Fig 2: Enforced expression of Krüppel-like factor 4 (KLF4) in the lung suppressed tumor metastasis and invasion in vivo. (A, B) Bioluminescence was analyzed in mice after tail vein injection of Lewis lung cancer (LLC) cells (n = 8 per group). Whole body bioluminescent imaging of animals was acquired on day 21 after tumor cell injection. (I) The total luminescence of lung between the control and AAV5 groups was compared by nonparametric tests (*p < 0.05). (C, D) Images of the lungs were taken after mice were sacrificed. (J) The number of metastatic nodules on the surface of the lungs was compared by nonparametric tests between the control adeno-associated viral vector (AAV5) and AAV5 expressing KLF4 groups (**p < 0.05). (E–H) Hematoxylin and eosin staining of mouse lungs at different magnifications. The arrow shows the location of the metastatic tumor. (G,H) The typical magnified image of a mouse metastatic tumor. (K) Immunofluorescence staining for KLF4 in paraffin sections from mouse lungs infected by control AAV5. (L) Immunofluorescence staining for KLF4 in paraffin sections from mouse lungs infected by AAV5 expressing KLF4. (M,N) The magnified image of the indicated area in (K,L) separately.

Fig 3: KLF4 inhibits the proliferation and migration ability of HCC cells. (A) Knockdown of KLF4 in SMMC-7721 and overexpression of KLF4 in PLC/PRF/5 was verified by western blot, and ß-actin was used as an internal control in western blot assays. (B) CCK8 assay was implemented to detect the proliferation rate of steadily transfected SMMC-7721 and PLC/PRF/5. (C) Cell wound scratch assay and (D) transwell assay was executed to evaluate the migration rate of steadily transfected SMMC-7721 and PLC/PRF/5. Student’s t-test was used in line charts and bar charts. P < 0.05 was considered statistically significant. * P < 0.05, ** P < 0.01, *** P < 0.001.

Fig 4: miR-21 delivered by EVs inhibited RA progression in vivo by TET1/KLF4 axis. (a) miR-21 expression in bone joint determined by RT-qPCR. (b) TET1 and KLF4 expression determined by western blot. (c) Clinical RA scores were recorded every 2 days before the end of the experiment. (d) Bone joints of RA mice analyzed and scored by HE staining. (e) microCT images of paws analyzed by BMD. (f) TRAP activity in plasma determined by ELISA. (g) TRAP expression in synovial tissues analyzed by IHC. (h) TNF-a, IL-1ß, and PGE2 levels in synovial tissues determined by ELISA. (i) TNF-a, IL-1ß, and PGE2 levels in plasma determined by ELISA. (j) NO and iNOS levels in plasma of RA mice determined by nitrate reductase assay.*p < 0.05, compared with oe-NC + EVs (mimic-NC).#p < 0.05, compared with oe-KLF4 + EVs (mimic-NC).Data are shown as the mean ± standard deviation. Statistical comparisons were performed by Tukey’s test-corrected one-way ANOVA when more than two groups were compared. The experiment was repeated three times. The number of mice in each group N = 12.EVs, extracellular vesicles; RA, rheumatoid arthritis; RT-qPCR, reverse transcription-polymerase chain reaction; BMD, Bone mineral density; TRAP, tartrate-resistant acid phosphatase; ELISA, enzyme-linked immuno sorbent assay; IHC, immunohistochemistry; oe-NC, overexpressed TET1 or KLF4 control.

Fig 5: Representative western blots of Slug (a), KLF4 (b) and vimentin (e) proteins in UPCI-SCC-90 cells in control, 1 ng/mL TGF-ß1 and in 50 ng/mL IL-6 treated conditions. TGF-ß1 induced a moderate but significant increase (p = 0.0499, by Mann Whitney test compared to control) in Slug protein level. Il-6 did not statistically upregulated Slug in UPCI- SCC-90 cells (a,c). KLF4 was not regulated by TGFß1, but it was upregulated by IL-6 (p = 0.021 by Student’s t-test compared to control) (b,d). TGF-ß1 upregulated vimentin at protein levels, but it was not statistically significant. IL-6 induced significant upregulation of vimentin (p = 0.012 by Student’s t-test compared to control) (e,f). Four western blot membranes were acquired digitally, and the band optical densities (ODs) of proteins of interest and of loading control (GAPDH) were measured using the Image Studio Lite of Li-cor. The ODs of proteins of interest were normalized to loading control. Mean normalized optical densities of control samples were set to “1”, and this control mean was used as reference (c,d,f). Loading control by western blot membrane reacted with GAPDH antibody is presented on panel (g).