Fig 2: Analysis of cSR cell line characteristics. (A) Growth of H28 and cSR cells. Cells are seeded at 2×105 cells/well and counted at the timepoints (3 to 17 days) as indicated. Each data point represents the mean ± SD of three independent experiment. (B) Morphology of H28 and cSR cells. Cells are seeded at 1×105 cells/well. After 24 h (low density) or 72 h (high density), cell morphology is observed. Scale bar, 100 µm. (C) Effect of cSBL, ONC, and DOX on H28 and cSR cells. Viability of cells treated with the drugs are measured by the WST-8 assay. IC50 values and RRs are calculated from the depicted dose-response curves. Each data point represents the mean ± SD of three independent experiment performed in triplicates. (D) Apoptosis induction in H28 and cSR-B1 cells treated with cSBL. Cells are treated with cSBL (1 or 5 µM), and the expression of cleaved caspase-3 is detected using western blotting. Densitometric quantification is performed using the results of three independent experiments (mean ± SD). The statistical significance of the bands compared to the non-treated control were shown. *P<0.05, **P<0.01 vs. 0 µM cSBL. cSR, cSBL-resistant; cSBL, bullfrog sialic acid-binding lectin; ONC, onconase; DOX, doxorubicin; RR, resistance rate; SD, standard deviation.

Fig 3: (A–D) HCT 116 cells and H9c2 cells were treated with sunitinib-loaded nanoparticles (SU-pSiNP) or pure sunitinib (SU) or unloaded nanoparticles (NP unloaded, (C), right panel) in the indicated concentrations (µM) and for the indicated time intervals. In (A) and (C), the representative WB of expression levels of apoptotic marker proteins PARP, caspase-3, and cleaved caspase (p19/17 fragments of caspase-3, p19/17 caspase-3) are shown. Gß and tubulin were used as loading controls. Blots were reproduced at least three times with similar results. (B) The histogram of flow cytometric analysis data for HCT 116 cells using subG1 assay; %: percent of SubG1 population. Results are shown as mean values ± standard deviation (SD), n = 3, *p < 0.05 vs. control 1 day, #p < 0.05 vs. control 2 days, §p < 0.05 vs. control 3 days, n.s. not significant. (D) MTT cell viability assay of H9c2 cells upon treatment with SU-pSiNPs and pure SU in indicated concentrations (µM) for 1 day. Results are presented as mean values ± standard deviation (SD), n = 3, *p < 0.05 vs. untreated control and pSiNPs-treated group. Ordinary one-way ANOVA was applied with Dunnett test to correct for multiple comparison.

Fig 4: RG2 cells evaded TMZ-induced apoptosis. Apoptosis was evaluated in U373-RGs and LN229-RGs after 72 h of TMZ (150 µM) treatment. (A) Apoptosis was detected by nuclear staining with Hoechst 33258. Cells exhibiting condensed chromatin or apoptotic bodies were counted as apoptotic cells. Quantitative analysis was performed in six random fields per well per group (in quadruplicate). Scale bar: 50 µm. ***, p < 0.001, compared with RG0. (B) Apoptosis assay by staining with Annexin V/PI followed by flow cytometry. Dot plots show apoptotic populations in RGs of U373 and LN229 cells (left) after 72 h of treatment with TMZ (150 µM) or vehicle. Bar graph shows the percentage of early and late apoptotic cells in total cells from the dot plots (right). (C) TMZ-induced caspase 3 activation was detected by western blot (left) and semi-quantitation (right). The results were reproduced in at least three independent experiments. ***, p < 0.001, compared with RG0. ###, p < 0.001, compared with a corresponding vehicle control (C).

Fig 5: Overexpression Fn14 alleviates cisplatin resistance in vivo. (a-c) Macrograph, volume and weight of tumors in both two groups. (d) Tunel assay was used to determine the CDDP-induced DNA damage in the tumors of each group. (e) Western blot analysis detecting the activation of Caspase-3 and the expression of p53 R248Q and Hsp90 in tumor tissues in both two groups. (f) and (g) Tumors of each group were immunohistochemically tested for Fn14, Hsp90 and p53 R248Q, *p < 0.05. Unpaired Student’s t test was performed for comparisons between two groups