Fig. 3: SFPQ knock-down increases basal and CDDP-induced cell death. | Oncogene

Fig. 3: SFPQ knock-down increases basal and CDDP-induced cell death.

From: Splicing factor proline- and glutamine-rich (SFPQ) protein regulates platinum response in ovarian cancer-modulating SRSF2 activity

Fig. 3

a Typical phase contrast images (10× objective) of MDAH cells stable transduced with sh-ctrl or sh-SFPQ#2, after few days in culture. b WB analysis evaluating the expression of SFPQ and of DNA damage (γ-H2AX) and apoptotic markers (PARP-1, caspase-8, Bcl-XL) in cells silenced for SFPQ as in (a). c Growth curve of MDAH and SKOV3 cells transiently transduced with sh-ctrl and sh-SFPQ#2 and #3 as indicated. Cells growth was monitored by Trypan Blue Exclusion test. Data are the mean (±SD) of three biological replicates. d Cell cycle distribution of MDAH cells transiently transduced with the indicated shRNAs (sh-ctrl and sh-SFPQ#3) and analyzed by FACS 72 h after transduction in untreated and CDDP-treated conditions (CDDP 50 µM, T0 = 16 h treatment; T24 = 16 h treatment + 24 h release). The percentages of cells in each phase of the cell cycle are indicated. Data are the mean (±SD) of three biological replicates. e WB analysis evaluating the expression of PARP-1, caspase-8, -9, -3, γ-H2AX and Bcl-XL in MDAH cells transduced with sh-ctrl and sh-SFPQ#2 and #3 and treated as in (d). f Representative RT-PCR analyses of pro (+) and anti (−) apoptotic isoforms of caspase-9 in MDAH cells transduced as indicated and treated with CDDP 50 µM for the indicated times. g Graphs reporting the quantification of the ratio between pro-/antiapoptotic isoforms of caspase-9, normalized on Gapdh expression and folded on untreated (UNT) condition. Data are the mean (±SD) of three independent experiments. h WB analysis evaluating PARP-1 cleavage in MDAH cells treated with CDDP 25 µM for 16 h, alone or in combination with Z-LEHD-FMK (caspase-9 inhibitor). i Dose-response curves evaluating cell viability of MDAH cells transiently transduced with sh-ctrl or SFPQ#3 shRNAs and treated with increasing doses of CDDP alone or in combination with 20 µM Z-LEHD-FMK. Data are expressed as percentage of viable cells compared with untreated cells and represent the mean (±SD) of three biological replicates. In the figure GAPDH was used as loading control and statistical significance was determined by a two-tailed, unpaired Student’s t test. (*p < 0.05; **p < 0.01; ***p < 0.001).

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