Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Affinity-based protein profiling to reveal targets of puerarin involved in its protective effect on cardiomyocytes.

doi: 10.1016/j.biopha.2020.111160

Figure Lengend Snippet: Fig. 5. Verification of the binding tar gets of Pue. A) The three enrichment targets of Pue- DA in the damaged cardiomyocytes were verified by western blotting. These experiments were performed in triplicate for each group, and data are represented as mean ± SEM, *p < 0.05 versus DMSO group. B) Cellular thermal shift assay was then used to confirm the stabilizing effect of Pue on the enrichment targets. Three independent experiments were performed for each group. C) Three siRNA strands of chaf1b were assessed by western-blotting assay. Three indepen dent experiments were performed for each group. Data are represented as mean ± SD, **p < 0.01 and ***p < 0.001 versus negative control group. D) AC16 cells were transfected with NC/CHAF1B- siRNA-2 (20 nM) and co-treated with equivalent volume DMSO or Pue (100 μM) for 48 h, and the cell survival was detected using CCK-8 kit. These experi ments were performed in triplicate for each group. Data are represented as mean ± SD, ***p < 0.001 versus nega tive control with DMSO group and ##p < 0.01 versus negative control with Pue group. E) AC16 cells were transfected with NC/ CHAF1B-siRNA-2 (20 nM) and co-treated with equivalent volume DMSO or Pue (100 μM) for 48 h, and the percent of apoptotic cells was determined by flow cytometry.

Article Snippet: The primary antibodies for UBE2C (1:1000, Rabbit), UBE2T (1:1000, Rabbit), CHAF1B (1:1000, Rabbit), and ATP5A1 (1:1000, Mouse) were purchased from Proteintech Group (Chicago, IL, USA).

Techniques: Binding Assay, Western Blot, Thermal Shift Assay, Negative Control, Transfection, CCK-8 Assay, Control, Flow Cytometry

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Affinity-based protein profiling to reveal targets of puerarin involved in its protective effect on cardiomyocytes.

doi: 10.1016/j.biopha.2020.111160

Figure Lengend Snippet: Fig. 6. CHAF1B has no effect on the expression of ATP5A1, UBE2T and UBE2C. A) Immunoprecipitation experiments were proceeded to reveal the binders of CHAF1B in triplicate for each group, and data are represented as mean ± SEM, *p < 0.05, **p < 0.01 versus DMSO group. B) The protein level of ATP5A1, UBE2T and UBE2C was detected by the western-blotting assay. AC16 cells were damaged by H2O2 (400 μM) or high glucose (4.5 g/L), then transfected with NC/CHAF1B-siRNA-2 (20 nM) for 24 h. These experiments were performed in triplicate for each group, and data are represented as mean ± SEM, *p < 0.05, **p < 0.01 versus NC group.

Article Snippet: The primary antibodies for UBE2C (1:1000, Rabbit), UBE2T (1:1000, Rabbit), CHAF1B (1:1000, Rabbit), and ATP5A1 (1:1000, Mouse) were purchased from Proteintech Group (Chicago, IL, USA).

Techniques: Expressing, Immunoprecipitation, Western Blot, Transfection

Journal: Stem cells (Dayton, Ohio)

Article Title: Serum Response Factor Reduces Gene Expression Noise and Confers Cell State Stability.

doi: 10.1093/stmcls/sxad051

Figure Lengend Snippet: Figure 3. Srf KO iPSCs display increased expression of marker genes for 2C-like state. (A) GSEA of the 229 upregulated genes in the Zscan4+ 2C-like cells.40 The x-axis shows the log2(fold change)-ranked Srf KO transcriptome. The enrichment score increases when a gene in the Srf KO transcriptome is also present in the 2C-like gene set, and a Black vertical bar is drawn at bottom; the enrichment score decreases when a gene is absent in the 2C-like gene set. The P-value was empirically determined on the basis of 1000 permutations of ranked gene lists. (B) GSEA of the 310 upregulated genes in the MERVL+/Zscan4+ 2C-like cells.40 (C) Expression of a panel of 2C-like genes between Srf KO and WT iPSCs. * |log2FC| > 1 and FDR < .05; **|log2FC| > 1 and FDR < .01. (D) GSEA of the top 500 most upregulated genes in p150 knockdown (KD) mESCs. (E) GSEA of the top 500 most upregulated genes in p60 KD mESCs.

Article Snippet: Primary antibodies used include β-tubulin (1:5000, Abcam Ab6046) and Caf1 (p60) (1:500, Santa Cruz, #sc-393662).

Techniques: Expressing, Marker, Knockdown

Journal: bioRxiv

Article Title: Lagging strand gap suppression connects BRCA-mediated fork protection to nucleosome assembly by ensuring PCNA-dependent CAF-1 recycling

doi: 10.1101/2021.11.08.467732

Figure Lengend Snippet: A. DNA fiber combing assays showing that CHAF1A depletion results in HU-induced fork degradation in wildtype cells, but suppresses fork degradation in 293T-PCNA K164R cells. The ratio of CldU to IdU tract lengths is presented, with the median values marked on the graph. The p-values (Mann-Whitney test) are listed at the top. A schematic representation of the DNA fiber combing assay conditions is also presented. B . Western blots showing CHAF1A depletion in 293T cells. C, D . Western blots showing CHAF1A depletion in BRCA2-knockout HeLa cells ( C ) and in BRCA1-knockout RPE1 cells ( D ). E, F . Western blots showing CHAF1A knockout in 293T ( E ) and HeLa ( F ) cells. G, H . Western blots showing BRCA2 depletion in CHAF1A-knockout 293T ( G ) and HeLa ( H ) cells. I . Western blots showing CHAF1A co-depletion with BRCA1 or BRCA2 in HeLa cells. J . Representative micrographs of the PARP1 SIRF experiment (scale bar represents 10μm). K . Western blots showing CHAF1A co-depletion with ZRANB3 in HeLa-BRCA2 KO cells.

Article Snippet: For CHAF1A gene knockout, the commercially available CHAF1A CRISPR/Cas9 KO plasmid was used (Santa Cruz Biotechnology sc-402472).

Techniques: MANN-WHITNEY, Western Blot, Knock-Out

Journal: bioRxiv

Article Title: Lagging strand gap suppression connects BRCA-mediated fork protection to nucleosome assembly by ensuring PCNA-dependent CAF-1 recycling

doi: 10.1101/2021.11.08.467732

Figure Lengend Snippet: A, B . DNA fiber combing assays showing that CHAF1A depletion results in HU-induced fork resection in wildtype cells, but suppresses this degradation in BRCA2-knockout HeLa cells ( A ) and in BRCA1-knockout RPE1 cells ( B ). The ratio of CldU to IdU tract lengths is presented, with the median values marked on the graph. The p-values (Mann-Whitney test) are listed at the top. Schematic representations of the DNA fiber combing assay conditions are also presented. Western blots confirming the knockdown are shown in . C, D . DNA fiber combing assays showing that CHAF1A knockout in 293T ( C ) or HeLa ( D ) cells results in HU-induced fork degradation, which is suppressed by BRCA2 knockdown. The ratio of CldU to IdU tract lengths is presented, with the median values marked on the graph. The p-values (Mann-Whitney test) are listed at the top. Schematic representations of the DNA fiber combing assay conditions are also presented. Western blots confirming the knockdown are shown in . E, F . RAD51 immunofluorescence experiment showing that CHAF1A depletion does not restore CPT-induced RAD51 foci in BRCA1 or BRCA2-depleted cells. HeLa cells were treated with 1μM CPT for 1h followed by media removal and chase in fresh media for 3h. Representative micrographs ( E ) and quantifications ( F ) are shown (scale bar represents 10μm). At least 50 cells were quantified for each condition. The median values are represented on the graph, and the p-values (Mann-Whitney test) are listed at the top. Western blots confirming the co-depletions are shown in . G, H . Inhibition of RAD51 by B02 treatment does not restore HU-induced fork degradation in CHAF1A-depleted HeLa-BRCA2 KO cells ( G ), or in BRCA2-depleted 293T-CHAF1A KO cells ( H ). The ratio of CldU to IdU tract lengths is presented, with the median values marked on the graph. The p-values (Mann-Whitney test) are listed at the top. Schematic representations of the DNA fiber combing assay conditions are also presented. I . SIRF assay showing that PARP1 binding to nascent DNA is increased upon CHAF1A depletion in HeLa-BRCA2 KO cells, indicating stabilization of reversed replication forks. At least 40 positive cells were quantified for each condition. The p-values (Mann-Whitney test) are listed at the top. A schematic representation of the SIRF assay conditions is also presented. Representative micrographs (scale bar represents 10μm) are shown in . Western blots confirming the co-depletions are shown in .

Article Snippet: For CHAF1A gene knockout, the commercially available CHAF1A CRISPR/Cas9 KO plasmid was used (Santa Cruz Biotechnology sc-402472).

Techniques: Knock-Out, MANN-WHITNEY, Western Blot, Knockdown, Immunofluorescence, Inhibition, Binding Assay

Journal: bioRxiv

Article Title: Lagging strand gap suppression connects BRCA-mediated fork protection to nucleosome assembly by ensuring PCNA-dependent CAF-1 recycling

doi: 10.1101/2021.11.08.467732

Figure Lengend Snippet: A, B . γ H2AX immunofluorescence experiment showing that CHAF1A depletion suppresses CPT-induced DNA damage accumulation in BRCA1 or BRCA2-depleted cells. HeLa cells were treated with 1μM CPT for 1h followed by media removal and chase in fresh media for 3h. Representative micrographs (scale bar represents 50μm) ( A ) and quantifications ( B ) are shown. At least 50 cells were quantified for each condition. The mean values are represented on the graph, and the p-values (t-test, two-tailed, unequal variance) are listed at the top. Western blots confirming the co-depletions are shown in . C, D . Neutral comet assay showing that CHAF1A depletion suppresses CPT-induced DSB formation in BRCA1 or BRCA2-depleted cells. HeLa cells were treated with 100nM CPT for 4h. Representative micrographs ( C ) and quantifications ( D ) are shown. At least 85 nuclei were quantified for each condition. The mean values are represented on the graph, and the p-values (t-test, two-tailed, unequal variance) are listed at the top. E, F . Clonogenic survival experiments showing that CHAF1A co-depletion in BRCA2-knockdown ( E ) or BRCA1-knockdown ( F ) HeLa cells promotes cisplatin resistance. The average of three experiments, with standard deviations indicated as error bars, is shown. Asterisks indicate statistical significance (two-way ANOVA).

Article Snippet: For CHAF1A gene knockout, the commercially available CHAF1A CRISPR/Cas9 KO plasmid was used (Santa Cruz Biotechnology sc-402472).

Techniques: Immunofluorescence, Two Tailed Test, Western Blot, Neutral Comet Assay, Knockdown

Journal: bioRxiv

Article Title: Lagging strand gap suppression connects BRCA-mediated fork protection to nucleosome assembly by ensuring PCNA-dependent CAF-1 recycling

doi: 10.1101/2021.11.08.467732

Figure Lengend Snippet: A . Western blots showing CHAF1A co-depletion with BRCA1 or BRCA2 in HeLa cells. B . Analyses of BRCA2-mutant ovarian TCGA cancer dataset showing that high CHAF1A levels are associated with increased survival, while low CHAF1A levels are associated with reduced survival. Mantel-Cox log ranked t test was used for statistical analyses (n=14, p=0.0590). The difference observed is not significant, likely because of the small number of BRCA2-mutant samples in the dataset.

Article Snippet: For CHAF1A gene knockout, the commercially available CHAF1A CRISPR/Cas9 KO plasmid was used (Santa Cruz Biotechnology sc-402472).

Techniques: Western Blot, Mutagenesis

Journal: bioRxiv

Article Title: Lagging strand gap suppression connects BRCA-mediated fork protection to nucleosome assembly by ensuring PCNA-dependent CAF-1 recycling

doi: 10.1101/2021.11.08.467732

Figure Lengend Snippet: A. DNA fiber combing assay showing that ASF1A depletion results in HU-induced fork degradation in wildtype cells, but does not affect this degradation in BRCA1 or BRCA2-depleted HeLa cells. The ratio of CldU to IdU tract lengths is presented, with the median values marked on the graph. The p-values (Mann-Whitney test) are listed at the top. A schematic representation of the DNA fiber combing assay conditions is also presented. Western blots confirming the co-depletion are shown in . B. DNA fiber combing assay showing that ASF1A co-depletion restores HU-induced fork degradation in CHAF1A-knockdown HeLa-BRCA2 KO cells. The ratio of CldU to IdU tract lengths is presented, with the median values marked on the graph. The p-values (Mann-Whitney test) are listed at the top. A schematic representation of the DNA fiber combing assay conditions is also presented. Western blots confirming the co-depletion are shown in . C. DNA fiber combing assay showing that inhibition of nucleases MRE11 (by treatment with mirin) or DNA2 (by treatment with C5) suppresses HU-induced fork degradation caused by CHAF1A or ASF1A loss in HeLa cells. The ratio of CldU to IdU tract lengths is presented, with the median values marked on the graph. The p-values (Mann-Whitney test) are listed at the top. A schematic representation of the DNA fiber combing assay conditions is also presented. D. DNA fiber combing assay showing that co-depletion of DNA translocases SMARCAL1 and ZRANB3 suppresses HU-induced fork degradation caused by CHAF1A knockdown in HeLa cells. The p-values (Mann-Whitney test) are listed at the top. A schematic representation of the DNA fiber combing assay conditions is also presented. Western blots confirming the co-depletion are shown in . E. DNA fiber combing assay showing that SMARCAL1 or ZRANB3 depletion suppresses HU-induced fork degradation in 293T-CHAF1A KO cells. The p-values (Mann-Whitney test) are listed at the top. A schematic representation of the DNA fiber combing assay conditions is also presented. Western blots confirming the knockdowns are shown in .

Article Snippet: For CHAF1A gene knockout, the commercially available CHAF1A CRISPR/Cas9 KO plasmid was used (Santa Cruz Biotechnology sc-402472).

Techniques: MANN-WHITNEY, Western Blot, Knockdown, Inhibition

Journal: bioRxiv

Article Title: Lagging strand gap suppression connects BRCA-mediated fork protection to nucleosome assembly by ensuring PCNA-dependent CAF-1 recycling

doi: 10.1101/2021.11.08.467732

Figure Lengend Snippet: A. Western blots showing ASF1A co-depletion with BRCA1 or BRCA2 in HeLa cells. B. Western blots showing ASF1A co-depletion with CHAF1A in HeLa-BRCA2 KO cells. C. Western blots showing CHAF1A co-depletion with SMARCAL1 or ZRANB3 in HeLa cells. D. Western blots showing ZRANB3 and SMARCAL1 depletions in 293T-CHAF1A KO cells.

Article Snippet: For CHAF1A gene knockout, the commercially available CHAF1A CRISPR/Cas9 KO plasmid was used (Santa Cruz Biotechnology sc-402472).

Techniques: Western Blot

Journal: bioRxiv

Article Title: Lagging strand gap suppression connects BRCA-mediated fork protection to nucleosome assembly by ensuring PCNA-dependent CAF-1 recycling

doi: 10.1101/2021.11.08.467732

Figure Lengend Snippet: A, B. Linear regressions of ( A ) HIRA Gene Effect (CERES) vs. CHAF1A Gene Effect (CERES) and ( B ) DAXX Gene Effect vs. CHAF1A Gene Effect (CERES) in Cancer Cell Line Encyclopedia (CCLE) cell lines containing either wildtype BRCA1 or BRCA1 with deleterious mutations are shown. A lower CERES score corresponds to greater survival dependency. C, D . SIRF assay showing that HIRA binding to nascent DNA is increased upon BRCA1 or BRCA2 depletion in HeLa cells. Representative micrographs (scale bar represents 10μm) ( C ) and quantifications ( D ) are shown. At least 40 positive cells were quantified for each condition. The p-values (Mann-Whitney test) are listed at the top. A schematic representation of the SIRF assay conditions is also presented. E. DNA fiber combing assay showing that HIRA co-depletion restores HU-induced nascent strand resection in CHAF1A-knockdown HeLa-BRCA2 KO cells. The ratio of CldU to IdU tract lengths is presented, with the median values marked on the graph. The p-values (Mann-Whitney test) are listed at the top. A schematic representation of the DNA fiber combing assay conditions is also presented. Western blots confirming the co-depletion are shown in .

Article Snippet: For CHAF1A gene knockout, the commercially available CHAF1A CRISPR/Cas9 KO plasmid was used (Santa Cruz Biotechnology sc-402472).

Techniques: Binding Assay, MANN-WHITNEY, Knockdown, Western Blot

Journal: bioRxiv

Article Title: Lagging strand gap suppression connects BRCA-mediated fork protection to nucleosome assembly by ensuring PCNA-dependent CAF-1 recycling

doi: 10.1101/2021.11.08.467732

Figure Lengend Snippet: A . SIRF assay confirm that the HIRA PLA signal is specific, since it is reduced upon HIRA depletion by siRNA. At least 20 positive cells were quantified for each condition. The p-values (Mann-Whitney test) are listed at the top. A schematic representation of the SIRF assay conditions is also presented. B . SIRF assay showing the impact of CHAF1A co-depletion on HIRA binding to nascent DNA in BRCA1 or BRCA2-knockdown HeLa cells. At least 65 cells were quantified for each condition. The p-values (Mann-Whitney test) are listed at the top. A schematic representation of the SIRF assay conditions is also presented. C . SIRF assay showing that DAXX binding to nascent DNA is decreased upon BRCA1 or BRCA2 depletion in HeLa cells. DAXX depletion by siRNA reduces the PLA signal, confirming its specificity. A schematic representation of SIRF assay conditions is also presented. At least 30 positive cells were quantified for each condition. The p-values (Mann-Whitney test) are listed at the top. A schematic representation of the SIRF assay conditions is also presented. D. Western blots showing HIRA co-depletion with CHAF1A in HeLa-BRCA2 KO cells. E. DNA fiber combing assays showing that co-depletion of HIRA or of H3.3-encoding genes H3F3A and H3F3B restores HU-induced nascent strand degradation in CHAF1A-depleted HeLa-BRCA2 KO cells. The ratio of CldU to IdU tract lengths is presented, with the median values marked on the graph. The p-values (Mann-Whitney test) are listed at the top. A schematic representation of the DNA fiber combing assay conditions is also presented. F . RT-qPCR experiment showing reduction in H3F3A and H3F3B mRNA levels upon siRNA-mediated knockdown. The average of two technical replicates is shown. (No antibody was available to us for verifying the depletion by Western blot.) G, H. DNA fiber combing assays showing the impact of the depletion of histone chaperones HIRA and DAXX on HU-induced nascent strand degradation in HeLa cells. The ratio of CldU to IdU tract lengths is presented, with the median values marked on the graph ( G ). The p-values (Mann-Whitney test) are listed at the top. A schematic representation of the DNA fiber combing assay conditions is also presented. Western blots confirming the knockdowns ( H ) are also presented.

Article Snippet: For CHAF1A gene knockout, the commercially available CHAF1A CRISPR/Cas9 KO plasmid was used (Santa Cruz Biotechnology sc-402472).

Techniques: MANN-WHITNEY, Binding Assay, Knockdown, Western Blot, Quantitative RT-PCR

Journal: bioRxiv

Article Title: Lagging strand gap suppression connects BRCA-mediated fork protection to nucleosome assembly by ensuring PCNA-dependent CAF-1 recycling

doi: 10.1101/2021.11.08.467732

Figure Lengend Snippet: A, B. SIRF assay showing that CHAF1A unloading from nascent DNA upon replication fork arrest is deficient in BRCA1 or BRCA2-depleted HeLa cells. To induce fork arrest, cells were treated with 4mM HU after EdU labeling. Representative micrographs (scale bar represents 10μm) ( A ) and quantifications ( B ) are shown. At least 30 positive cells were quantified for each condition. The p-values (Mann-Whitney test) are listed at the top. A schematic representation of the SIRF assay conditions is also presented. C. SIRF assay showing that, in BRCA1 or BRCA2-depleted HeLa cells, CHAF1A is retained on nascent DNA behind the replication fork after recovery from replication stress. Cells were labeled with EdU in the presence of low-dose HU (0.4mM for 30mins) to induce replication stress, washed, and chased for 4h in fresh media containing 50μM thymidine. At least 35 positive cells were quantified for each condition. The p-values (Mann-Whitney test) are listed at the top. A schematic representation of the SIRF assay conditions is also presented. D. DNA fiber combing assay showing that CHAF1A knockdown restores fork protection to wildtype HeLa cells treated with the RAD51 inhibitor B02. The ratio of CldU to IdU tract lengths is presented, with the median values marked on the graph. The p-values (Mann-Whitney test) are listed at the top. A schematic representation of the DNA fiber combing assay conditions is also presented. E. SIRF assay showing that prior replication stress reduces the levels of CHAF1A at ongoing replication forks in BRCA1 or BRCA2-depleted HeLa cells. Cells were subjected to low-dose HU (0.4mM for 30 mins) to induced replication stress, chased in fresh media for 4h to recover from replication stress, then labeled with EdU. At least 40 positive cells were quantified for each condition. The p-values (Mann-Whitney test) are listed at the top. A schematic representation of the SIRF assay conditions is also presented. F. DNA fiber combing assay showing that CHAF1A overexpression suppresses HU-induced fork degradation in BRCA1 or BRCA2-depleted HeLa cells. CHAF1A expression is under the control of the tetracycline responsive element (TRE), and is induced upon doxycycline (DOX) treatment. The ratio of CldU to IdU tract lengths is presented, with the median values marked on the graph. The p-values (Mann-Whitney test) are listed at the top. A schematic representation of the DNA fiber combing assay conditions is also presented. Western blots showing CHAF1A overexpression are presented in .

Article Snippet: For CHAF1A gene knockout, the commercially available CHAF1A CRISPR/Cas9 KO plasmid was used (Santa Cruz Biotechnology sc-402472).

Techniques: Labeling, MANN-WHITNEY, Knockdown, Over Expression, Expressing, Control, Western Blot

Journal: bioRxiv

Article Title: Lagging strand gap suppression connects BRCA-mediated fork protection to nucleosome assembly by ensuring PCNA-dependent CAF-1 recycling

doi: 10.1101/2021.11.08.467732

Figure Lengend Snippet: A. SIRF assay showing that, in the absence of replication stress, CHAF1A recycling is not impaired in BRCA1 or BRCA2-depleted HeLa cells. Cells were labeled with EdU, washed, and chased for 4h in fresh media containing 50μM thymidine. At least 25 positive cells were quantified for each condition. The p-values (Mann-Whitney test) are listed at the top. A schematic representation of the SIRF assay conditions is also presented. B. Western blots showing doxycycline-induced CHAF1A overexpression, and BRCA1 or BRCA2 knockdown in these cells.

Article Snippet: For CHAF1A gene knockout, the commercially available CHAF1A CRISPR/Cas9 KO plasmid was used (Santa Cruz Biotechnology sc-402472).

Techniques: Labeling, MANN-WHITNEY, Western Blot, Over Expression, Knockdown

Journal: bioRxiv

Article Title: Lagging strand gap suppression connects BRCA-mediated fork protection to nucleosome assembly by ensuring PCNA-dependent CAF-1 recycling

doi: 10.1101/2021.11.08.467732

Figure Lengend Snippet: A, B . BrdU alkaline comet assay showing that PRIMPOL depletion reduces, but does not abolish replication stress-induced ssDNA accumulation in BRCA2-knockout HeLa cells, indicating the presence of PRIMPOL-independent gaps. Representative micrographs ( A ) and quantifications ( B ) are shown. At least 50 nuclei were quantified for each condition. The median values are represented on the graph, and the p-values (Mann-Whitney test) are listed at the top. A schematic representation of the assay conditions is also presented. Western blots confirming PRIMPOL depletion are presented in . C, D. SIRF assay showing that, upon replication stress, LIG1 knockdown induces PAR chain formation in wildtype HeLa cells, but not in BRCA1 or BRCA2-depleted HeLa cells upon treatment with a low dose of HU (0.4mM), indicating the prevalence of lagging strand gaps in these cells. Representative micrographs (scale bar represents 10μm) ( C ) and quantifications ( D ) are shown. At least 35 positive cells were quantified for each condition. The p-values (Mann-Whitney test) are listed at the top. A schematic representation of the SIRF assay conditions is also presented. Western blots confirming the co-depletion are shown in . E, F. SIRF assay showing that Pol α inhibition suppresses the increased CHAF1A retention in BRCA1 or BRCA2-depleted HeLa cells. Representative micrographs (scale bar represents 10μm) ( E ) and quantifications ( F ) are shown. At least 35 positive cells were quantified for each condition. The p-values (Mann-Whitney test) are listed at the top. A schematic representation of the SIRF assay conditions is also presented. G, H. SIRF assay showing that RPA1 co-depletion restores HIRA levels to the same levels in wildtype and BRCA1 or BRCA2-knockodown HeLa cells. Representative micrographs (scale bar represents 10μm) ( G ) and quantifications ( H ) are shown. At least 30 positive cells were quantified for each condition. The p-values (Mann-Whitney test) are listed at the top. A schematic representation of the SIRF assay conditions is also presented. Western blots confirming the co-depletion are shown in .

Article Snippet: For CHAF1A gene knockout, the commercially available CHAF1A CRISPR/Cas9 KO plasmid was used (Santa Cruz Biotechnology sc-402472).

Techniques: Alkaline Single Cell Gel Electrophoresis, Knock-Out, MANN-WHITNEY, Western Blot, Knockdown, Inhibition

Journal: bioRxiv

Article Title: Lagging strand gap suppression connects BRCA-mediated fork protection to nucleosome assembly by ensuring PCNA-dependent CAF-1 recycling

doi: 10.1101/2021.11.08.467732

Figure Lengend Snippet: A, B. SIRF assay showing deficient PCNA unloading from nascent DNA upon replication fork arrest in BRCA-depleted HeLa cells. Pol α inhibition by treatment with 10μM ST1926 suppresses this defect, indicating that this PCNA retention on nascent DNA occurs at lagging strand gaps. Representative micrographs (scale bar represents 10μm) ( A ) and quantifications ( B ) are shown. At least 50 positive cells were quantified for each condition. The p-values (Mann-Whitney test) are listed at the top. A schematic representation of the SIRF assay conditions is also presented. C. DNA fiber combing assay showing that co-depletion of BRD3 or BRD4 restores HU-induced fork degradation in CHAF1A-knockdown HeLa-BRCA2 KO cells. The ratio of CldU to IdU tract lengths is presented, with the median values marked on the graph. The p-values (Mann-Whitney test) are listed at the top. A schematic representation of the DNA fiber combing assay conditions is also presented. Western blots confirming the co-depletion are shown in . D, E. Chromatin fractionation experiments showing that ATAD5 ( D ) or LIG1 ( E ) knockdown increases the chromatin levels of wildtype PCNA, but not those of PCNA C81R and D150E variants. MRE11 is used as a control for the chromatin fraction. LIG1 depletion is confirmed by western blot. ATAD5 depletion is confirmed by RT-qPCR, since no antibody was available to use to verify depletion by western blot. The average of two technical replicates is shown. F. DNA fiber combing assay showing that BRCA2 knockdown causes HU-induced fork degradation in 293T cells expressing wildtype PCNA, but not in 293T cells expressing PCNA variants with deficient chromatin retention. The ratio of CldU to IdU tract lengths is presented, with the median values marked on the graph. The p-values (Mann-Whitney test) are listed at the top. A schematic representation of the DNA fiber combing assay conditions is also presented. Western blots confirming the knockdown are shown in . G. DNA fiber combing assay showing that CHAF1A knockdown suppresses HU-induced fork degradation caused by B02 treatment in 293T cells expressing wildtype PCNA, but not in 293T cells expressing PCNA variants with deficient chromatin retention. The ratio of CldU to IdU tract lengths is presented, with the median values marked on the graph. The p-values (Mann-Whitney test) are listed at the top. A schematic representation of the DNA fiber combing assay conditions is also presented. Western blots confirming the knockdown are shown in . H. Schematic model outlining the proposed mechanism for fork degradation in BRCA-deficient cells caused by PCNA unloading defects. The failure of BRCA-deficient cells to restrain fork progression during replication stress causes gap accumulation due to repriming by PRIMPOL (on the leading strand) and Polα (on the lagging strand). Polα-mediated repriming results in formation of lagging strand gaps which preclude PCNA unloading. The persistence of PCNA behind replication forks sequesters CAF-1 away from active replication factories, thus interfering with nucleosome assembly at forks and priming them for degradation upon stalling. A more detailed version of this model figure is provided in .

Article Snippet: For CHAF1A gene knockout, the commercially available CHAF1A CRISPR/Cas9 KO plasmid was used (Santa Cruz Biotechnology sc-402472).

Techniques: Inhibition, MANN-WHITNEY, Knockdown, Western Blot, Fractionation, Control, Quantitative RT-PCR, Expressing

Journal: bioRxiv

Article Title: Lagging strand gap suppression connects BRCA-mediated fork protection to nucleosome assembly by ensuring PCNA-dependent CAF-1 recycling

doi: 10.1101/2021.11.08.467732

Figure Lengend Snippet: A. Western blots showing CHAF1A co-depletion with BRCA3 or BRD4 in HeLa-BRCA2 KO cells. B. Alignment of yeast and human PCNA, indicating the PCNA mutations performed. C . Western blots showing the expression of PCNA variants in PCNA-hypomorph 293T cells. D. Western blots showing BRCA2 depletion in 293T cells expressing PCNA variants. E. Western blots showing CHAF1A depletion in 293T cells expressing PCNA variants.

Article Snippet: For CHAF1A gene knockout, the commercially available CHAF1A CRISPR/Cas9 KO plasmid was used (Santa Cruz Biotechnology sc-402472).

Techniques: Western Blot, Expressing

Journal: Nucleic Acids Research

Article Title: BESST: a novel LncRNA knockout strategy with less genome perturbance

doi: 10.1093/nar/gkad197

Figure Lengend Snippet: The determination of the molecular mechanism of the BESST knockout-induced RNA degradation. ( A ) Schematic representation of pathways and associated factors for degradation of lncRNAs. ( B ) The real-time qPCR results of DHRS4-AS1 level of the Cap-RIP samples using DHRS4-AS1 knockout cells versus control. The BESST strategy was applied to produce the knockout cell line. ( C ) The RNA level of DHRS4-AS1 in BESST-mediated DHRS4-AS1 knockout cells upon knocking down of cap-binding complex ( NCBP1 and ARS2 ), 5′ decapping enzyme ( DCP2 , DXO , and NUTD16 ) and 5′-3′ exonuclease ( XRN2 ) by siRNA duplexes. ( D ) The RNA level of DHRS4-AS1 in BESST-mediated DHRS4-AS1 knockout cells upon knocking down of endonuclease ( RNASEH1 ) by RNA silencing. ( E, F ) The RNA level of DHRS4-AS1 in BESST-mediated DHRS4-AS1 knockout cells upon knocking down the critical components of PAXT/PPC-NEXT-TRAMP complex ( ZCCHC7 , ZC3H18 , ZCCHC8 , and ZFC3H1 ) and RNA exosomes ( EXOSC3 , EXOSC10 , DIS3 , and MTR4 ) by siRNA duplexes. ( G ) The poly(A) tail length assay using BESST-mediated DHRS4-AS1 knockout cell line versus wildtype control. ( H, I ) The RNA level of DHRS4-AS1 in BESST-mediated DHRS4-AS1 knockout cells upon knocking down the essential proteins of 3′-5′ exonuclease and poly(A) deadenylation complex ( CNOT7 , CNOT8 , PAN2 , and PARN ) and poly(A) binding protein PABPN1 by RNA silencing. ( J ) A schematic diagram of BESST-KO strategy to reduce lncRNAs through poly(A) deadenylation and decapping. At least three biological replicates and three technical replicates for each sample were performed. One-way ANOVA performed the statistical significance, ** indicates P < 0.01, * indicates P < 0.05, and the ‘ns’ indicates no significance.

Article Snippet: The nitrocellulose membranes (PALL, #66485) were blocked with 5% non-fat milk in 1× TBST buffer for 1 h and incubated on an orbital shaker for 1hr at room temperature with primary antibodies: anti-B2M antibody (Cell Signaling Technology, #12851), anti-GAPDH antibody (Proteintech, #10494-1-AP), anti-lamin B1 antibody (Novus, #NBP1-19804), anti-CNOT7 antibody (Proteintech, #14102-1-AP), and anti-CNOT8 antibody (Proteintech, #10752-1-AP).

Techniques: Knock-Out, Control, Binding Assay

Journal: Scientific Reports

Article Title: Bone secreted factors induce cellular quiescence in prostate cancer cells

doi: 10.1038/s41598-019-54566-4

Figure Lengend Snippet: Secreted factors unique in bone-conditioned media.

Article Snippet: Antibodies for Ki67 (Dako #MIB-1), p27Kip1 (Cell Signaling #D69C12), phospho-p38MAPK (Thr180/Tyr182) (Cell Signaling #4511), DKK3 (Proteintech #10355-1-AP), BMP1 (abcam #ab205394), MIA (Santa Cruz #sc-37735), NGAL (Santa Cruz #sc-515876), and PEDF (R&D #AF-1177) were obtained from commercial sources.

Techniques: Sequencing

Journal: Scientific Reports

Article Title: Bone secreted factors induce cellular quiescence in prostate cancer cells

doi: 10.1038/s41598-019-54566-4

Figure Lengend Snippet: Bone-secreted factors induce cellular quiescence in PCa cells. ( A ) Dose response. C4-2B4 PCa cells were treated without or with various recombinant human bone-secreted factors at different concentrations and analyzed by live-cell imaging as in Fig. . About 100 cells were monitored for each factor at each concentration. Using this approach, we obtained an empirically-derived optimal concentration to be used for each factor (arrowheads): DKK3 (10 µg/mL), BMP1 (0.4 µg/mL), VASN (0.25 µg/mL), NEO1 (0.5 µg/mL), MIA (0.1 µg/mL), and NGAL (0.25 µg/mL). ( B ) (Left) Phase contrast brightfield images. Cells were plated in a Q4 glass-bottom dish, treated with and without various recombinant proteins, and analyzed by live-cell imaging. Representative images are shown for control cells and cells treated with recombinant human DKK3 (cell a), vasorin (cell b), neogenin (cell c), and BMP1 (cell d). Asterisks (*) follow a control cell through 3 cell divisions. Round cells are cells undergoing mitosis. (Right) Immunofluorescence images. At the end of live-cell imaging, cells were immediately fixed and co-immnostained for Ki67 (proliferation marker) and p27 (dormancy marker), and merged with phase contrast images. Cell outlines are traced for ease of view. All bars, 20 µm. ( C ) C4-2B4 cells were treated with various bone-secreted factors, using concentrations as determined in (A), and analyzed by live-cell imaging. PEDF was used at 0.25 µg/ml . The dormancy factor BMP7 (0.4 µg/ml) was used as a positive control. % quiescent cells that did not divide relative to total cells counted were quantified (mean ± s.e.m), except for PEDF and BMP7 (mean ± s.d.). n , number of cells monitored. N, number of independent experiments for control (N = 24), DKK3 (N = 10), BMP1 (N = 10), vasorin (N = 9), neogenin (N = 4), MIA (N = 4), NGAL (N = 6), PEDF (N = 2), BMP7 (N = 2). P values were by t test. ns, not significant. ( D ) C4-2b cells were treated with various bone-secreted factors as indicated, monitored by live-cell imaging, and analyzed as in ( C ). n , number of cells monitored. N, number of independent experiments for control (N = 3), DKK3 (N = 3), BMP1 (N = 3), vasorin (N = 2), neogenin (N = 2). P values were by t test.

Article Snippet: Antibodies for Ki67 (Dako #MIB-1), p27Kip1 (Cell Signaling #D69C12), phospho-p38MAPK (Thr180/Tyr182) (Cell Signaling #4511), DKK3 (Proteintech #10355-1-AP), BMP1 (abcam #ab205394), MIA (Santa Cruz #sc-37735), NGAL (Santa Cruz #sc-515876), and PEDF (R&D #AF-1177) were obtained from commercial sources.

Techniques: Recombinant, Live Cell Imaging, Concentration Assay, Derivative Assay, Control, Immunofluorescence, Marker, Positive Control