Journal: Cell Death & Disease

Article Title: Inhibition of BAD-Ser99 phosphorylation synergizes with PARP inhibition to ablate PTEN -deficient endometrial carcinoma

doi: 10.1038/s41419-022-04982-8

Figure Lengend Snippet: Chou-Talalay synergy analysis for NPB (N) in combination with three PARP inhibitors, namely Olaparib (O), Rucaparib (R) or Talazoparib (T), in a panel of EC cells including KLE ( PTEN- proficient), AN3CA ( PTEN- deficient), Ishikawa ( PTEN- deficient) and RL95-2 ( PTEN- deficient). Cells were treated with the indicated concentration (log 10 scale) of NPB and mentioned PARP inhibitor for 6 days. The survival fraction was assessed using a total cell number assay. The logarithmic combination index (CI) value corresponding to cell fraction affected (Fa) was determined using the CompuSyn software ( http://www.combosyn.com ) as described in “Materials and methods”. CI value indicates: <1 synergism; =1 additive Synergy; >1 antagonism ( n = 3). Dose-response curves for a panel of cells treated with the indicated concentration of PARP inhibitors with or without 1 µM NPB in total cell number assays. Arrow indicates fold reduction in respective PARP inhibitor IC 50 in the presence of NPB ( n = 3).

Article Snippet: KLE ( PTEN -proficient) and Ishikawa, RL-95-2, or AN3CA ( PTEN -deficient) EC cell lines were purchased from Procell Life Science & Technology Co. Ltd (Wuhan, China).

Techniques: Concentration Assay, Software

Journal: Cell Death & Disease

Article Title: Inhibition of BAD-Ser99 phosphorylation synergizes with PARP inhibition to ablate PTEN -deficient endometrial carcinoma

doi: 10.1038/s41419-022-04982-8

Figure Lengend Snippet: A EC cells, KLE, AN3CA, Ishikawa, and RL95-2 were incubated with the indicated drug concentrations of NPB (N) and Olaparib (O), Rucaparib (R) or Talazoparib (T) or combinations thereof for foci formation assays and stained with 0.2% crystal violet ( n = 3). B Microscopic visualization of calcein-AM (green) stained spheroids (live) and BOBO-3 Iodide (red) stained cell debris (dead) generated by EC cells (KLE, Ishikawa, RL95-2, and AN3CA) cultured in 3D Matrigel after exposure to NPB (N) and PARP inhibitors (Olaparib = O, Rucaparib = R, Talazoparib = T) and combinations thereof ( n = 3). Red-fluorescence (staining of ethidium homodimer-1 indicating loss of plasma membrane integrity) and decreased green-fluorescence (staining of calcein-AM to indicate intercellular esterase activity). Scale bars, 100 µm. Right: Cell fraction (SF) was evaluated using AlamarBlue® in EC cells treated with NPB (N), PARP inhibitors (Olaparib = O, Rucaparib = R, Talazoparib = T) and in combination with the indicated drug concentrations for 14 days in 3D Matrigel. ( n = 3). The white color arrow highlights the apoptotic cell death. Columns are mean of triplicate experiments; bars, ±SD. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: KLE ( PTEN -proficient) and Ishikawa, RL-95-2, or AN3CA ( PTEN -deficient) EC cell lines were purchased from Procell Life Science & Technology Co. Ltd (Wuhan, China).

Techniques: Incubation, Staining, Generated, Cell Culture, Fluorescence, Clinical Proteomics, Membrane, Activity Assay

Journal: Cell Death & Disease

Article Title: Inhibition of BAD-Ser99 phosphorylation synergizes with PARP inhibition to ablate PTEN -deficient endometrial carcinoma

doi: 10.1038/s41419-022-04982-8

Figure Lengend Snippet: A Western blot analysis was used to assess the expression of PTEN and downstream effectors PI3K, AKT, BAD, and CHK1 protein; and levels of pAKT, pBADS99, and pCHK1 were determined in KLE- WT, and PTEN-KO cells. Soluble whole-cell extracts were run on an SDS-PAGE and immunoblotted as described in materials and methods. β-ACTIN was used as input control for cell lysates. The sizes of detected protein bands in kDa are shown on the left side Densitometries of protein bands were subsequently determined using ImageJ software ( https://imagej.nih.gov/ij/ ). B Dose-response curves for a panel of KLE-WT and KLE-PTEN-KO cells treated with the indicated concentration of Olaparib with or without a constant concentration of NPB (1μM) in total cell number assays. Arrow indicates fold reduction in respective PARP inhibitor IC 50 in the presence of NPB ( n = 3). C KLE-WT and KLE-PTEN-KO cells were incubated with the indicated concentrations of NPB (N), Olaparib (O), or a combination thereof for foci formation and stained with 0.2% crystal violet ( n = 3). D Western blot analysis was used to assess the expression of BAD, CHK1, cle-CASP, and cle-CASP7 protein and the level of pBADS99 in KLE-WT and KLE-PTEN-KO cells after treatment with NPB (N), Olaparib (O) or combination thereof. Soluble whole-cell extracts were run on an SDS-PAGE and immunoblotted as described in materials and methods. β-ACTIN (ACTB) was used as input control for cell lysate. The sizes of detected protein bands in kDa are shown on the left side. E Representative immunofluorescent images of RAD51 in KLE-WT and KLE-PTEN-KO cells. Cells were treated with NPB (N), Olaparib (O), or a combination (N + O). Scale bars, 50 µm. Below: Quantification of RAD51 foci positive AN3CA cells treated with NPB (N), Olaparib (O), or a combination (N + O) are shown. Cells with more than 5 RAD51 foci per nucleus were counted as RAD51 positive cells. 100 cells were analyzed in 2–3 separate fields for each sample ( n = 3). Representative EC cells with positive RAD51 foci are highlighted using the white color arrow. Columns and points are the mean of triplicate determinations; bars, ±SD. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: KLE ( PTEN -proficient) and Ishikawa, RL-95-2, or AN3CA ( PTEN -deficient) EC cell lines were purchased from Procell Life Science & Technology Co. Ltd (Wuhan, China).

Techniques: Western Blot, Expressing, SDS Page, Control, Software, Concentration Assay, Incubation, Staining