Fig 1: Prediction and verification of miR‐23b‐3p target gene and the expression of EGR1 in Hep3B or Hep3B/So cells. A, Prediction of miR‐23b‐3p target gene. B, Verification of miR‐23b‐3p target gene detected by dual‐luciferase reporter assay. *P < .05, **P < .001, vs Mimic. C‐E, EGR1 protein and gene expression in Hep3B and Hep3B/So cells determined by qRT‐PCR, GAPDH served as internal reference. *P < .05, **P < .001, vs Hep3B. F‐H, EGR1 protein and gene expressions in Hep3B/So cells detected by qRT‐PCR, with the group divided into control, NC, si‐SNHG16, si‐SNHG16 + inhibitor and inhibitor, GAPDH served as internal reference. **P < .001, vs control, ## P < .001, vs NC, ^^ P < .001, vs si‐SNHG16, △△ P < .001, vs Inhibitor
Fig 2: LINC01569 recruits YBX1 to target mRNAs in the GMD complex.(A) Stretched HSFBs were treated with TA and transfected with the control or LINC01569 siRNAs. The cells were used in an IP experiment performed using an anti-YBX1 antibody (rabbit IgG as a negative control). EGR1, CITED2, and BMP7 mRNA levels were evaluated. (B) Schematic representation of the predicted binding sites of LINC01569 on the UTRs of EGR1, CITED2, and BMP7. (C) TA-treated and stretched HSFBs were transfected with empty vectors or those expressing wild-type or truncated LINC01569 and were further treated with ACD. The levels of indicated mRNAs were measured using qRT-PCR. (D) TA-treated and stretched HSFBs were transfected with the constructs for wild-type LINC01569 or LINC01569 harboring mutations (mut) in the sites responsible for binding to the corresponding mRNAs. RIP was performed using an anti-YBX1 antibody. The immunoprecipitated RNA was extracted and subjected to reverse transcription. (E) HSFBs were transfected with empty vectors or those overexpressing wild-type LINC01569 or LINC01569 harboring mutations in the mRNA binding sites, followed by treatment with ACD. The mRNAs of mechanosensors were measured using qRT-PCR. (F) Schematic representation of the UTR regions of the mechanosensors cloned into the luciferase plasmid. (G) Luciferase vectors containing wild-type or mutated LINC01569-binding sequences of EGR1, CITED2, and BMP7 UTRs and siRNAs were introduced into TA-treated and stretched HSFBs. pCDNA expressing EGR1, CITED2, and BMP7 UTRs were cotransfected as competitors on binding to LINC01569 with luciferase transcripts containing EGR1, CITED2, and BMP7 UTRs. Luciferase mRNA expression was determined by qPCR 48 hours after transfection. Data were normalized to GAPDH mRNA expression. For (A), (C), (D), (E), and (G), data are presented as means ± SD. *P < 0.05 and **P < 0.01, by Student’s t test for two groups or ANOVA for more than two groups.
Fig 3: Overexpression of EGR1 partially reversed the effects of overexpression of miR‐23b‐3p on cell viability, apoptosis and autophagy sorafenib‐resistant Hep3B cells. A, CCK‐8 assay was used to detect the viability of sorafenib‐resistant Hep3B cells. B and C, Flow cytometry was used to detect the apoptosis of sorafenib‐resistant Hep3B cells. D‐F, Western blot was used to detect the expression of LC3I and LC3II in sorafenib‐resistant Hep3B cells. *P < .05, **P < .001, vs control, # P < .05, ## P < .001, vs mimic control, ^ P < .05, ^^ P < .001, vs mimic, △ P < .05, △△ P < .001, vs EGR1, ‡ P < .05, ‡‡ P < .001, vs NC
Fig 4: Tumorigenesis in mice injected with Hep3B or Hep3B/So cells and the expressions of EGR1 and LC3II and LC3I, with group divided into Hep3B, Hep3B/So, Hep3B + So, Hep3B/So + So, Hep3B/So + si‐SNHG16 + inhibitor+So, Hep3B/So + si‐SNHG16 + inhibitor+So, Hep3B/So + inhibitor+So. A and B, Tumor volume from scarified mice. C‐E, EGR gene and protein expression levels detected by qRT‐PCR and western blot. F‐H, LC3II, LC3I, and LC3II/LC3I expressions determined by western blot. * P < .05, ** P < .001, vs Hep3B, # P < .05, ## P < .001, vs Hep3B/So, ^ P < .05, ^^ P < .001, vs Hep3B + So, △ P < .05, △△ P < .001, vs Hep3B/So + So, ▲ P < .05, ▲▲ P < .001, vs Hep3B/So + si‐SNHG16 + So, ‡ P < .05, ‡‡ P < .001, vs Hep3B/So + si‐SNHG16 + inhibitor + So
Fig 5: CD80 expression level is increased EGR1-deficient DCs(A) Levels of IL-12 p40, TNF-a, and IL-6 secreted by BMDCs stimulated with LPS (100 ng/mL) for 24 hr were measured by ELISA. Data are expressed as mean ± SD from two independent experiments (n = 3–5).(B) RNA from BMDCs from WT and Egr1-/- mice was subjected to microarray analysis. The log2 ratio was determined for the corresponding genes categorized in cell adhesion molecules within KEGG pathways and the results were arranged in descending order. Genes that overlapped with TLR signaling pathways are in red boxes.(C) Flow cytometric analyses of CD80 and CD86 in CD11c+ DCs within splenocytes from WT and Egr1-/- mice. Representative plots are shown (left). Data are expressed as mean ± SD from two independent experiments (right, n = 3). MFI, median fluorescence intensity.(D) BMDCs from WT or Egr1-/- mice were stimulated with LPS (100 ng/mL) and/or lactate (Lac, 20 mM) for 24 hr and the MFI of CD80 was assessed (n = 3). Data are expressed as mean ± SD.(E) CFSE-labeled CD8+ T cells from OT-I transgenic mice were co-cultured with BMDCs from WT or Egr1-/- mice pulsed with Ova 257–264 peptide. Proliferation of OT-I CD8+ T cells was assessed after 72 hr by flow cytometry.(F and G) Tumor growth kinetics in WT or Egr1-/- mice subcutaneously injected with 1 × 106 B16-F1 melanoma cells and treated with anti-PD-L1 antibody or control antibody (Ctrl), Lac and/or PBS as indicated. On days 6, 9, and 12 after injection of B16-F1 cells, 200 µg of control IgG Ab or anti-PD-L1 Ab were injected intraperitoneally (F), and Lac (5 mM, 200 µL) or PBS control (200 µL) was intratumorally administered (G). Data are shown as mean ± SD of 3–4 mice per group.(H) Tumor growth kinetics in diphtheria toxin (DTX)-treated CD11c-DTR bone marrow chimeric mice with subcutaneous injection with 1 × 106 B16-F1 melanoma cells and treated intratumorally with 1×106 WT or Egr1-/- BMDCs. Data are shown as mean ± SD (n = 4 mice per group).Significance was analyzed using a two-tailed Student's t-test, Welch's t test, or Mann–Whitney's U test. *p < 0.05; **p < 0.01; n.s., not significant (p > 0.05). See also Figures S10 and S11.
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