on chemotherapy response and apoptosis induction of PDAC cell lines.Figure 8A: Cell lysates of the indicated human pancreatic tumor cell lines (S2-007, PaTu8988t, MiaPaCa-2, Panc1) were resolved by SDS-PAGE and analyzed by immunoblotting for p14ARF (α-p14ARF) and PRMT1 (α-PRMT1) protein levels. GAPDH staining served as loading control. B: PaTu8988t cells were treated with 0, 1, 3, and 10 μM gemcitabine (GEM). After 48 hours, cell lysates were analyzed by immunoblotting using the indicated antibodies. The p14ARF signals were densitometrically quantified and normalized to the respective GAPDH signal, as specified by the numbers below the blots, with the untreated condition (- GEM) set to 1. The percentage of PARP cleavage was also densitometrically quantified and is indicated below the blot. C: PaTu8988t cells were treated with 3 μM gemcitabine (GEM) or left untreated (0 hour). After 0, 6, 12, 24 and 48 hours, methylation of endogenous p14ARF was analyzed by immunoblotting using α-me-p14ARF, α-p14ARF and α-GAPDH (loading control) antibodies. The methylated p14ARF and the p14ARF signals were densitometrically quantified and normalized to the respective GAPDH signal, as specified by the numbers below the blots, with the untreated condition (0 hour) set to 1. D, PaTu8988t and MiaPaCa-2 cells were treated (+) with 20 μM MS023 or left untreated (-) for 3 days. For the last 2 days, the cells were additionally exposed to 0, 1, or 3 μM gemcitabine (GEM). Subsequently, cell death was quantified by flow cytometry (in (D)) using FITC-labeled AnnexinV and propidium iodide (PI) for four independent experiments (mean ± SD, *: p-value ≤ 0.05 using Welch´s t-test). E: Additionally, PaTu8988t cell lysates were analyzed by immunoblotting using the indicated antibodies. A representative result is displayed in (E). The p14ARF signals were densitometrically quantified and normalized to the respective GAPDH signal, as specified by the numbers below the blots, with the untreated condition (- GEM, -MS013) set to 1. The percentage of PARP cleavage was also densitometrically quantified and is indicated below the blot. F, PaTu8988t were transfected with the indicated siRNAs (two control/non-targeting siRNAs and two p14ARF-specific siRNAs) and treated (+) with 20 μM MS023 or left untreated (-) for 3 days. For the last 2 days, the cells were additionally exposed 1 μM gemcitabine (+ GEM) or not (- GEM). p14ARF depletion was monitored by immunoblotting using the indicated antibodies. A representative result is displayed in (F). The p14ARF signals in the siControl conditions were densitometrically quantified and normalized to the respective CDK2 signal, as specified by the numbers below the blot, with the untreated condition (- GEM) of the siControl_2 sample set to 1. G: Apoptosis was quantified by flow cytometry (G) using FITC-labeled AnnexinV and propidium iodide (PI) for six independent experiments (mean ± SD, **: p-value ≤ 0.005, *: p-value ≤ 0.05, ns: not significant using Welch´s t-test). H, MiaPaCa-2 cells were transfected with empty vector (e.v., control) or Flag-tagged wild type (wt) p14ARF-containing plasmid and treated (+) with 20 μM MS023 or left untreated (-) for 3 days. For the last 2 days, the cells were additionally exposed to 1 μM gemcitabine (+ GEM) or not (- GEM). p14ARF overexpression was monitored by immunoblotting using the indicated antibodies. A representative result is displayed in (H). The p14ARF signals in the overexpression conditions were densitometrically quantified and normalized to the respective GAPDH signal, as specified by the numbers below the blot, with the untreated condition (- GEM) set to 1. I: Apoptosis was analyzed by flow cytometry (I) using FITC-labeled AnnexinV and propidium iodide (PI). The increase in the apoptotic cell fraction upon gemcitabine and/or MS023 was quantified for four independent experiments (mean ± SD, ***: p-value ≤ 0.001, *: p-value ≤ 0.05 using Welch´s t-test).