Fig 2: IL-1ß signaling was responsible for PD-L1 expression in HCC cells induced by M1 macrophages. (A) Co-expression of IL-1ß and PD-L1 gene in HCC samples from TCGA database. (B) HCC cells were transfected with siNC or siIL-1R1, then cultured in the presence or absence of M1S. The PD-L1 expression in these HCC cells was determined by quantitative RT-PCR assay. (C) Huh7 cells pretreated with IL-1Ra were cultured in the presence or absence of M1S. The PD-L1 expression was detected by quantitative RT-PCR. (D) HCC (BEL-7402 or Huh7) cells were stimulated with IL-1ß. The PD-L1 expression was detected by quantitative RT-PCR. (E) Huh7 cells were stimulated with IL-1ß. The PD-L1 expression was detected by flow cytometry. (F) Huh7 cells were transfected with siNC, siP65, or siIRF1, then cultured in the presence or absence of IL-1ß. The PD-L1 expression was determined by quantitative RT-PCR assay. (G,H) HCC cells were transfected with siNC, siP65, or siIRF1, then cultured in the presence or absence of M1S. The PD-L1 expression was determined by quantitative RT-PCR assay. (I,J) BEL-7402 cells were treated with or without M1S, then, CHIP assay was performed to assess the occupancy of transcription factor P65 or IRF1 on the PD-L1 promoter. (K,L) BEL-7402 cells were co-transfected with the mixture of internal control plasmid pRL-TK and luciferase reporter plasmid pGL3-PD-L1, pGL3-mut-p65A, pGL3-mut-p65B, pGL3-mut-p65A+B, or pGL3-mut-IRF1, then treated with M1S. The relative luciferase activities were measured by dual-luciferase reporter assay. The experiment was carried out in triplicate and repeated at least twice. Data are shown as means ± SD. *P < 0.05, **P < 0.01, ***P < 0.001.

Fig 3: Inhibiting autophagy in SV-HUC-1 cells activates progressive canonical followed by non-canonical NF-κB signaling. (A) Immunocytochemistry of 200 nM BafA1-treated SV-HUC-1 cells stained for RelA (red) showed nuclear translocation as early as 6 h, with sustained localization observed at 24 h and 48 h. (B) Immunocytochemistry of 200 nM BafA1-treated SV-HUC-1 cells demonstrated that nuclear translocation of the non-canonical NF-κB signaling constituent p52 (red) was not observed at 6 h but appeared later at 24 h and 48 h. (C) TWEAK-treated SV-HUC-1 cells stained for RelA (red) and p52 (red) demonstrated that both canonical and non-canonical NF-κB signaling were active at 6 h. (D) Quantification of the percentage of cells showing nuclear translocation of RelA and p52 in response to TWEAK, VPS34i or BafA1 treatment at 0, 6, 24 and 48 h. Scale bars: 10 µm.

Fig 4: Fxyd5 regulates cell viability, apoptosis and ECM degradation via activating the NF-κB signaling pathway in ATDC5 cells. Following cell treatment with 10 nM BA, a NF-κB activator, for an additional 48 h (A) the phosphorylation levels of p65 and IκBα were increased, as demonstrated by western blot analysis. (B) Cell Counting Kit 8 assay showed that cell viability was increased in Fxyd5-depleted ATDC5 cells co-treated with LPS and BA compared with cells treated only with LPS. (C and D) Western blotting was used to evaluate the expression of proteins involved in cell apoptosis and ECM degradation. Untreated and untransfected ATDC5 cells served as the control group. GAPDH served as the loading control. ***P<0.001 vs. the control group; ##P<0.01 and ###P<0.001 vs. the LPS + shNC group; $P<0.05, $$P<0.01 and $$$P<0.001 vs. the LPS + shFxyd5-2 + BA group. Fxyd5, FXYD domain containing ion transport regulator 5; ECM, extracellular matrix; BA, betulinic acid; LPS, lipopolysaccharide; shRNA, short hairpin RNA; NC, negative control.

Fig 5: p38 MAPK and p44/42 MAPK were the main signaling pathways downstream of ROS in IS-treated rats and NRVMs.(A and B) Representative immunoblots of phosphorylated p38 (P-p38) MAPK, P-p44/42 MAPK, P-NF-κB, p38 MAPK, p44/42 MAPK, and NF-κB in sham, CKD, and CKD plus BB536 groups (A) and in vehicle and IS treatment groups (B). (C and D) Average data of P-p38 MAPK/p38 MAPK, P-p44/42 MAPK–p44/42 MAPK, and P-NF-κB/NF-κB in sham, CKD, and CKD plus BB536 groups (n = 5 per group) (C) and in vehicle and IS treatment groups (n = 6 per group) (D). **P < 0.01. (E and F) Representative immunoblots of P-p38 MAPK, P-p44/42 MAPK, P-NF-κB, p38 MAPK, p44/42 MAPK, and NF-κB (E) and average data (F) of P-p38 MAPK p38 MAPK, P-p44/42 MAPK–p44/42 MAPK and P-NF-κB/NF-κB in NRVMs treated with different concentrations of IS (n = 3 per group,*P < 0.05 versus control, **P < 0.01 versus control). (G) Translocation of NF-κB–p65 from cytoplasm to nucleus measured by immunofluorescence. White arrows indicate the nuclear localization of NF-κB–p65. Scale bar: 10 μm. (H) Nuclear/cytoplasmic fluorescence intensities of NF-κB in control and IS groups (n = 5 per group). (I and J) Representative immunoblots of P-p38 MAPK, P-p44/42 MAPK, P-NF-κB, p38 MAPK, p44/42 MAPK, and NF-κB (I) and average data (J) of P-p38 MAPK/p38 MAPK, P-p44/42 MAPK–p44/42 MAPK, and P-NF-κB/NF-κB in control, NAC, IS, and IS plus NAC groups (n = 3 per group). NRVMs in IS and IS plus NAC groups were treated with 10 μM IS. Data are presented as mean ± SEM. Statistical analysis was performed using 2-tailed Student’s t test (D and H) and 1-way ANOVA, followed by Bonferroni post hoc test (C, F, and J). *P < 0.05, **P < 0.01.