Fig. 2: The potential target gene mediating the SOX6-induced autophagy.

A Scatter plot of top 20 enriched KEGG pathways regulated by SOX6, according to the differentially expressed genes (DEGs) of microarray data. B Western blotting analysis on the protein levels of key molecules within MAPK/ERK and PI3K-Akt signaling pathways in HeLa and CaSki cells transfected with plex-MCS (CTRL) or plex-HA-SOX6 expression plasmid. α-tubulin protein was used as the internal control. C Overview of the SOX6-regulated DEGs by RNA sequencing. D Venn diagram of the DEGs enriched in MAPK and PI3K-Akt signaling pathways between microarray and RNA-sequencing. E Heatmap of the 32 DEGs merged between microarray and RNA-sequencing analysis. The color scale beside the heatmap represents the raw Z-score ranging from blue (low expression) to red (high expression). F The RT-qPCR (SYBR Green) analyses on the levels of MAP4K4 mRNA in HeLa-HA-SOX6-tet and HeLa-HA-SOX6ΔHMG-tet cells treated with or without Dox (4 μg/mL) for 48 h, respectively. G Western blotting analyses on the levels of MAP4K4 protein in HeLa-HA-SOX6-tet and HeLa-HA-SOX6ΔHMG-tet cells treated with or without Dox (4 μg/mL) for 48 h, respectively. H Experimental design of the steps used to produce xenograft in BALB/c nude mice. I Growth curve of tumors formed by subcutaneous injection of HeLa-HA-SOX6-tet or HeLa-HA-SOX6ΔHMG-tet cells into the left flank of nude mice, after which the mice were daily intraperitoneally injected with Dox (20 mg/kg, PBS as solvent control) for 3 weeks. J Representative tumor blocks that were collected from the mice sacrificed under anesthesia at 3 weeks post-injection. K Average tumor weights (g) of tumor blocks. L Immunofluorescence staining of SOX6 (green) and MAP4K4 (red), or SOX6 (green) and p62/SQSTM1 (red) in the frozen section of xenograft tumor tissues. Data are mean ± SEM of at least three fields. M Relative expression level of SOX6 (green) and MAP4K4 (red) or N SOX6 (green) and p62/SQSTM1 (red) in the frozen section of xenograft tumor tissues. All data are mean ± SEM (*P < 0.05, ***P < 0.001, one-way ANOVA and post hoc Tukey tests). IOD integral optical density.