Fig. 2: PPARγ-mediated modulation of NECTIN4 expression.

a NECTIN4 mRNA levels in RT112 cells treated with 1μM rosiglitazone and T0070907 (T007) for 72 h. Data are presented as mean ± SEM, n = 6 for DMSO and rosiglitazone, n = 4 for T0070907, all biological replicates. b Western blot for NECTIN4 and HPGD in RT112 cells treated with T0070907, rosiglitazone, and pioglitazone at indicated concentrations for 72 h. This was repeated n = 3 independent times with similar results. c, d NECTIN4 surface staining (c) and quantification of median fluorescence intensify (MFI) (d) in RT112 cells treated with 1μM rosiglitazone and T0070907 for 72 h. Data are presented as mean ± SEM, n = 4 for DMSO and T0070907, n = 5 for rosiglitazone, all biological replicates. e Western blot for NECTIN4 and HPGD in RT112 cells treated with rosiglitazone for 72 h across a dose series (starting at 1 μM on the right most lane with serial 2-fold dilutions to the left). This was repeated n = 3 independent times with similar results. f Dose response curves of total NECTIN4 and HPGD protein expression after 72 h rosiglitazone treatment in RT112 cells. Data are presented as mean values. g NECTIN4 surface staining in RT112 cells treated with rosiglitazone for 72 h across a dose series. Dose series starts at 1μM with serial 2-fold dilutions. h Dose response curves of surface NECTIN4 and TROP2 expression for rosiglitazone in RT112 cells after 72 h. Data are presented as mean ± SEM, n = 2 biological replicates. i Western blots for NECTIN4, PPARγ and FABP4 in RT112 cells expressing sgRNAs against GAL4 (control), NECTIN4, and two unique PPARG guides. This was repeated n = 3 independent times with similar results. j Schematic of select predicted PPARG binding sites (labeled 1 and 2) on the NECTIN4 promoter region (500 to −2k) by FIMO using transcription binding motif matrices JASPAR 2018. Created with BioRender. Chang (2025) https://BioRender.com/k3j6fgi. k Chromatin from RT112 cells was precipitated using antibodies against PPARG or IgG. Primers targeting putative PPARG binding motifs 1 and 2 from (j) were used for PCR and the PCR product was visualized by gel electrophoresis. l Quantitative analysis of ChIP-qPCR experiments. Results are represented as fold-enrichment relative to IgG control. Data are presented as mean ± SEM, n = 3 biological replicates. For panels (a) and (d), ordinary one-way ANOVA with Sidak’s multiple comparison was used. For panel (l), two-sided, unpaired Student’s t test was used for each primer set. Vinculin shown as a loading control for panels (b) and (e) and GAPDH for panel (i). Source data are provided as a Source Data file.