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. 2022 May 20;298(7):102059. doi: 10.1016/j.jbc.2022.102059

Figure 5.

Figure 5

RORα inhibits Snail expression by directly repressing Snail transcription.A, SNAI1 mRNA levels in control and RORα-expressing MDA-MB 157 cells from the microarray data. B, SNAI1 mRNA levels were quantified by real-time PCR in control and RORα-silenced MCF10A cells; n = 4. ∗∗p < 0.01. C, SNAI1 mRNA levels were examined by real-time PCR in control and RORα-expressing MDA-MB 157 and BT549 cells; n = 3. ∗∗p < 0.01. D, protein expression of Snail was examined by immunoblot of RORα-expressing MDA-MB 157 cells. E, confocal images showed Snail and RORα immunofluorescence staining in RORα-expressing MDA-MB 157 cells. Bar represents 10 μm. F, Snail protein levels were examined by Western blot in control or RORα-expressing MCF10A cells after treatment with the proteasome inhibitor bortezomib (1 μM) for 24 h. G, potential ROREs were identified in SNAI1 gene. H, agarose gel images of ChIP PCR. I, ChIP PCR quantification data showed the binding of RORα to ROREs in the SNAI1 gene in RORα-expressing MDA-MB 231 cells; n = 3. ∗p < 0.05. J, luciferase reporter data showed that RORα inhibited the transcription driven by RORE2 in HEK 293 cells transfected with renilla luciferase vector, pCDH1-RORA-FLAG and pGL4-RORE2; n = 4. ∗∗p < 0.01. K, luciferase report data showed that deletion of ROREs in the pGL4-RORE2 vector released the RORα-suppressed transcription in HEK 293 FT cells. The cells were transfected with renilla luciferase vector, pCDH1-RORA-FLAG, and wildtype/mutant pGL4-RORE2 vectors (pGL4-RORE2-WT/pGL4-RORE2-MT); n = 3. ∗∗p < 0.01. ChIP, chromatin immunoprecipitation; HEK 293, human embryonic kidney 293 cell line; RORα, retinoid orphan nuclear receptor alpha; RORE, ROR response element.