Figure 6.

Chemotherapy induces the expression of circMRPS35 and translation of circMRPS35-168aa

(A) RNA-seq (GEO: GSE140202) analyses of circMRPS35 in sorafenib-treated cells compared to non-treated cells. (B) Quantitative real-time PCR analysis of circMRPS35 after DOX, etoposide, ACTD, and cisplatin treatment or non-treated Huh-7 and HCC-LM3 cells. (C) Schematic illustration showing that IRESs in circMRPS35 were cloned between R-Luc and F-Luc reporter genes with independent start and stop codons (upper). The relative luciferase activity of F-Luc/R-Luc in the above vectors was tested in Huh-7 cells (lower). The encephalomyocarditis virus (EMCV) IRES was used as a positive control. (D) Polysome fractionation and RT-PCR analysis of Huh-7 and HCC-LM3 cell lysate, with GAPDH as the positive control. (E and F) IP by FLAG antibody and SDS-PAGE separation of protein bands stained by Coomassie brilliant blue (CBB) and the band (red frame) (left) analyzed by LC-MS (right). (G) Western blot analysis of the expression of circMRPS35-168aa after treatment with five chemotherapy drugs in Huh-7 and HCC-LM3 cells, with GAPDH as the control. Error bars represent the means ± SEM of three independent experiments. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.