anti phospho 53bp1  (Cell Signaling Technology Inc)

Journal: bioRxiv

Article Title: The SF3B1 inhibitor pladienolide B massively inhibits DNA damage signaling and repair and counteracts resistance to platinum salts in Non-Small Cell Lung Cancer

doi: 10.64898/2026.02.17.706284

Figure Lengend Snippet: (a) H460S/R or A549S/R cells were treated or not with 5 nM pladienolide B for indicated times. Cell cycle distribution (%) in the different phases of the cell cycle. Mean ± SD. n = 3. Unpaired t test. * p< 0.05, ** p< 0.01, **** p< 0.0001. (b) Percentage of EdU-positive H460R cells in untreated or pladienolide B condition. Mean ± SD. n = 3. Unpaired t test. * p< 0.05. (c, d, e) H460R and H460S cells were treated or not with 5 nM pladienolide B for 6 hours. Upper panels: representative immunofluorescence stainings of RPA32 (c), γH2AX (d), or phospho-53BP1(Ser1778) (e). DAPI was used to counterstain the nucleus. Scale bar = 10 µm. Lower panels: quantification of the number of RPA32 (c), γH2AX (d), or phospho-53BP1(Ser1778) (e) foci/nucleus. Mean ± SD. n = 3. Unpaired t test. ** p< 0.01, *** p< 0.001, **** p<0.0001. (f) Upper panels: representative immunoblots of γH2AX protein in H460S/R cells treated with 5 nM pladienolide B for indicated times. KU80 was used as a loading control. Lower panels: densitometric quantification (fold change) of γH2AX signal normalized to KU80 signal. Mean ± SD. n=3. Mann-Whitney t test. * p< 0.05. (g, h) H460R cells were treated or not for 6 hours with 5 nM pladienolide B. (g) SIRF analysis of PCNA recruitment at nascent replication forks. Lefts panels: representative images. Right panel: quantification of EdU-PCNA fluorescent intensity (ratio signal intensity/area)/nucleus. Mean. n = 3. Unpaired t test. **** p<0.0001. (h) PCNA immunoblot. Tubulin was used as a loading control.

Article Snippet: After three washes with 1X PBS for 5 min at RT, non-specific binding sites were blocked with 1X PBS, 10% bovine serum, and 0.3% Triton X-100 for 1 hour at RT before overnight incubation at 4°C with an anti-γH2AX antibody (cat# 05-636, Millipore) together with an anti phospho-53BP1(Ser1778) antibody (cat#2675, Cell Signaling Technology), or an anti RPA32 antibody (cat#52448, Santa-Cruz), overnight at 4°C.

Techniques: Immunofluorescence, Western Blot, Control, MANN-WHITNEY

Journal: Nature Communications

Article Title: Non-canonical dihydrolipoyl transacetylase promotes chemotherapy resistance via mitochondrial tetrahydrofolate signaling

doi: 10.1038/s41467-025-63892-3

Figure Lengend Snippet: a Effect of targeting DLAT on cellular uptake of cisplatin and cisplatin-mediated DNA damage. Cells were treated with sublethal doses of cisplatin for 24 h, followed by staining with fluorescently labeled antibodies against cisplatin-DNA adducts, phospho-Histone H2A.X, and phospho-53BP1.Effect of DLAT loss and cisplatin treatment on bioenergetics, biosynthesis, and ROS levels. Cells were treated with cisplatin, and ATP, RNA, protein synthesis levels ( b ) and cellular ROS levels ( c ) were measured by luminescent assay, metabolic labeling, and DCFDA staining, respectively. d Cisplatin-treated cells with DLAT knockdown were treated with antioxidant NAC (0.5 mM). Cellular ROS (upper), apoptosis (middle), and cell viability (lower) were measured. Effect of mitochondria-targeted antioxidant mito-TEMPO ( e ) or catalase overexpression ( f ) on ROS levels, apoptosis, and cell viability in cells with DLAT knockdown and cisplatin. Mito-TEMPO (10 μM) or flag-tagged catalase was introduced in cisplatin-treated cells. Mitochondrial ROS and cytoplasmic hydrogen peroxide were measured by mitoSOX staining and luminescence detection, respectively. g Immunoblotting of apoptosis-associated proteins. Cells with or without DLAT knockdown were treated with sublethal doses of cisplatin for 24 h. h Effect of DLAT loss and NAC treatment on Bcl-xL expression. i Scatter plot of drug sensitivity in KB-3-1 cisR and A549 cisR representing ROS level, apoptotic rate, or growth inhibition vs -log 10 ( P value). Cells were treated with sublethal doses of drugs (2 μg/ml cisplatin, 50 μM carboplatin, 10 nM gemcitabine, 10 μM etoposide, 0.5 μM pemetrexed, 3 nM paclitaxel, 1 μM erlotinib) for 48 h followed by DCFDA staining (left), annexin V staining (middle), and CellTiter-Glo viability assay (right). Fold changes were obtained by comparing the DLAT knockdown group to the control group. Data are mean ± SD from 3 independent biological replicates for ( a – f , i ). P values were determined by two-tailed Student’s t -test ( a , i ) and one-way ANOVA for the rest. Source data are provided as a Source Data file.

Article Snippet: Antibodies against MTHFD2 (41377/D8W9U), myc-Tag (2278/71D10), phospho-Histone gamma H2AX S139 (9718/20E3), phospho-53BP1 S1778 (2675), COX1/MT-CO1 (62101), COX2/MT-CO2 (31219), COX IV (4850/3E11), acetyl-lysine (9441), Bcl-xL (2762), Bcl2 (15071), Mcl-1 (39224/D5V5L), Bad (9268/11E3), Bim (2933/C34C5), PARP (9542), Histone H3 (4499/D1H2), LC3A/B-I/II (4108), and p62 (5114) were purchased from Cell Signaling Technology.

Techniques: Staining, Labeling, Luminescence Assay, Knockdown, Over Expression, Western Blot, Expressing, Inhibition, Viability Assay, Control, Two Tailed Test

Journal: EMBO Reports

Article Title: Inhibition of the minor spliceosome restricts the growth of a broad spectrum of cancers

doi: 10.1038/s44319-025-00511-8

Figure Lengend Snippet: ( A ) RT-PCR analysis of cell cycle related MIG splicing changes in A549 cells after 72 h treatment with 10 nM ASO. Schematic depictions of the obtained amplicons are shown on the right with the minor intron in red and the upstream and downstream exons coloured blue and orange, respectively. Exons not separated by a minor intron are grey. ( B ) Representative FACS plots of yH2AX expression from A549 cells after 72 h treatment with 10 nM ASO. ( C ) Quantification of yH2AX FACS analysis. Data are represented by the mean ± SEM ( n = 4). Significance was assessed by one-way ANOVA with Tukey’s multiple comparison test. ( D ) Two representative images of 53BP1 staining in A549 cells 72 h after 10 nM ASO treatment. Scale bar is 10 µm. ( E ) P53 immunoblot of nuclear and cytoplasmic fractions from A549 cells exposed to 10 nM ASO for 72 h ( n = 3 biological replicates/condition). ( F ) Flow cytometry histograms of FITC senescence probe in A549 cells 72 h after 10 nM ASO treatment. ( G ) Quantification of senescence FACS analysis. MFI = median fluorescence intensity. Data are represented by mean ± SEM ( n = 4). Significance was assessed by one-way ANOVA with Tukey’s multiple comparison test. ( H ) Representative FACS plots of cell cycle analysis from A549 cells after 72 h treatment with 10 nM ASO. Proliferating cells stained for incorporated EdU against total DNA content measured by DAPI. ( I ) Quantification of FACS cell cycle analysis. Data are represented by mean ± SEM ( n = 4). Significance was assessed by one-way ANOVA with Tukey’s multiple comparison test. U12 ASO-treated samples were significantly different to NT ASO samples at two cell cycle stages: S P = 0.0009, G2 P < 0.0001. ( J ) Quantification of A549 cell growth over 72 h treatment across a titration of U12 ASO (0.625 nM–10 nM) compared to 10 nM non-targeted (NT) ASO. ( K ) Schematic diagram depicting the sequence of molecular and cellular events linking minor splicing disruption with reduced tumour burden. Data are represented as mean ± SEM ( n = 4, 16 images per well, every hour). .

Article Snippet: IF: Rabbit anti-phospho 53BP1 (ser1778) , Cell Signaling Technologies , Cat#2675; RRID: AB_490917.

Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Comparison, Staining, Western Blot, Flow Cytometry, Fluorescence, Cell Cycle Assay, Titration, Sequencing, Disruption