Fig 1: MEK-162 treatment reduces TR4 expression and inhibits binding to POMC promoterA., Q-PCR was used to quantitate TR4 mRNA in murine corticotroph tumor AtT20 cells following MEK-162 treatment (20 µM and 40 µM for 24 h). B., TR4 protein expression was detected by Western Blotting in murine corticotroph tumor AtT20 cells following 40 µM MEK-162 treatments for 12 h and 24 h, and quantified using densitometric analyses of the TR4 protein bands vs. the individual loading controls. C., AtT20 cells were transiently transfected with V5-TR4 plasmid. 24 h later, cells were treated with 20 µM and 40 µM MEK for 24 h, after which ChIP assay was performed to measure TR4 binding to the POMC promoter (-854bp~ -637bp). Each bar indicates the mean ± standard error of triplicate tests. * p < 0.05; ** p < 0.01, *** p < 0.005. Data shown were representative of at least three independently repeated experiments.
Fig 2: Effect of TR4 on viability in BC cell lines: (A) After transfecting Vector and TR4 in BC cell lines, the proliferation ability was measured by the EdU assay. Quantification of positive cells was determined by microscopy with ×40 magnification in three randomly chosen fields. Data are presented as mean ± SD. *p < 0.01. (B) After transfecting scr and siTR4 in BC cell lines, quantification of positive cells was determined in three randomly chosen fields. *p < 0.05, **p < 0.01.
Fig 3: Bexarotene decreased TR4’s transactivation via regulating the transcription from a natural promoter: a Diagram of the PITCh system with CRISPR-CAS9 technique to establish stable cell lines with luciferase inserted at the genomic location in the oxytocin gene in 293T cells. b Twelve single clones were expanded upon selection with 2 µM puromycin, six of which had luciferase value increases in response to oeTR4 through lentiviral transfection, and three clones (#6, #11, and #12) had the correct genomic structure indicating that luciferase construct was indeed successfully inserted at the intended genomic location through genomic PCR assays (lower panel). c Selected cloned cells, 1 × 103 of cells/well, were plated in 24-well plates and transfected with vector or oeTR4 for 24 h. After transfection, cells were treated with DMSO or 8 µM Bex for 24 h. Renilla luciferase ORF was measured by the Dual-Luciferase Assay. The results showed TR4 could significantly increase the luciferase activity, which can be reduced with Bex treatment in the PITCh system. In b and c, data are presented as mean ± SD, *p < 0.05
Fig 4: TR4 antagonist bexarotene (Bex) increased chemo-sensitivity of docetaxel in PCa cells: a Chemical structure of Bex, a retinoid derivative. b PC-3 and Du-145 cells were treated with different concentrations (0–20 µM for PC-3 and 0–24 µM for Du-145) of Bex within the clinical dosage range for 72 h and we found Bex concentrations below 8 µM have little influence on PC-3 and Du-145 proliferation, excluding the possibility that Bex may function via regulating its purported target, RAR/RXR. c Bex at 8 µM increased the chemo-sensitivity of docetaxel through TR4 in Du-145 and PC-3 cells treated with 2 nM DTX and oeTR4. d Parental 22RV-1 and C4-2 cells were treated with 8 µM Bex for 72 h and DTX-resistant 22RV-1R and C4-2R cells were treated with 8 µM Bex for 5 days. The 22RV-1R cells were maintained in 20 nM DTX and C4-2R dells maintained in 6 nM DTX. Cell proliferation results showed reduced cell proliferation in DTX-resistant cells, with no significant change in parental cells. Data are presented as mean ± SD, *p < 0.05
Fig 5: Targeting TR4/lncTASR/AXL signaling with tretinoin can increase sunitinib sensitivity to better suppress the RCC progression. a Tretinoin alone was not toxic to RCC cells. b Tretinoin can enhance the sensitivity of RCC to sunitinib when combined with low dose sunitinib. c Tretinoin actually has synergetic effects when combined with sunitinib in the treatment of RCC cells. d Western blotting (left) with quantification (right) shows that the expression of both TR4 and AXL decreased after treated with tretinoin. e Tretinoin can reverse the resistance of RCC cells to sunitinib caused by overexpressed TR4. f Viability of OSRC-2 cells in sunitinib with/without tretinoin and with/without TR4-shRNA. g DID (difference in difference) analysis shows that TR4-knockdown OSRC-2 cells have less difference change in sunitinib resistance with/without tretinoin compared with OSRC-2 cells without TR4-knockdown. h Tretinoin decreases both TR4 and AXL protein expressions in OSRC-2 cells with pLKO vehicle, but does not decrease TR4 and AXL expressions in TR4-knockdown OSRC-2 cells. Data is presented as Mean ± s.e.m., **p < 0.01, ***p < 0.001, ns not significant
Supplier Page from Abcam for Anti-TR4 antibody [EPR1773(2)]