anti pc4  (Santa Cruz Biotechnology)

Journal: EMBO Reports

Article Title: Human PC4 supports telomere stability and viability in cells utilizing the alternative lengthening of telomeres mechanism

doi: 10.1038/s44319-024-00295-3

Figure Lengend Snippet: Reagents and tools table

Article Snippet: Rabbit polyclonal anti-PC4 , Bethyl Laboratories , Cat # A301-161A-M.

Techniques: In Vitro, Recombinant, shRNA, Control, Plasmid Preparation, Sequencing, Negative Control, Magnetic Beads, Protease Inhibitor, Blocking Assay, Software, Microscopy

Journal: EMBO Reports

Article Title: Human PC4 supports telomere stability and viability in cells utilizing the alternative lengthening of telomeres mechanism

doi: 10.1038/s44319-024-00295-3

Figure Lengend Snippet: ( A ) Volcano plot derived from the analysis of the Project Achilles database. The plot shows the differential dependency, as Log fold change lethality, for 14 ALT cell lines versus the remaining ones. The horizontal dashed line identifies the statistical significance threshold of 0.05 (Log10 Benjamini Hochberg FDR-adjusted P value). The genes in the upper right quadrant, defined by the dashed lines, are the ones with the most significant impact on ALT cells. The analysis was performed once. ( B ) Violin plot of the gene effect scores for PC4 across all available cell lines. ALT cell lines are indicated by their names. Boxed cell lines are derived from bone cancers. The analysis was performed once. ( C ) The indicated cell lines were transfected with siRNAs against PC4 (siPc1 and siPc2) over a time course of 13 days or infected with lentiviruses expressing an shRNA against PC4 (shPc1) over a time course of 15 days. Cell counts at the indicated time points are normalized against control siRNA/shRNA samples and values for the first day of counting are set to 1. Bars and error bars are means and SDs from three biological replicates. ( D ) Cells as in ( C ) were collected on day 9, stained with PI without fixation, and FACS analyzed. The fraction of PI-positive (permeable) cells is represented. siCt and shCt: control si/shRNAs; siBlm: siRNA against Bloom. Bars and error bars are means and SDs from three biological replicates. P 1 = 0.00411; P 2 = 0.0529; P 3 = 0.0053; P 4 = 0.0439; P 5 = 0.0383; P 6 < 0.0001; P 7 = 0.0003; P 8 = 0.0026; P 9 = 0.005; P 10 = 0.0135; P 11 = 0.0013; P 12 = 0.0234; P 13 = 0.0005; P 14 = 0.0172; P 15 = 0.0335; P 16 = 0.0159 (Student’s t -test). .

Article Snippet: Extracts were centrifuged at 800× g at 4 °C for 5 min and then incubated with 1 μg of rabbit anti-PC4 antibody or with 3 μg of mouse monoclonal anti-Myc antibody (Cell Signaling Technology) for 3 h at 4 °C on a rotating wheel.

Techniques: Derivative Assay, Transfection, Infection, Expressing, shRNA, Control, Staining

Journal: EMBO Reports

Article Title: Human PC4 supports telomere stability and viability in cells utilizing the alternative lengthening of telomeres mechanism

doi: 10.1038/s44319-024-00295-3

Figure Lengend Snippet: ( A ) Western blot analysis of PC4, BLM, and ATRX protein levels. Cells were transfected with siRNAs or infected with shRNA lentiviruses, and total proteins were extracted 72 h after siRNA transfections or 5 days after shRNA infections. Beta Actin (βAct), Vinculin (Vinc), and Helic2 serve as loading controls. Marker molecular weights are on the left of the gels in kDa. ( B ) Examples of FACS profiles of cells stained with PI without permeabilization. The indicated cell lines were depleted of PC4 for 9 days. Cell counts (y-axis) are plotted against PI intensity (x-axis) for one representative experiment. Numbers are percentages of cells positive to PI staining as defined by the indicated gates (vertical bars). ( C ) Examples of FACS profiles of Saos-2 and HOS cells ethanol-fixed and stained with PI. Cells were transfected with siRNAs every 72 h for a total of 9 and 15 days, respectively. Cell counts (y-axis) are plotted against PI intensity (x-axis). The percentages of cells with different DNA contents, including sub-G1 (

Article Snippet: Extracts were centrifuged at 800× g at 4 °C for 5 min and then incubated with 1 μg of rabbit anti-PC4 antibody or with 3 μg of mouse monoclonal anti-Myc antibody (Cell Signaling Technology) for 3 h at 4 °C on a rotating wheel.

Techniques: Western Blot, Transfection, Infection, shRNA, Marker, Staining

Journal: EMBO Reports

Article Title: Human PC4 supports telomere stability and viability in cells utilizing the alternative lengthening of telomeres mechanism

doi: 10.1038/s44319-024-00295-3

Figure Lengend Snippet: ( A ) Dot-blot hybridization of PC4 ChIPs using radiolabeled probes to detect telomeric (Telo) or Alu-repeat DNA. In: Input (1%), Bd: only beads control (50%), IP: PC4 immunoprecipitation (50%). Signals were quantified and graphed (bottom) as the fraction of input DNA found in the corresponding IP samples, after subtraction of Bd-associated signals. Bars and error bars are means and SDs from three biological replicates. ( B ) Examples of PC4 (green) and TRF2 (red) double IF in cells transfected with an siRNA-depleting FANCM (siFM) or siCt. Cells were harvested 48 h after transfection. In the merge panel, DAPI-stained DNA is in blue. Arrowheads point to co-localization events. ( C ) Quantifications of the percentage of cells as in ( B ) showing two or more co-localization events. At least 100 nuclei were analyzed for each sample in each of the three biological replicates. Bars and error bars are means and SDs. P 1 = 0.0021; P 2 = 0.0005 (Student’s t -test). Scale bar: 10 μm. .

Article Snippet: Extracts were centrifuged at 800× g at 4 °C for 5 min and then incubated with 1 μg of rabbit anti-PC4 antibody or with 3 μg of mouse monoclonal anti-Myc antibody (Cell Signaling Technology) for 3 h at 4 °C on a rotating wheel.

Techniques: Dot Blot, Hybridization, Control, Immunoprecipitation, Transfection, Staining

Journal: EMBO Reports

Article Title: Human PC4 supports telomere stability and viability in cells utilizing the alternative lengthening of telomeres mechanism

doi: 10.1038/s44319-024-00295-3

Figure Lengend Snippet: ( A ) Dot-blot hybridization of endogenous PC4 ChIPs in U2OS or HOS cells using radiolabeled probes to detect telomeric (Telo) DNA. Cells were either transfected with siPc2 and harvested 72 h after transfection, or treated with 0.2 mM hydroxyurea (HU) for 16 h. In: Input (1%), Bd: only beads control (50%), IP: PC4 immunoprecipitation (50%). Signals were quantified and graphed (bottom) as the fraction of input DNA found in the corresponding IP samples, after subtraction of Bd-associated signals. For U2OS, results are from one biological replicate. For HOS, bars and error bars are means and SDs from three biological replicates. The disappearance of telomeric DNA signal in the IP fraction of PC4-depleted U2OS cells confirms the specificity of the antibody. ( B ) Examples of PC4 (green) immunostaining in U2OS cells transfected with siPc2 or siCt and harvested 72 h after transfection. Cells were either permeabilized with mild detergent prior to fixation (right panels) or left untreated (left panels), in order to visualize chromatin-bound PC4 and total PC4, respectively. The substantial decrease in staining in PC4-depleted cells confirms the specificity of the antibody. Scale bar: 30 μm.

Article Snippet: Extracts were centrifuged at 800× g at 4 °C for 5 min and then incubated with 1 μg of rabbit anti-PC4 antibody or with 3 μg of mouse monoclonal anti-Myc antibody (Cell Signaling Technology) for 3 h at 4 °C on a rotating wheel.

Techniques: Dot Blot, Hybridization, Transfection, Control, Immunoprecipitation, Immunostaining, Staining

Journal: EMBO Reports

Article Title: Human PC4 supports telomere stability and viability in cells utilizing the alternative lengthening of telomeres mechanism

doi: 10.1038/s44319-024-00295-3

Figure Lengend Snippet: ( A ) Western blot analysis of PC4, RNAPII phosphorylated at CTD Serine 2 (pSer2) and 5 (pSer5), and total RNAPII. Total proteins were extracted 72 h after siRNA transfection. Beta Actin (βAct) serves as a loading control. Marker molecular weights are on the left of the gels in kDa. ( B ) pSer2 and pSer5 signals were quantified and graphed after normalization using the corresponding total RNAPII signals. siCt values are set to 1. Bars and error bars are means and SDs from three biological replicates. P 1 = 0.0071; P 2 = 0.0077 (Student’s t -test). .

Article Snippet: Extracts were centrifuged at 800× g at 4 °C for 5 min and then incubated with 1 μg of rabbit anti-PC4 antibody or with 3 μg of mouse monoclonal anti-Myc antibody (Cell Signaling Technology) for 3 h at 4 °C on a rotating wheel.

Techniques: Western Blot, Transfection, Control, Marker

Journal: EMBO Reports

Article Title: Human PC4 supports telomere stability and viability in cells utilizing the alternative lengthening of telomeres mechanism

doi: 10.1038/s44319-024-00295-3

Figure Lengend Snippet: ( A ) Western blot analysis of endogenous (Endo) and ectopic (Ecto) PC4 proteins in U2OS cell lines expressing siRNA-resistant PC4 proteins, either wt or W89A. Cells were grown in the presence or absence of doxycycline (dox), transfected with siRNAs, and harvested 72 h after transfection. Ectopic PC4 variants run slower because they carry C-terminal Myc and Flag tags. Beta Actin (βAct) serves as a loading control. Marker molecular weights are on the left in kDa. ( B ) Examples of pS33 (green) and TRF2 (red) double IF (left panels) and of PML IF (green) combined with telomeric DNA FISH (red; right panels) in cells as in ( A ). Only cells treated with dox are shown. Arrowheads point to co-localization events. The plots show the co-localization events in experiments as above. Each dot represents one nucleus, black bars are medians. At least 100 nuclei were analyzed for each sample in each of the three biological replicates. For the plot on the left: P 1 < 0.0001; P 2 = 0.031; P 3 < 0.0001; P 4 = 0.0002; for the plot on the right: P 1 < 0.0001; P 2 < 0.0001; P 3 < 0.0001; P 4 < 0.0001 (Mann–Whitney U -test). Scale bars: 10 μm. ( C ) Dot-blot hybridization of anti-Myc ChIPs in cells as in ( A ) using radiolabeled probes to detect telomeric (Telo) DNA. In: Input (2%), Bd: only beads control (50%), IP: Myc immunoprecipitation (50%). Signals were quantified and graphed (bottom) as the fraction of input DNA found in the corresponding IP samples, after subtraction of Bd-associated signals. Bars and error bars are means and SDs from three biological replicates. P 1 = 0.0470 (Student’s t -test). ( D ) Electrophoretic mobility shift assays with recombinant PC4wt and W89A proteins and ssDNA oligonucleotides. The underlined C in the oligonucleotide on the right eliminates the formation of G4 structures. w: wells; B: bound probe; F: free probe. The graph on the right shows quantifications of bound oligonucleotides graphed as a fraction of the total signal within each lane. Data points and error bars are means and SDs from three biological replicates. .

Article Snippet: Extracts were centrifuged at 800× g at 4 °C for 5 min and then incubated with 1 μg of rabbit anti-PC4 antibody or with 3 μg of mouse monoclonal anti-Myc antibody (Cell Signaling Technology) for 3 h at 4 °C on a rotating wheel.

Techniques: Western Blot, Expressing, Transfection, Control, Marker, MANN-WHITNEY, Dot Blot, Hybridization, Immunoprecipitation, Electrophoretic Mobility Shift Assay, Recombinant

Journal: EMBO Reports

Article Title: Human PC4 supports telomere stability and viability in cells utilizing the alternative lengthening of telomeres mechanism

doi: 10.1038/s44319-024-00295-3

Figure Lengend Snippet: Reagents and tools table

Article Snippet: Extracts were centrifuged at 800× g at 4 °C for 5 min and then incubated with 1 μg of rabbit anti-PC4 antibody or with 3 μg of mouse monoclonal anti-Myc antibody (Cell Signaling Technology) for 3 h at 4 °C on a rotating wheel.

Techniques: In Vitro, Recombinant, shRNA, Control, Plasmid Preparation, Sequencing, Negative Control, Magnetic Beads, Protease Inhibitor, Blocking Assay, Software, Microscopy