Fig. 5

LINC00909 enhances the stemness of pancreatic ductal adenocarcinoma (PDAC) by inhibiting SMAD4 expression. (A–C) RT-qPCR analysis was performed to detect the mRNA levels of SMAD4 in LINC00909-overexpressing cells (A) and LINC00909-knockdown cells (B, C). (D–F) Western blotting analysis was conducted to examine the protein expression of SMAD4 in PDAC cells. (G, H) The efficiency of SMAD4 knockdown in PANC-1 (G) and AsPC-1 cells (H) was detected by RT-qPCR. (I, J) Representative images of sphere-formation assays in PANC-1/KD and AsPC-1/KD cells transfected with siSMAD4-2. (K, L) The protein levels of SMAD4, KLF4, c-Myc, JNK, p-JNK, JUN and p-JUN in PANC-1/LINC00909-KD (K) and AsPC-1/LINC00909-KD cells (L) transfected with siSMAD4-2 were analyzed by western blotting. (M) PANC-1 cells transfected with EV or vector encoding LINC00909 were treated with actinomycin D (1 mg/ml) at the indicated time point. (N, O) PANC-1 or AsPC-1 cells transfected with siNC, siLINC00909-3, or siLINC00909-6 were treated with actinomycin D (1 mg/ml) at the indicated time point. Total RNA was extracted and analyzed by RT-qPCR to examine the relative levels of SMAD4 mRNA (M–O). All *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. P-values were assessed using ANOVA and two-tailed t-tests, followed by Dunnett’s tests for multiple comparisons in (A–O).