Journal: Cell Death Discovery

Article Title: KDM4C silencing inhibits cell migration and enhances radiosensitivity by inducing CXCL2 transcription in hepatocellular carcinoma

doi: 10.1038/s41420-023-01418-w

Figure Lengend Snippet: A Huh7 and HepG2 cells were treated as indicated, and the proportions of cells in each phase of the cell cycle were analyzed using ModFit software. Results are shown in histograms ( n = 3). B KDM4C silencing led to reduced rad51 foci formation by Huh7 and HepG2 cells 4 h post-irradiation. Representative immunofluorescence images and statistical diagrams are shown ( n = 3). Scale bar, 10 μm. C Knockdown of KDM4C contributed to enhanced radiosensitivity in HCC cells ( n = 3).

Article Snippet: After 4 h, the cells were fixed with 4% paraformaldehyde for 15 min and permeabilized with 0.2% Triton X-100 for 15 min. After blocking for 1 h, the cells were incubated with γ-H2AX (Abcam, ab26350, 1:1000) and Rad51 (Abcam, ab133534, 1:1000) antibodies overnight and the corresponding secondary antibody for 1 h. DNA was stained with DAPI for 10 min.

Techniques: Software, Irradiation, Immunofluorescence

Journal: Cell Death Discovery

Article Title: KDM4C silencing inhibits cell migration and enhances radiosensitivity by inducing CXCL2 transcription in hepatocellular carcinoma

doi: 10.1038/s41420-023-01418-w

Figure Lengend Snippet: A Huh7 cells were transfected with the indicated siRNAs, seeded in six-well plates and counted every other day ( n = 3). B Cell proliferation was examined by an EdU assay ( n = 3). Scale bar, 50 μm. C Transwell assays were performed to evaluate the migration of Huh7 cells ( n = 3). Scale bar, 50 μm. D A neutral comet assay was conducted in Huh7 cells to assess single-cell post-irradiation DNA damage in each group ( n = 3). Scale bar, 50 μm. E Rad51 foci formation was used to analyze DNA repair. Representative immunofluorescence images and statistical diagrams are shown ( n = 3). Scale bar, 10 μm.

Article Snippet: After 4 h, the cells were fixed with 4% paraformaldehyde for 15 min and permeabilized with 0.2% Triton X-100 for 15 min. After blocking for 1 h, the cells were incubated with γ-H2AX (Abcam, ab26350, 1:1000) and Rad51 (Abcam, ab133534, 1:1000) antibodies overnight and the corresponding secondary antibody for 1 h. DNA was stained with DAPI for 10 min.

Techniques: Transfection, EdU Assay, Migration, Neutral Comet Assay, Irradiation, Immunofluorescence

Journal: Cancers

Article Title: The RAD51-FFPE Test; Calibration of a Functional Homologous Recombination Deficiency Test on Diagnostic Endometrial and Ovarian Tumor Blocks

doi: 10.3390/cancers13122994

Figure Lengend Snippet: From RECAP to RAD51-FFPE test. (A) EC and OC tumor specimens obtained during surgery were used for both the RECAP test (left) and RAD51-FFPE test (right). The RECAP test requires ex vivo irradiation of fresh tumor specimens (5 Gy) and incubation prior to tissue fixation. The RAD51-FFPE test is performed directly on (archival) diagnostic FFPE specimens. ( B ) Diagnostic FFPE specimens matched with RECAP specimens that were previously published [ , ] were subjected to a three-step quality control (QC). First, diagnostic FFPE specimens with >70% tumor tissue were included (QC1). Second, the γH2AX score based on the percentage of γH2AX+/GMN+ (≥ 2 foci) cells was calculated (QC2). If this score was ≥ 25%, specimens were analyzed with a co-IF for the presence of GMN and RAD51. If ≥ 40 GMN+ cells could be counted, the RAD51-FFPE specimen was included for analysis (QC3). Abbreviations: EC = endometrial carcinoma; OC = ovarian carcinoma; RECAP = REcombination CAPacity; FFPE = formalin-fixed paraffin-embedded; GMN = geminin.

Article Snippet: Subsequently, the slides were blocked with blocking buffer (DAKO wash buffer with 1% BSA) for ten minutes and incubated with primary antibodies for RAD51 (rabbit, monoclonal, Abcam, Cambridge, UK, cat. ab133534) and GMN (mouse, monoclonal, NovoCastra, Leica Biosystems, Buffalo Grove, IL, USA, cat. NCL-L) (1:1000 and 1:60, respectively) for 60 min at RT.

Techniques: Ex Vivo, Irradiation, Incubation, Diagnostic Assay, Formalin-fixed Paraffin-Embedded

Journal: Cancers

Article Title: The RAD51-FFPE Test; Calibration of a Functional Homologous Recombination Deficiency Test on Diagnostic Endometrial and Ovarian Tumor Blocks

doi: 10.3390/cancers13122994

Figure Lengend Snippet: Microscopy illustration of RECAP versus RAD51-FFPE IF stained slides of EC and OC. Tumor specimens were stained for geminin, a marker for the G2/S phase, and RAD51 (Diagnostic FFPE: geminin-Novocastra and RAD51-Abcam; RECAP: geminin-ProteinTech and RAD51-GeneTex) . Case numbers correspond with case numbers in de Jonge et al. and van Wijk et al. [ , ]. Abbreviations: EC = endometrial carcinoma; OC = ovarian carcinoma; RECAP = REcombination CAPacity; FFPE = formalin-fixed paraffin-embedded; HRP = homologous recombination proficient; HRD = homologous recombination deficient.

Article Snippet: Subsequently, the slides were blocked with blocking buffer (DAKO wash buffer with 1% BSA) for ten minutes and incubated with primary antibodies for RAD51 (rabbit, monoclonal, Abcam, Cambridge, UK, cat. ab133534) and GMN (mouse, monoclonal, NovoCastra, Leica Biosystems, Buffalo Grove, IL, USA, cat. NCL-L) (1:1000 and 1:60, respectively) for 60 min at RT.

Techniques: Microscopy, Staining, Marker, Diagnostic Assay, Formalin-fixed Paraffin-Embedded, Homologous Recombination

Journal: Cancers

Article Title: The RAD51-FFPE Test; Calibration of a Functional Homologous Recombination Deficiency Test on Diagnostic Endometrial and Ovarian Tumor Blocks

doi: 10.3390/cancers13122994

Figure Lengend Snippet: Concordance in HRD classification between RECAP and RAD51-FFPE test results when applying various HRD thresholds and RAD51 foci number cut-offs. The HR group classification of the RECAP test was based on a 20% HRD threshold with a RAD51 foci cut-off of ≥ 5 (HRD ≤ 20%; HRP ≥ 20%). RECAP-HRI cases were considered as HRP in this analysis. The test parameters with the highest sensitivity and specificity for all cases are highlighted in bold. Sub analysis of EC and OC cases is represented in Table S4 .

Article Snippet: Subsequently, the slides were blocked with blocking buffer (DAKO wash buffer with 1% BSA) for ten minutes and incubated with primary antibodies for RAD51 (rabbit, monoclonal, Abcam, Cambridge, UK, cat. ab133534) and GMN (mouse, monoclonal, NovoCastra, Leica Biosystems, Buffalo Grove, IL, USA, cat. NCL-L) (1:1000 and 1:60, respectively) for 60 min at RT.

Techniques:

Journal: Cancers

Article Title: The RAD51-FFPE Test; Calibration of a Functional Homologous Recombination Deficiency Test on Diagnostic Endometrial and Ovarian Tumor Blocks

doi: 10.3390/cancers13122994

Figure Lengend Snippet: Correlation RAD51-FFPE scores with RECAP scores in EC and OC. RECAP scores were calculated as the percentage of GMN + cells with ≥5 RAD51 foci. RAD51-FFPE scores were calculated as the percentage of GMN + cells with ≥2 RAD51 foci. The 20% HRD threshold for HR classification of RECAP specimens is indicated with a dotted vertical line. RAD51-FFPE scores significantly correlated with RECAP scores for EC and OC combined ( A ) ( n = 70; Pearson R 2 = 0.20, p = 0.0001), for EC ( B ) ( n = 23; Pearson R 2 = 0.22, p = 0.025) and for OC ( C ) ( n = 47; Pearson R 2 = 0.19, p = 0.0023). One OC case (OC-45) with a pathogenic variant in BRCA1 and classified as HRD by the RECAP test (RECAP score 5%) was a clear outlier in our study, as it had a RAD51-FFPE score of 59%. Details for this case can be found in . Abbreviations: EC = endometrial carcinoma; OC = ovarian carcinoma; RECAP = REcombination CAPacity; FFPE = formalin-fixed paraffin-embedded; GMN = geminin; BRCA deficient = cases with pathogenic variants in BRCA1/2 or BRCA1 promotor hypermethylation.

Article Snippet: Subsequently, the slides were blocked with blocking buffer (DAKO wash buffer with 1% BSA) for ten minutes and incubated with primary antibodies for RAD51 (rabbit, monoclonal, Abcam, Cambridge, UK, cat. ab133534) and GMN (mouse, monoclonal, NovoCastra, Leica Biosystems, Buffalo Grove, IL, USA, cat. NCL-L) (1:1000 and 1:60, respectively) for 60 min at RT.

Techniques: Variant Assay, Formalin-fixed Paraffin-Embedded

Journal: Science Advances

Article Title: A methylation-phosphorylation switch determines Plk1 kinase activity and function in DNA damage repair

doi: 10.1126/sciadv.aau7566

Figure Lengend Snippet: ( A ) Asynchronous cells stably expressing Flag-Plk1 were treated with VP-16 (40 μM) or CPT (2 μM) for 1 hour. Plk1 were immunoprecipitated using an α-Flag antibody, and samples were probed with the indicated antibodies. ( B ) Inhibition of ATR pathway by using the ATR inhibitor VE-821 (10 μM) resulted in premature mitotic entry. Synchronized G 2 cells were treated with VP-16 (40 μM) or CPT (2 μM) for 1 hour and an addition with or without VE-821 treatment for 2 hours. The changes of K209me1 and pT210 levels were examined by Western blot. ( C , F , and H ) The indicated cells were treated with VP-16 (40 μM for 1 hour) and allowed to release into normal medium for the indicated times and analyzed using immunofluorescent staining with an α-γH2A.X, α-RPA2, or α-RAD51 antibody, respectively. ( D ) Quantification of γH2A.X-positive cells in (C) using ImageJ. The data represent means ± SD ( n > 100 each) from three independent experiments. ( E ) The indicated cells that were treated as described in (C) were analyzed using Western blotting. ( G and I ) Quantification of RPA2 or RAD51 foci numbers in individual cells described in (F) or (H) using ImageJ. The boxes designate cells with more than 10 foci, whose percentage is indicated above each box. *** P < 0.001. ( J ) The indicated cells were treated as described in (C), the chromatin fractions were collected, and chromatin-bound RPA2 and RAD51 levels were examined using Western blotting.

Article Snippet: The following antibodies and reagents were obtained from commercial sources: The α-Plk1 (F-8) (sc-17783), α-HA (Y-11) (sc-805), and α-Wee1 (B-11) (sc-5285) antibodies were purchased from Santa Cruz Biotechnology; α-Flag (F7425-2MG) and α-Aurora A (A1231) antibodies were purchased from Sigma-Aldrich; α-G9a/EHMT2(C6H3) (3306S) and α-pCdc2 (Y15) (4539T) antibodies were purchased from Cell Signaling Technology; α-cyclin B1 (1495-1), α-phospho histone H3 pS10 (1173-1), and α-phospho Plk1 pT210 (3646-1) antibodies were purchased from Epitomics; α-BubR1 (ab172581), α-RAD51 (ab133534), and α-H3K9me2 (ab1220) antibodies were purchased from Abcam; α-eGFP (50430-2-AP), α-RPA2 (10412-1-AP), α-Myc (60003-2-Ig), and α-Actinβ (60008-1-Ig) antibodies were purchased from Proteintech; α-H3K9me2 (A2359) and mouse α-GAPDH (glyceraldehyde phosphate dehydrogenase) (AC002) antibodies were purchased from ABclonal; α-H3 (39163) antibody was purchased from Active Motif; pan-mono/dimethyl (TM602) antibody was purchased from PTM Biolabs; α-HEC1(9G3.23) (GTX70268) antibody was purchased from GeneTex; α-Plk1 K209me1 antibody was generated by ABclonal by immunizing rabbits with a K209-monomethylated peptide (DGERKK(me1)TLC) conjugated with keyhole limpet hemacyanin; secondary horseradish peroxidase–conjugated α-mouse or α-rabbit antibodies were purchased from Jackson ImmunoResearch Laboratories; the α-FLAG M2 affinity gel (A2220), thymidine, and nocodazole were purchased from Sigma-Aldrich; the α–hemagglutinin (HA) affinity gel (B23301) was purchased from BioTool; BIX-01294 was purchased from Selleck; VP-16 and VE-821 were purchased from TargetMol; and CPT was purchased from Aladin.

Techniques: Stable Transfection, Expressing, Immunoprecipitation, Inhibition, Western Blot, Staining

Journal: Science Advances

Article Title: A methylation-phosphorylation switch determines Plk1 kinase activity and function in DNA damage repair

doi: 10.1126/sciadv.aau7566

Figure Lengend Snippet: ( A ) Asynchronous cells stably expressing Flag-Plk1 were treated with VP-16 (40 μM) or CPT (2 μM) for 1 hour. Plk1 were immunoprecipitated using an α-Flag antibody, and samples were probed with the indicated antibodies. ( B ) Inhibition of ATR pathway by using the ATR inhibitor VE-821 (10 μM) resulted in premature mitotic entry. Synchronized G 2 cells were treated with VP-16 (40 μM) or CPT (2 μM) for 1 hour and an addition with or without VE-821 treatment for 2 hours. The changes of K209me1 and pT210 levels were examined by Western blot. ( C , F , and H ) The indicated cells were treated with VP-16 (40 μM for 1 hour) and allowed to release into normal medium for the indicated times and analyzed using immunofluorescent staining with an α-γH2A.X, α-RPA2, or α-RAD51 antibody, respectively. ( D ) Quantification of γH2A.X-positive cells in (C) using ImageJ. The data represent means ± SD ( n > 100 each) from three independent experiments. ( E ) The indicated cells that were treated as described in (C) were analyzed using Western blotting. ( G and I ) Quantification of RPA2 or RAD51 foci numbers in individual cells described in (F) or (H) using ImageJ. The boxes designate cells with more than 10 foci, whose percentage is indicated above each box. *** P < 0.001. ( J ) The indicated cells were treated as described in (C), the chromatin fractions were collected, and chromatin-bound RPA2 and RAD51 levels were examined using Western blotting.

Article Snippet: The following antibodies and reagents were obtained from commercial sources: The α-Plk1 (F-8) (sc-17783), α-HA (Y-11) (sc-805), and α-Wee1 (B-11) (sc-5285) antibodies were purchased from Santa Cruz Biotechnology; α-Flag (F7425-2MG) and α-Aurora A (A1231) antibodies were purchased from Sigma-Aldrich; α-G9a/EHMT2(C6H3) (3306S) and α-pCdc2 (Y15) (4539T) antibodies were purchased from Cell Signaling Technology; α-cyclin B1 (1495-1), α-phospho histone H3 pS10 (1173-1), and α-phospho Plk1 pT210 (3646-1) antibodies were purchased from Epitomics; α-BubR1 (ab172581), α-RAD51 (ab133534), and α-H3K9me2 (ab1220) antibodies were purchased from Abcam; α-eGFP (50430-2-AP), α-RPA2 (10412-1-AP), α-Myc (60003-2-Ig), and α-Actinβ (60008-1-Ig) antibodies were purchased from Proteintech; α-H3K9me2 (A2359) and mouse α-GAPDH (glyceraldehyde phosphate dehydrogenase) (AC002) antibodies were purchased from ABclonal; α-H3 (39163) antibody was purchased from Active Motif; pan-mono/dimethyl (TM602) antibody was purchased from PTM Biolabs; α-HEC1(9G3.23) (GTX70268) antibody was purchased from GeneTex; α-Plk1 K209me1 antibody was generated by ABclonal by immunizing rabbits with a K209-monomethylated peptide (DGERKK(me1)TLC) conjugated with keyhole limpet hemacyanin; secondary horseradish peroxidase–conjugated α-mouse or α-rabbit antibodies were purchased from Jackson ImmunoResearch Laboratories; the α-FLAG M2 affinity gel (A2220), thymidine, and nocodazole were purchased from Sigma-Aldrich; the α–hemagglutinin (HA) affinity gel (B23301) was purchased from BioTool; BIX-01294 was purchased from Selleck; VP-16 and VE-821 were purchased from TargetMol; and CPT was purchased from Aladin.

Techniques: Stable Transfection, Expressing, Immunoprecipitation, Inhibition, Western Blot, Staining