figure Lengend Snippet: ( A ) Left: Representative PAR (red) immunofluorescence images of siA1depleted (siA1) wild-type or Fancd2 −/− cells exposed to 5hmdC (10 μM) for 3 hours. DAPI (blue) stains nuclear DNA and EdU (green) stains S-phase cells. Right: Plot depicting PAR mean intensity signal per EdU + nucleus. ( B ) Left: Representative images of EdU-PAR foci (red) by SIRF assay from wild-type, siA1 Fancd2 −/− , or Fancd2 −/− siA1 cells exposed to 5hmdC (10 μM) for 3 hours. DAPI (blue) stains nuclear DNA and EdU (green) stains S-phase cells. Right: Plot depicting EdU-PAR foci per nucleus. ( C ) Left: Representative images of alkaline comet assay of APEX1 knockdown wild-type or Fancd2 −/− cells exposed to 5hmdC (10 μM) for 3 hours. Right: Plot depicting comet tail moment per cell. ( D ) Top left: Scheme of DNA fiber assay. Bottom left: Representative images of DNA fibers from Fancd2 −/− and Fancd2 −/− siA1 cells untreated or exposed to 5hmdC (40 μM) for 30 min. Right: Box plot of the frequency of IdU/CldU ratio of siA1-depleted wild-type or Fancd2 −/− cells upon 5hmdC treatment ( n = 200 of each of the two biological replicates). ( E ) Top: MTT cell proliferation assay of siA1-depleted wild-type or Fancd2 −/− cells exposed to the indicated dose of 5hmdC for 3 days ( n = 9; means ± SD). Bottom: MTT cell proliferation assay of APEX1- or siA2-depleted wild-type or Fancd2 −/− cells exposed to the indicated dose of 5hmdC for 3 days. ( F ) Plot depicting EdU-PAR foci per nucleus by SIRF assay from wild-type, Fancd2 −/− , siA1, siA2, Fancd2 −/− siA1, and Fancd2 −/− siA2 cells exposed to 5hmdC (10 μM) for 3 hours ( n = 2).

Article Snippet: The antibodies used were against PAR (Millipore, MABE1016, Merck Millipore, Burlington, MA, USA), ERCC1 (sc-8408), PCNA (sc-56), Lamin A/C (sc-376248), BrdU (BU1/75, Abcam, Cambridge, England), IdU (Sigma-Aldrich, SAB3701448), Biotin (B7653, Sigma-Aldrich), HMCES (NPB2-14410, Novus Biologicals, Centennial, CO, USA), SMUG1 (E7P4I, Cell Signaling Technology, Danvers, MA, USA), APEX1 (A1117, ABclonal Technology, Woburn, MA, USA), XRCC1 (A0442, ABclonal), DCK (A1794, ABclonal), DCTD (16784-1-AP, Proteintech, Rosemont, IL, USA), FLAG (F1804, Sigma-Aldrich), FLAG (D6W5B, Cell Signaling Technology), PARP1 (46D11, Cell Signaling Technology), and PRIMPOL ( https://pubmed.ncbi.nlm.nih.gov/36152632/ ).

Techniques: Immunofluorescence, Alkaline Single Cell Gel Electrophoresis, Knockdown, MTT Cell Proliferation

Journal: Science Advances

Article Title: HMCES corrupts replication fork stability during base excision repair in homologous recombination–deficient cells

doi: 10.1126/sciadv.ads3227

Figure Lengend Snippet: ( A ) Left: Representative PAR (red) immunofluorescence images of XRCC1-depleted wild-type or Fancd2 −/− cells exposed to 5hmdC (10 μM) for 3 hours. DAPI (blue) stains nuclear DNA and EdU (green) stains S-phase cells. Right: Plot depicting PAR mean intensity signal per nucleus. ( B ) Left: Representative images of EdU-PAR foci (red) by SIRF assay of XRCC1-depleted wild-type or Fancd2 −/− cells exposed to 5hmdC (10 μM) for 3 hours. DAPI (blue) stains nuclear DNA and EdU (green) stains S-phase cells. Right: Plot depicting EdU-PAR foci per nucleus. ( C ) Top: MTT cell proliferation assay of wild-type or Fancd2 −/− cells lacking XRCC1 exposed to the indicated dose of 5hmdC for 3 days ( n = 9; means ± SD). Bottom: MTT cell proliferation assay of wild-type, siPARP1, Fancd2 −/− , and Fancd2 −/− siPARP1 cells exposed to the indicated dose of 5hmdC for 3 days ( n = 5; means ± SD). ( D ) Top left: Scheme of DNA fiber assay. Bottom left: Representative images of DNA fibers from Fancd2 −/− and Fancd2 −/− siXRCC1 cells untreated or exposed to 5hmdC (40 μM) for 30 min. Right: Box plot of the frequency of IdU/CldU ratio of XRCC1-depleted wild-type or Fancd2 −/− cells upon 5hmdC treatment ( n = 200 of each of the two biological replicates). ( E ) Top right: Scheme of the SCE assay. Left: Bar plot of SCE/chromosome per metaphase from SMUG1-, APEX1-, XRCC1-, or PARP1-depleted Fancd2 −/− cells exposed to 5hmdC (10 μM) for 12 hours ( n = 50 of each of the two biological replicates; bar represents means ± SD).

Article Snippet: The antibodies used were against PAR (Millipore, MABE1016, Merck Millipore, Burlington, MA, USA), ERCC1 (sc-8408), PCNA (sc-56), Lamin A/C (sc-376248), BrdU (BU1/75, Abcam, Cambridge, England), IdU (Sigma-Aldrich, SAB3701448), Biotin (B7653, Sigma-Aldrich), HMCES (NPB2-14410, Novus Biologicals, Centennial, CO, USA), SMUG1 (E7P4I, Cell Signaling Technology, Danvers, MA, USA), APEX1 (A1117, ABclonal Technology, Woburn, MA, USA), XRCC1 (A0442, ABclonal), DCK (A1794, ABclonal), DCTD (16784-1-AP, Proteintech, Rosemont, IL, USA), FLAG (F1804, Sigma-Aldrich), FLAG (D6W5B, Cell Signaling Technology), PARP1 (46D11, Cell Signaling Technology), and PRIMPOL ( https://pubmed.ncbi.nlm.nih.gov/36152632/ ).

Techniques: Immunofluorescence, MTT Cell Proliferation

Journal: Science Advances

Article Title: HMCES corrupts replication fork stability during base excision repair in homologous recombination–deficient cells

doi: 10.1126/sciadv.ads3227

Figure Lengend Snippet: ( A ) Left: Representative PAR (red) immunofluorescence images from Fancd2 −/− cells upon depletion of HMCES, APEX1, or their combinations exposed to 5hmdC (10 μM, 3 hours). DAPI (blue) stains nuclear DNA and EdU (green) stains S-phase nuclei. Right: Plot depicting PAR mean intensity signal per nucleus. ( B ) Left: Representative PAR (red) immunofluorescence images from Fancd2 −/− cells upon depletion of HMCES, APEX2, or their combinations exposed to 5hmdC (10 μM, 3 hours). DAPI (blue) stains nuclear DNA and EdU (green) stains S-phase cells. Right: Plot depicting PAR mean intensity signal per nucleus. ( C ) MTT cell proliferation assay of HMCES-, APEX1-, or HMCES siA1–depleted wild-type or Fancd2 −/− cells exposed to the indicated dose of 5hmdC for 3 days ( n = 5; means ± SD). ( D ) MTT cell proliferation assay of HMCES-, APEX2-, or HMCES siA2–depleted wild-type or Fancd2 −/− cells exposed to the indicated dose of 5hmdC for 3 days.

Article Snippet: The antibodies used were against PAR (Millipore, MABE1016, Merck Millipore, Burlington, MA, USA), ERCC1 (sc-8408), PCNA (sc-56), Lamin A/C (sc-376248), BrdU (BU1/75, Abcam, Cambridge, England), IdU (Sigma-Aldrich, SAB3701448), Biotin (B7653, Sigma-Aldrich), HMCES (NPB2-14410, Novus Biologicals, Centennial, CO, USA), SMUG1 (E7P4I, Cell Signaling Technology, Danvers, MA, USA), APEX1 (A1117, ABclonal Technology, Woburn, MA, USA), XRCC1 (A0442, ABclonal), DCK (A1794, ABclonal), DCTD (16784-1-AP, Proteintech, Rosemont, IL, USA), FLAG (F1804, Sigma-Aldrich), FLAG (D6W5B, Cell Signaling Technology), PARP1 (46D11, Cell Signaling Technology), and PRIMPOL ( https://pubmed.ncbi.nlm.nih.gov/36152632/ ).

Techniques: Immunofluorescence, MTT Cell Proliferation

Journal: Science Advances

Article Title: HMCES corrupts replication fork stability during base excision repair in homologous recombination–deficient cells

doi: 10.1126/sciadv.ads3227

Figure Lengend Snippet: ( A ) Left: Representative images of EdU-PAR foci (red) by SIRF assay from wild-type, siPRIMPOL, Fancd2 −/− , and Fancd2 −/− siPRIMPOL cells exposed to 5hmdC (10 μM, 3 hours). DAPI (blue) stains nuclear DNA and EdU (green) stains S-phase cells. Right: Plot depicting EdU-PAR foci per nucleus. ( B ) Plot depicting comet tail moment per cell from PRIMPOL-depleted wild-type or Fancd2 −/− cells following 5hmdC treatment (10 μM, 3 hours). ( C ) Top: MTT cell proliferation assay of wild-type or Fancd2 −/− cells knocked down for APEX1, PRIMPOL, or APEX1 PRIMPOL and exposed to the indicated dose of 5hmdC for 3 days. Bottom: MTT cell proliferation assay of wild-type or Fancd2 −/− cells knocked down for APEX1, PRIMPOL, or APEX1 PRIMPOL exposed to the indicated dose of 5hmdC for 3 days. ( D ) Left: Representative images of SCEs from Fancd2 −/− , Fancd2 −/− siHMCES, Fancd2 −/− siPRIMPOL, and Fancd2 −/− siHMCES/siPRIMPOL cells exposed to 5hmdC (10 μM, 12 hours). Top right: Scheme of SCE assay. Bottom right: Bar plot of SCE/chromosome per metaphase spread ( n = 50 of each of the two biological replicates; bar represents means ± SD). ( E ) Working model depicting the mechanism of replication fork instability by HMCES during the removal of 5hmdU in the absence of the replication fork maintenance FA pathway. RF, replication fork.

Article Snippet: The antibodies used were against PAR (Millipore, MABE1016, Merck Millipore, Burlington, MA, USA), ERCC1 (sc-8408), PCNA (sc-56), Lamin A/C (sc-376248), BrdU (BU1/75, Abcam, Cambridge, England), IdU (Sigma-Aldrich, SAB3701448), Biotin (B7653, Sigma-Aldrich), HMCES (NPB2-14410, Novus Biologicals, Centennial, CO, USA), SMUG1 (E7P4I, Cell Signaling Technology, Danvers, MA, USA), APEX1 (A1117, ABclonal Technology, Woburn, MA, USA), XRCC1 (A0442, ABclonal), DCK (A1794, ABclonal), DCTD (16784-1-AP, Proteintech, Rosemont, IL, USA), FLAG (F1804, Sigma-Aldrich), FLAG (D6W5B, Cell Signaling Technology), PARP1 (46D11, Cell Signaling Technology), and PRIMPOL ( https://pubmed.ncbi.nlm.nih.gov/36152632/ ).

Techniques: MTT Cell Proliferation