Fig 2: The TGF-β2/Smad signaling pathway activation is required for KDM4C-mediated effects on tumor growth and radioresistance in lung cancer.a SPC-A1 cells transfected with indicated siRNAs in the absence or presence of 4 ng/ml TGF-β2 for 24 h were collected and analyzed by Western blotting using indicated antibodies (n = 3). b SPC-A1 cells treated with LY2109761 (10 μM), SD70 (3 μM) or a combination of both were collected and analyzed by immunoblotting (n = 3). c, d SPC-A1 cells treated with indicated siRNAs or drugs were seeded at low density in 6-well plates. Cell numbers were counted every other day. **P < 0.01, ***P < 0.001 (n = 3). e, f SPC-A1 cells treated with indicated siRNAs or drugs were plated in triplicates. Histograms representing the numbers of colonies are depicted. ***P < 0.001 (n = 3). n. s. indicates no statistically significant difference (P > 0.05, n = 3). g, h Neutral comet assay performed after exposure to irradiation. Left panel: Representative images show comet tail. Scale bar, 10 μm. Right panel: the olive tail moment was quantified and graphed for each group. ** P < 0.01, ***P < 0.001 (n = 3). i, j SPC-A1 cells treated with indicated siRNAs or drugs were irradiated using indicated doses. Colonies containing more than 50 cells were recorded after 2 weeks. **P < 0.01, ***P < 0.001 (n = 3).

Fig 3: KDM4C expression level in cancer cells. (A) Average KDM4C expression level in cancer tissues relative to matching normal tissues, by qRT-PCR. (B) Ratios of KDM4C expression levels in cancers and the matching controls in seven tissues by our qRT-PCR compared with data from the GENT database of microarray data. (C) KDM4C expression in HT-29 cells expressing shRNA targeting KDM4C or a control shRNA, by qRT-PCR (top) and by Western blot (bottom). (D) ChIP for KDM4C at two target genes, MYC and RNMT, following KDM4C knockdown in HT-29 cells. (E) The ratio of H3K9me2 to H3K9me3 levels at promoters of MYC and RNMT, as determined by ChIP in HT-29 cells following KDM4C knockdown relative to controls. (F) Expression changes in KDM4C target genes following KDM4C knockdown in HT-29 cells (PDIA6 is the control gene). (G) Growth curve analysis (measured in quadruplicate; P = 7 × 10−8, ANOVA), BrdU incorporation (H; measured in triplicate) and changes in the proportion of cells at different stages of the cell cycle (I) are shown for HT-29 cells with and without KDM4C knockdown. Ctr indicates control cells ([*] P < 0.05, t-test). Error bars, SEM.

Fig 4: Enhanced antitumor immunity caused by KDM4C inhibition is mediated by CXCL10. (A) C57BL/6 mice were injected subcutaneously with 1×106 Lewis cells. When the tumor volume reached an average of 100 mm3, the mice were given a vehicle either alone or in combination with SD70 and/or anti-CXCL10. Tumor volume was measured every 3 days (mean±SEM; n=8; ***p<0.001; two-tailed t-test). (B) Tumor wet weights at 22 days’ postinoculation. n=8, **p<0.01. (C, D) Flow cytometry analyses showing the changes in the percentages of CD8+ T cells (C) and IFN-γ+ CD8+ T cells (D) in each group. n=8/group, *p<0.05, ***p<0.001. (E) Changes in Ki67+ CD8+ T cells with treatment. n=3, **p<0.01, ***p<0.001. (F) CFSE staining of proliferating CD8+ T cells measured by flow cytometry. Each experiment was repeated three times independently. (G) Changes in the in vitro migration of CD8+ T cells. n=3, ***p<0.001. (H) Schematic illustration of the in vivo migration experiment. (I, J) IHC was used to analyze the infiltration of CD8+ T cells in subcutaneously transplanted tumors for each group. Scale bar: 100 µm; n=8/group; ***p<0.001. (K, L) Flow cytometry was used to detect the expression of cytotoxicity markers (IFN-γ, GZMB, Perforin, and CD107a) and exhaustion markers (PD-1 and CD39) in CD8+ T cells in each group. n=3; *p<0.05, **p<0.01, ***p<0.001. (M) Lewis cells in 24-well plates were cocultured with pretreated CD8+ T cells in the absence or presence of an anti-CXCL10 neutralizing antibody or control antibody for 24 hours. Crystal violet staining identified the surviving tumor cells. Each group of experiments was carried out three times.

Fig 5: Inhibition of KDM4C with SD70 impairs cell proliferation and radioresistance in lung cancer in vitro and in vivo.a, b The inhibition efficacy of SD70 is time and concentration-dependent (n = 3). c SPC-A1 and H460 cells were treated with SD70 (3 μM) for 24 h and 48 h, respectively. Cells were then seeded and cultured for 2 weeks. The colonies were stained using crystal violet solution and then counted. Representative pictures are shown. **P < 0.01, ***P < 0.001 (n = 3). d Inhibition of KDM4C using indicated doses of SD70 impairs tumor growth in lung cancer in vivo (n = 5 mice for each group). ***P < 0.001. e Changes in body weight of xenografts post-inoculation were represented. f Neutral comet assay shows the effect of SD70 treatment on tail formation. The olive tail moment was quantified and graphed. ***P < 0.001 (n = 3). Scale bar, 20 μm. g Quantification of Rad51 foci intensity in SPC-A1 and H460 cells after treatment with SD70. Representative images are shown. Scale bar, 10 μm. ***P < 0.001 (n = 3). h The effects of SD70 treatment on the radiosensitivity of lung cancer cells were determined by clonogenic survival assay. **P < 0.01, ***P < 0.001 (n = 3). i Schematic representation of the xenograft study design and experimental workflow. j Growth curves of xenograft tumors in each group. Mice were treated with SD70 at the concentration of 10 mg/kg (n = 8 mice for each group). ***P < 0.001. k Kaplan–Meier analysis of mice survival due to treatments in (j).