Fig 1: Efficient delivery of both siPTPN13 and siNOX4 into (myo)fibroblasts by Fab'-conjugated dual siRNA-loaded micelles with potent transfection efficiency in vitro. (A) The viability of (myo)fibroblasts incubated with different concentrations of siRNA and siRNA-loaded micelles for 24 h as measured by the CCK-8 assay. The data are presented as the mean ± SD (*p < 0.05 vs control group). (B) Intracellular distribution of siRNA-loaded micelles in (myo)fibroblasts as determined by immunofluorescence assay. siRNA was labelled with Alexa 647. (C) The percentage of micelle+ cells was analyzed by flow cytometry. Rright panels: Quantified data of Alexa 647+ cells. Results are expressed as the mean ± SD (**p < 0.01). (D) PTPN13 and NOX4 mRNA levels in (myo)fibroblasts incubated with Fab'-conjugated micelles loaded with different concentrations of siRNA (PTPN13 or NOX4) were measured by qRT-PCR. Data are shown as the mean ± SD (*p < 0.05 compared to the control group). (E) The PTPN13, NOX4, and α-SMA expression levels in (myo)fibroblasts transfected with an siRNA concentration of 5 µg/mL for both free siRNA and Fab'-conjugated siRNA-loaded micelles were determined by western blot. (F) (Myo)fibroblasts were challenged with FasL (100 ng/mL) for 12 h. Apoptosis was evaluated by flow cytometry detecting Annexin V and PI. Results are expressed as the mean ± SD (*p < 0.05 compared with the PBS group). (G) The expression of α-SMA in cells treated with siRNA-loaded micelles.
Fig 2: Compound 2 treatment reduces caspase activation and improves 661W cell survival in an in vitro model of apoptosis. (a) 661W cells express PKM2 under 5.5 mM or 25 mM glucose concentration, the latter of which was and has been [9,36,38] used in the caspase and cell viability experiments that follow. Caspase 8 activation (b), caspase 3 and 7 activation (c), and cell viability (d) assayed in cells treated with DMSO or different concentrations of compound 2 (Cmpd 2) under standard tissue culture conditions or in the presence of 500 ng/mL FasL and 250 ng/mL hemagglutinin. Two hours before treatment with FasL, 661W cells were pre-treated with compound 2 or DMSO, and caspase activation and viability were measured 8 and 48 h after, respectively. n = 6; Mean ± SEM.
Fig 3: ML-265 treatment blocks caspase activation and improves cell survival in a model of apoptosis in vitro. Caspase 8 activity (a), caspase 3/7 activity (b), and cell viability (c) assayed in the presence of DMSO or different concentrations of ML-265 under standard tissue culture conditions or 500 ng/mL FasL and 250 ng/mL hemagglutinin. Cells were pre-treated with ML-265 or DMSO for 2 hours prior to treatment with FasL, and caspase activity and cell viability were measured 8 and 48 hours, respectively, thereafter. n = 8; Mean ± SEM; *p < 0.05; **p < 0.01; ***p < 0.005.
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