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. Author manuscript; available in PMC: 2019 Mar 25.
Published in final edited form as: Adv Biol Regul. 2018 Sep 16;71:183–193. doi: 10.1016/j.jbior.2018.09.008

Fig. 5.

Fig. 5.

ATX and LPA2 play key roles in resistance against radiation-induced programmed cell death. (A) LPA protects HPAF II human pancreatic cancer cells against gemcitabine-induced apoptosis. HPAF II cells were cultured in growth media (DMEM F12 supplemented with heat inactivated 10% FBS, 200 μM L-glutamine, 100 U penicillin and 100 μg/ml streptomycin). Next day, cells were serum starved for 24 h followed by exposure to 10 μM Gemcitabine with or without LPA (at 3 μM or 10 μM). Caspase 3/7 activity was measured after 24 h of treatment using the CaspaseGlo® Kit (Promega). (B) LPA2 and (C) ATX expression are upregulated in 4T1 CSC upon exposure to Paclitaxel or γ-irradiation. 4T1 CSC were generated as described in Fig. 3 and spheroids were allowed to form for 3 days. At day 4, CSCs were either treated with 80 nM of Paclitaxel (PCTXL) or fractionated doses of γ-irradiation (6Gy/day for 4 days). CSCs were harvested for QPCR analysis after 4 days of PCTXL treatment or 24 h after the last dose of fractionated radiation. GraphPad Prism was used to perform one-factor ANOVA with Bonferroni’s post-test to determine the significance in LPA2 or ATX expression in vehicle versus PCTXL or radiation treatment, respectively (*** = p < 0.001; n = 3). (D) The ATX inhibitor BMP-22 enhances 4T1 cell killing by PCTXL. 4T1 cells were plated in quadruplicate in 96-well plates at a density of 5 × 103 cells per well in RPMI with 1% (v/v) charcoal-stripped FBS and incubated overnight at 37 °C with 5% CO2. Cells were then treated for 24 h with 3 μM BMP-22 followed by 48 h treatment with a dose range of 0–1 μM PT in RPMI with 1% (v/v) charcoal-stripped FBS. After 48 h incubation, cells were allowed to recover in RPMI with 1% (v/v) charcoal-stripped FBS for an additional 72 h. Viability was then assessed via Promega CellTiter Blue (as per manufacturer’s instructions) and normalized to % vehicle control samples. Data were plotted using GraphPad Prism v. 5.0a, and non-linear regression analysis was performed in a variable slope model in order to determine LD50 concentrations for PCTXL in the presence and absence of BMP-22.