Fig 1: Aloperine inhibits IL-6 production in ESCC Cells. (A) Schematic of experimental design. TE1 cells were treated with DMSO (Ctrl) or ALO for 6 h before RNA sequencing. (B) Heatmap of differentially expressed genes (DEGs) between Ctrl and ALO-treated groups. n = 3 per group. (C) Volcano plot of differentially expressed genes. Red dots indicate significantly upregulated genes, green dots indicate downregulated genes, and gray dots indicate non-significant genes. (D) KEGG pathway enrichment analysis of downregulated genes after ALO treatment. (E–G) Heatmapsof representative genes in the TNF signaling, NF-κB signaling, and JAK-STAT signaling pathways. (H–L) RT-qPCR validation of key genes from the above pathways. Data are presented as the mean ± SD from three independent experiments. ** denotes p < 0.01, and # denotes p < 0.001 as compared to Ctrl (one-way ANOVA with Tukey’s post-test).
Fig 2: ALO and cisplatin exert synergistic antitumor effects in vitro and in vivo. (A) Effects of ALO and cisplatin (CDDP) on ESCC cell viability. AKR cells were treated with increasing concentrations of CDDP alone or in combination with ALO for 24 h, and cell viability was measured by CCK-8 assay. Data are presented as the mean ± SD, n = 3. (B) Schematic representation of the in vivo tumor model and treatment schedule. Mice were inoculated with AKR cells, allowed to grow for 7 days, randomized into four groups (Ctrl, CDDP, ALO, and ALO + CDDP) and treated as every 2 days for 20 days. Tumor size was measured every 5 days, and tissues were collected on day 27 for analysis. (C) Tumor growth curves. Data are presented as the mean tumor volume ± SD, n = 6 per group. (D) Representative images of excised tumors from each group at the endpoint. (E) Tumor weight quantification. Data are presented as the mean ± SD, n = 6 per group. (F) Representative Ki67 immunohistochemical staining and TUNEL staining of tumor sections. Scale bar is 100 μm. (G) Quantification of Ki67-positive cells. (H) Quantification of TUNEL-positive cells. Data are presented as the mean ± SD, n = 3. (I) Western blot analysis of IL-6, STAT3, phosphorylated STAT3, caspase-3, cleaved caspase-3, and GAPDH in tumor lysates. (J–L) Quantification of IL-6, phosphorylated STAT3, and cleaved caspase-3 protein levels normalized to GAPDH. Data are presented as the mean ± SD, n = 3. * denotes p < 0.05, ** denotes p < 0.01, and # denotes p < 0.001 (one-way ANOVA with Tukey’s post-test). Original western blot images can be found in the Supplementary Materials.
Fig 3: ALO Inhibits IL-6/JAK-STAT Signaling Pathway via Targeting AP-1. (A) ELISA analysis of IL-6 levels in the culture supernatant of TE-1 cells treated with ALO for 24 h. Data are presented as mean ± SD. (B,C) RT-qPCR analysis of cFOS and cJUN mRNA levels in TE-1 cells treated with ALO (600 μM) for the indicated times. Data are presented as the mean ± SD. (D) Western blot analysis of cFOS, cJUN, and phosphorylated cJUN (p-cJUN) in TE-1 cells treated with ALO for 24 h. GAPDH was used as a loading control. (E–G) Quantification of cFOS, cJUN and p-cJUN protein levels from (D), normalized to GAPDH. (H) Chromatin immunoprecipitation assay showing cJUN enrichment at the IL-6 promoter in control and ALO-treated cells. Enrichment was quantified by qPCR and expressed as the percentage of input. (I) AP-1 luciferase reporter assay in TE-1 cells. Cells were co-transfected with an AP-1-responsive Firefly reporter and a Renilla internal control, then treated with DMSO, ALO (600 μM), or the specific AP-1 inhibitor SR11302 (2 μM) for 12 h. AP-1 activity was calculated as the Firefly/Renilla ratio. (J) Western blot analysis of IL-6, total STAT3, and phosphorylated STAT3 (p-STAT3), BCL2 and c-Myc in TE-1 cells treated with ALO for 24 h. GAPDH was used as a loading control. (K,L) Quantification of IL-6 and p-STAT3 protein levels normalized to GAPDH. (M) Viability changes of TE-1 cells treated with ALO (600 μM) in the presence or absence of exogenous IL-6 (20 ng/mL) for 12 h, measured by CCK-8. Data are presented as the mean ± SD. ** denotes p < 0.01, and # denotes p < 0.001 (one-way ANOVA with Tukey’s post-test). Original western blot images can be found in the Supplementary Materials.
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