Fig 1: Effects of CUL3 overexpression on SHH pathway-related proteins and Nrf2 in PDLSCs treated with pg-LPS. (a) Western blot analysis was used to detect the effect of CUL3 overexpression on SHH, Gli1 and CUL3 in PDLSCs treated with 1 μg/mL pg-LPS. (b) Western blot analysis was used to detect the effect of CUL3 overexpression on Nrf2 and NQO1 in PDLSCs treated with 1 μg/mL pg-LPS. *p < 0.05 and **p < 0.01 vs. pcDNA-NC. ***p < 0.001 vs Control, #p < 0.05, ##p < 0.01 vs LPS+pcDNA-NC. CUL3: Cullin3.CUL3: Cullin3. CUL3: Cullin3
Fig 2: TMP treatment blocks the Hh signaling pathway in C33A cells. Protein expression levels of Hh signaling pathway-related proteins in C33A cells following TMP treatment were detected via western blotting. *P<0.05, **P<0.01 and ***P<0.001 vs. 0 µM. N≥3. TMP, tetramethylpyrazine; PTCH1, patched 1; SMO, smoothened homolog precursor; GLI1, GLI family zinc finger 1; Shh, sonic hedgehog.
Fig 3: Interfering CHOP of AECII abrogates HH activation of fibroblast. MRC5 was incubated with AECII CM for 72 h. A Expression of GLI1, GLI2, and PTCH1 was examined by Western blotting. Shh (100 ng/mL) was used as the positive control. β-actin was used as the loading control. Each group has 3 replicates. B Quantification of protein level relative to control. C Expression of nuclear GLI1 and GLI2 by Western blotting. Lamin B was used as the loading control. D Quantification of protein level of nuclear GLI1 and GLI2 relative to control. E mRNA levels of GLI1, GLI2, and PTCH1 in MRC5 relative to control
Fig 4: Downregulation of GLI1 enhanced cell sensitivity to gefitinib. PC9 cells were exposed to 40 nM gefitinib, gefitinib + small interfering (si)RNA negative control (NC), and gefitinib+siGLI1; untreated cells were considered the control. Cell apoptosis was determined by flow cytometry assay. *P < 0.05.
Fig 5: GLI1 was a potential target gene of miR‐873. (a) miR‐873 messenger RNA (mRNA) expression was evaluated by quantitative real‐time (qRT)‐PCR. qRT‐PCR and Western blot assays were performed to assess GLI1 (b) mRNA and (c) protein levels. *P < 0.01 versus control; **P < 0.01 versus mock. (d) The binding site of GLI1 to miR‐873 was predicted. The GLI1 3’ untranslated region (UTR)‐mutant (mut) is presented. (e) The 3’UTR of GLI1 with affinity for miR‐873 and a mutant reporter were cloned to the downstream of firefly luciferase of psiCHECK‐2 vector. miR‐873 was transfected into HEK293T cells. Luciferase activity was examined. *P < 0.01 versus GLI1 3’UTR; **P < 0.01 miR‐873 + GLI1–3’UTR + mut. NS, not significant.
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