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. 2021 Feb 10;13(4):725. doi: 10.3390/cancers13040725

Figure 1.

Figure 1

CFL1 in pancreatic cancer. (a) Quantitative real-time polymerase chain reaction (qRT-PCR) analyses showed overexpression of CFL1 mRNA in tissue samples of pancreatic ductal adenocarcinoma (Cancer, n = 12) compared to chronic pancreatitis (C.P., n = 9) or healthy donor (Donor, n = 8) pancreas tissues. (b) CFL1 knockdown was performed by three independent siRNAs in four pancreatic cancer cell lines (Panc-1, S2-007, IMIM-PC1, and S2-028) and one control cell line (HEK-293). CFL1 knockdown was confirmed on the protein level (left panel). β-actin was used as an equal loading control (right panel). (c) BrdU incorporation assays were performed 48 h after the transfection of three independent siRNAs and relative light units per second (Rlu/s) were measured. The number of samples (n) represents biological replicates. (d) Panc1 and S2-007 cells were propidium-iodide-stained 48 h post-transfection with CFL1-specific or non-silencing siRNAs and subjected to flow cytometric analysis. Both cell lines treated with siRNA1 or siRNA3 show a decrease of cells in S-phase accompanied by an increase of cells in the G1 phase. S2-007 cells transfected with siRNA2 show a dramatic increase of cells in the G2 phase, consistent with G2/M phase arrest. Results depict the average of three independent experiments. (e) S2-007 cells with doxycycline-inducible CFL1 knockdown were injected into nude mice. One-half of the mice (n = 6) were treated with doxycycline via drinking water to induce CFL1 repression. After the removal of the tumors RNA and protein were extracted from tissues and analyzed for the CFL1 level. Quantitative RT-PCR as well as western blots confirmed repression of CFL1 in the treatment group. β-Actin served as a loading control and mean values of expression were normalized to RPLP0 housekeeping gene. Statistics were performed using Wilcoxon-test. (f) Tumor size measurements revealed a significant decrease in tumor growth in doxycycline-treated animals (n = 6) compared to controls (n = 6). All mice developed tumors. Displayed are data points with a smoothing line and the corresponding confidence interval. Boxplots depict the median with the first and third quartiles.