Fig 2: (A) Transwell migration assay of shCtrl- and shKIF11-infected MH7A cells at 24 h and 48 h. Representative images (40 × magnification) and migrated cell quantification (right). Data are mean ± SD (n = 3). *P < 0.05, **P < 0.01 vs. CtrlshRNA group. (B) The expression of IL-1β, IL-6, and IL-8 was detected by ELISA assay in cell supernatant. ***P < 0.001. (C) Rescue of KIF11 expression in knockdown cells. MH7A cells with stable KIF11 knockdown (shKIF11) were transfected with either a human KIF11 cDNA overexpression plasmid (KIF11-OE) or the empty control vector (Pcmv3 vector). After 24 hours, KIF11 protein expression was analyzed by Western blotting. β-actin served as a loading control. (D) The relative KIF11 protein levels were shown in histogram. Data are presented as mean ± SD (n = 3). ***P < 0.001 vs. the vector-transfected shKIF11 group. (E) Rescue of pro-inflammatory cytokine secretion. The cell culture supernatants from the experiment described in (D) were collected, and the concentrations of TNF-α, IL-1β, IL-6, and IL-8 were measured by ELISA. Data are presented as mean ± SD (n = 3). ***p < 0.001 vs. the control vector-transfected shKIF11 group.

Fig 3: Hub genes correlated with KIF11 and their expression in RA tissues.A. Network analyzed using STRING with interaction threshold of 0.40. B. Hub genes were predicted using Cytoscape. Top 10 nodes were ranked using MCC. First-stage nodes are shown here. C. The expression of top10 hub genes in RA tissues. Log FC values were shown in histogram in five datasets including GSE55457, GSE55235, GSE2053, GSE12021, and GSE1919.

Fig 4: Knockdown of KIF11 in THP-1-derived M1 macrophages suppresses the secretion of inflammatory factors by M1 macrophages.A. QPCR assay was performed to test the relative expression of IL-1β, IL-6, iNOS and TNF-α in shKIF11 lentivirus infected M1 macrophages and control lentivirus infected M1 macrophages. ***P < 0.001. B. ELISA assay. THP-1-derived M1 macrophages were infected with shKIF11 lentivirus or negative control lentivirus for 48 h. The concentration of IL-1β, IL-6 and TNF-α in cell culture supernatant were tested in shKIF11 lentivirus -infected M1 cells or negative control lentivirus-infected M1 cells. ***P < 0.001, compared with the negative control group.

Fig 5: KIF11 regulates the NF-κB signaling pathway in MH7A cells.(A) KIF11 knockdown inhibited NF-κB p65 phosphorylation. MH7A cells were infected with control shRNA (shCtrl) or KIF11 shRNA (shKIF11) lentivirus for 24 hours. Whole-cell lysates were subjected to Western blotting using antibodies against phospho-p65 (p-p65), total p65, and KIF11. β-actin served as a loading control. (B) The histogram shows the relative ratio of p-p65 to total p65. Data are presented as mean ± SD (n = 3). ***P < 0.001 vs. shCtrl group. (C) KIF11 knockdown impaired nuclear translocation of NF-κB p65. Cytoplasmic and nuclear fractions were extracted from shCtrl- and shKIF11-infected MH7A cells. The distribution of p65 in the cytoplasm (Cyto) and nucleus (Nuc) was analyzed by Western blotting. Lamin B1 and α-tubulin were used as markers for nuclear and cytoplasmic fractions, respectively. (D)The relative abundance of p65 in the nuclear and cytoplasmic fractions were shown in histograms, normalized to their respective loading controls. Data are presented as mean ± SD (n = 3). ***P < 0.001 vs. shCtrl group.