Fig 2: The effects of WNQP on 35 types of cytokines in nude mice tumors, detected using an Apoptosis Array Kit (n = 3). The arrows indicate factors marked for further detection: 1. Bax, 2. Bcl-2, 3. Bcl-xL, 4. cleaved caspase-3, 5. cIAP, 6. clusterin, 7. TRAIL R2/DR5, 8. FADD, 9. Fas, 10. HIF-1α, 11. HO-1, 12. HSP 60, 13. HtrA2, 14. livin, 15. SMAC, 16. survivn, 17. XIAP. (A, B) Scatter diagram of 35 cytokines. The relative density is the ratio of the absolute value and reference spot value. The red dots indicate factors with a change of >20% relative to control mice. (C, D) WNQP strongly enhanced the expression levels of Bax and suppressed the expression levels of Bcl-xL, HSP27, HSP60, and HSP70 in tumor tissues. Quantitative protein expression data were normalized to the corresponding GAPDH levels, and the average fold changes in band intensity are marked (E, F) (n = 3).

Fig 3: Nrf2 signaling is involved in the improvement of immune function during WNQP-mediated inhibition of tumor growth. WNQP strongly enhanced the expression levels of Nrf2, HO-1, HO-2 and SOD-1 in the spleen tissues of BALB/c mice bearing (A, B) HepG2- and (C, D) SMMC-7721-xenograft tumors. (E, F) In Nrf2-siRNA-transfected primary cultured spleen cells, WNQP-induced increase in the levels of Nrf2, HO-1, HO-2 and SOD-1 were abolished. Quantitative protein expression data were normalized to the corresponding GAPDH levels, and the average fold changes in band intensity are marked. Data are expressed as the mean ± SD (n = 3). *P < 0.05, **P < 0.01 and ***P < 0.001 versus the control group.

Fig 4: METTL3-dependent m6A methylation regulates the processing of mmu-miR-873-5p by DGCR8. (A) Co-IP of the METTL3-interacting protein DGCR8. mREECs were crosslinked before the immunoprecipitation. IgG antibody was used as control. (B) MiR-126 and pri-miR126 were quantified by qPCR in all groups. (C) IP of DGCR8-associated RNA from control or METTL3-overexpressing cells followed by qPCR to detect pri-mmu-miR-873-5p binding toDGCR8. Immunoprecipitation of m6A modified RNA in control or METTL3-overexpressing cells followed by qPCR to assess the pri-mmu-miR-873-5p m6A modification levels. (D) Bioinformatics analysis of predicted interactions of mmu-miR-873-5p with its binding sites in Keap1. Luciferase activity was measured by dual-luciferase reporter assay. Luciferase activity was normalized to Renilla luciferase activity. (E) Western blot and qPCR analysis of Keap1, Nrf2, and HO-1 expression. The values that are significantly different from the values relative to control group are indicated by asterisks as follows: ∗∗p < 0.01. Values that are significantly different from the values of colistin-treated group are indicated as follows: ##p < 0.01.