fitc- h2ax antibody  (Cell Signaling Technology Inc)

Journal: Journal of Translational Medicine

Article Title: Exploring the role of PARP1 inhibition in enhancing antibody–drug conjugate therapy for acute leukemias: insights from DNA damage response pathway interactions

doi: 10.1186/s12967-024-05838-9

Figure Lengend Snippet: Effect of INO, GO and γ-calicheamicin on cell viability, cell-cycle perturbation and G2/M checkpoint activation in ALL and AML cell lines. A Reduction of the cell viability of ALL (blue) and AML (red) cell lines treated for 24, 48 and 72 h with increasing concentration of INO and GO, respectively. Each point represents the mean ± standard deviation of normalized cell viability obtained from at least three independent experiments. Dashed lines represent the indicative values of inhibitory concentration 50 (IC 50 ). B Expression of CD33 and CD22 in AML (red) and ALL (blue) cell lines by flow cytometry. Histograms represent the mean and standard deviation of the mean fluorescence intensity (MFI) of AML and ALL cell lines obtained from at least three independent experiments. The table reports the Pearson correlation coefficient in AML and ALL cell lines between CD22 expression and IC 50 values after 24, 48 and 72 h of GO and INO, respectively. C Reduction of the cell viability of ALL (blue) and AML (red) cell lines treated for 24, 48 and 72 h with increasing concentrations of γ-calicheamicin. Each point represents the mean ± standard deviation of normalized cell viability obtained from at least three independent experiments. Dashed lines represent the indicative values of inhibitory concentration 50 (IC 50 ). D Cell-cycle profile analysis of ALL and AML cell lines treated for 18, 24 and 48 h with subtoxic concentrations of INO (near IC 50 values after 24 h) and GO (near IC 50 values after 24 h), respectively. E , F Representative immunoblotting analysis of ALL and AML cell lines treated with INO (near IC 50 values after 24 h) or GO (near IC 50 values after 24 h) for 18 h. β-actin was used for loading normalization. G Histograms representing the mean ± standard deviation the relative bands intensity of CCNB1, phospho-CDK1 Tyr15 and phospho-H2AX Ser139 proteins in ALL (blue) and AML (red) cell lines treated INO or GO for 18 h. H Immunoblotting analysis of ALL and AML cell lines treated with γ-calicheamicin (IC 50 values after 24 h) for 18 h. β-actin was used for loading normalization. I Histograms represent the mean and standard deviation the relative bands intensity of phospho-ATM Ser1981 , phospho-ATR Ser428 , phospho-CHK2 Thr68 , phospho-CHK1 Ser345 and phospho-WEE1 Ser642 proteins in ALL (blue) and AML (red) cell lines treated INO or GO for 18 h. All error bars represent the mean ± SD

Article Snippet: Cells were exposed or not to UVC-ray (15 J/m 2 × 10′′) using BLX-e254 UV-Crosslinker (Vilber Lourmat, Eberhardzell, Germany) for 3 h and then 0.5X10 6 cells were harvested and fixed with 95% ethanol/5% acetic acid followed by 1% formaldehyde, 0.25% Triton X-100 in TBS and stained with phospho-H2AX Ser139 -FITC conjugated antibody (Merck-Millipore) following manufacturer’s instructions.

Techniques: Activation Assay, Concentration Assay, Standard Deviation, Expressing, Flow Cytometry, Fluorescence, Western Blot

Journal: Journal of Translational Medicine

Article Title: Exploring the role of PARP1 inhibition in enhancing antibody–drug conjugate therapy for acute leukemias: insights from DNA damage response pathway interactions

doi: 10.1186/s12967-024-05838-9

Figure Lengend Snippet: Effect of PARP1 inhibition on cell viability, cell-cycle regulation and DDR in ALL and AML cell lines. A Reduction of the cell viability of ALL (blue) and AML (red) cell lines treated for 24, 48 and 72 h with increasing concentration of veliparib, olaparib or talazoparib. Each point represents the mean ± standard deviation of normalized cell viability obtained from at least three independent experiments. Dashed lines represent the indicative values of inhibitory concentration 50 (IC 50 ). B Representative immunoblot showing the basal expression level of proteins from different DDR pathways. β-actin was used for loading normalization. C Box-plots showing the distribution of the normalized protein levels (normalization: bands intensity of protein of interest/band intensity of β-actin) in ALL (n = 5; blue) and AML (n = 5; red) cell lines. In the graph, the box-plots represent the distribution of the mean values of at least two independent experiments. D Histograms show the percentages of phospho-H2AX Ser139 expressing cells after 3 h from the exposure to UVC ray in ALL (blue) and AML (red) cells. The left panel of the figure represents the effect of all cell lines together while the right panel represents the effect of each cell lines. In the graph, histograms represent the results of at least three independent experiments. E Cell viability of AML and ALL cell lines after 24 h from UV-ray exposure. The left panel of the figure represents the normalized cell viability (UV-treated/untreated cells) of all cell lines together while the right panel represents the effect of each cell line. Histograms represent the results of at least three independent experiments. F Effect of subtoxic concentrations of talazoparib on cell-cycle profiles of ALL (RS4;11 = 333.3 nM, REH = 1000 nM, KOPN8 = 111.1 nM and SUP-B15 = 37.0 nM) and AML (KASUMI-1 = 3000 nM, MOLM-13 = 3000 nM, OCI-AML3 = 3000 nM, MV4-11 = 1000 nM and KG-1 = 1000 nM) cell lines treated for 18, 24 and 48 h. The different cell-cycle-phases are represented as percentage of cells. All error bars represent the mean ± SD

Article Snippet: Cells were exposed or not to UVC-ray (15 J/m 2 × 10′′) using BLX-e254 UV-Crosslinker (Vilber Lourmat, Eberhardzell, Germany) for 3 h and then 0.5X10 6 cells were harvested and fixed with 95% ethanol/5% acetic acid followed by 1% formaldehyde, 0.25% Triton X-100 in TBS and stained with phospho-H2AX Ser139 -FITC conjugated antibody (Merck-Millipore) following manufacturer’s instructions.

Techniques: Inhibition, Concentration Assay, Standard Deviation, Western Blot, Expressing