Fig. 7.

CXCL5 played a critical role in Cr(VI)-induced oncogenic transformation and carcinogenesis. (A) Soft agar assay of BALB/3 T3 cells that were cultured with or without Cr (VI)(2 μM) and CXCL5 (100 ng/mL) for 6 weeks. Images of colonies were taken 2 weeks after seeding. (B) Immunoblotting was used to confirm the levels of CRISPR/Cas9-mediated CXCL5 deletion. (C) Soft agar assay of wild-type (WT) Cr-T cells or Cr-T cells in which stable knockout (KO) of CXCL5 was achieved using CRISPR/Cas9-mediated deletion (left). Right panel depicts the relative number of colonies formed for each cell line. (D) B2B cells were infected with a lend virus stably expressing an empty vector CXCL5, respectively. MTT assay was conducted to determine relative proliferation rates of B2B cells (left). Cr-T cells were transfected with a control siRNA (siSCR) and an siRNA targeting CXCL5, respectively. The MTT assay determined the relative proliferation rates of Cr-T cells (right). (E) B2B and Cr-T cells were treated as above in Fig. 7C and D, and morphological changes were tracked by microscopy. Scale bar = 100 μM (× 400 magnification). (F) CXCL5 wild type and CXCL5 KO Cr-T cells (4 × 10⁶) stably expressing the firefly luciferase were resuspended in 50 μL of DMEM basal medium plus 50 μL of Matrigel and injected into the lung of a nude mouse (n = 10/group). Bioluminescence imaging was performed at post-implantation time points in mice. (G) Representative tumors from CXCL5 wild type and CXCL5 KO Cr-T cells. (H) After the mice were euthanized, xenografts were trimmed out and tumor weight was measured. (I) Kaplan-Meier survival curves of mice that received CXCL5 wild type and CXCL5 KO Cr-T cells for tumor growth assay. (J) CXCL5 levels in the tumor tissues were detected using RT-qPCR. (K) The expression levels of CXCL5 and GAPDH in xenografts. Data were presented as means ± SEM of three independent experiments. ** and ***, p < 0.01 and p < 0.001 compared to CXCL5 wild type group.