Fig 2: IPO4 augments nuclear translocation of CEBPD in response to CDDP by NLS.a The Venn diagram of screened karyopherins in CC. We first analyzed 25 karyopherins in the chemotherapy database of CC (GSE3578), and found that 10 karyopherins fitting the filtering criteria that the correlation between karyopherins and PRKDC was increased after chemotherapy. Next, we identified only IPO4, among above 10 karyopherins, could interact with CEBPD through bioGRID (https: //thebiogrid.org). b, c Correlation of IPO4 with PRKDC prior to/after the chemotherapy in GSE3578 dataset. (Spearman rank correlation test). d, e Siha cells were transfected with flag-tagged CEBPD expression constructs and treated with CDDP (1 µg/ml) for 24 h. Lysates were immunoprecipitated (IP) with anti-CEBPD antibody, anti-IPO4 antibody and IgG control. Interaction of IPO4 and CEBPD proteins and quantitative analysis in triplicate were performed. f, g SiHa cells were stably transfected with shRNA against IPO4 with shRNA as control. CDDP (1 µg/ml) was added 24 h later, and cells were incubated for another 24 h before preparation of cytoplasmic (CE) or nuclear (NE) cell extracts. Data shown are mean ± SD. Tubulin and Lamin B were used as internal controls for CE and NE. h SiHa cell was co-transfected CEBPD NLS-deficient mutant (p.195-222del) or wild-type plasmids with siRNA-CEBPD, while siRNA-CEBPD target at 3’UTR (target sequences: sense: 5‘-CAGCUAAGGUACAUUUGUATT-3', anti-sense: 5‘-UACAAAUGUACCUUAGCUGTT-3'), which did not interfere with the expression of wild-type and NLS-mutant plasmids. The impact of CEBPD NLS-deficient mutant plasmid on PRKDC level in response to CDDP was detected by western blotting. i SiHa cells expressing shNC or shIPO4 were treated with CHX (100 mg/ml) for the indicated time points, 0, 0,5, 1, 2, 4, 8 h respectively. SiHa cells was transfected with either control shRNA or shIPO4 for 48 h, followed by CHX (100 mg/ml) treatment for the indicated times. The cell extracts were detected by WB.

Fig 3: Silencing C/EBPd enhances proliferation of Capan-2 cells. (A) Quantitative RT-PCR shows decreased levels of CEBPD mRNA in Capan-2 cells stably expressing shRNAs against CEBPD. (p = **0.0058, *0.0429, *0.0338 and **0.001 for shCTRL vs. shCEBPD #1, #2, #2 and #4, respectively). (B) Proliferation of CEBPD-silenced Capan-2 cells is significantly increased compared to control cells. (p = **0.0013, *0.011, *0.0146 and **0.0013 for shCTRL vs. shCEBPD #1, #2, #2 and #4, respectively).

Fig 4: CDDP enhances the transcriptional activity of CEBPD to PRKDC promoters and CEBPD attenuates CDDP sensitivity in vitro and in vivo.a Western blot analysis for CEBPD level was conducted with lysates harvested from different concentration of CDDP-treated SiHa for 24 h. b The potential PRKDC binding sites (1, 2, 3) in SiHa cell lines were predicted by the program IGV_2.5.3. c A ChIP assay was used to verify the potential CEBPD co-binding sites in the PRKDC promoter regions in SiHa cell with or without CDDP treatment (1 µg/ml, 24 h). Sonicated input DNA and IgG were used as controls. d Diagram of 3 binding site mutant plasmids including mutant 1(M1), mutant 2 (M2) and mutant 3 (M3). e Luciferase activities in luciferase reporter plasmids containing wild-type and M1, M2 and M3 PRKDC promoters were indicated in SiHa cell with or without CDDP treatment (1 µg/ml, 24 h). The pGL3 promotor vector without enhancer sequence was used as control construct. Values are mean ± SD. (Student’s t test). f Knockdown of CEBPD and control shRNA SiHa cell were treated with 1 µg/ml CDDP for 24 h followed by western blotting. (Student’s t test). g Cell viability assay in SiHa/shNC and shCEBPD-1 cells at different concentration of CDDP. Values are mean ± SD. (Student’s t test). h Apoptotic assay in SiHa/shNC and shCEBPD-1 cells after treatment with 0.1%DMSO or 3 µg/ml CDDP for 24 h. Values are mean ± SD. (Student’s t test). i ?H2AX assay in SiHa/shNC and shCEBPD-1 cells with CDDP treatment (6 µg/ml, 1 h). Refer to Fig. 1j. j Tumor volumes of xenograft tumors from SiHa/shNC+saline, shNC+CDDP, shCEBPD-1+saline, shCEBPD-1+CDDP groups (n = 5). Scale bars = 1 cm. groups of shNC+saline and shNC+CDDP were taken the same as previous mentioned. Values are mean ± SEM. (Student’s t test). Refer to Fig. 1l. k Tumor weight in above four groups. (Student’s t test). l TUNEL assay in above four groups. Scale bar indicated 100 µm. (Student’s t test). NS no significance, *p < 0.05, **p < 0.01 and ***p < 0.001.

Fig 5: The schematic diagram of IPO4-CEBPD-PRKDC axis in modulating CC chemosensitivity.Upon the treatment of CDDP, CEBPD is induced and the interaction of CEBPD and IPO4 is strengthened. Importin 4 (IPO4) augment the nuclear translocation of CEBPD, and CEBPD transcriptionally upregulated PRKDC, which lead to DNA damage repair and thus blunted CDDP sensitivity.