Fig 2: Treatment of glomeruli with vit D and calcipotriol leads to increased protein levels of NPHS1, CFP and VDR after 9 days. (A,C,E,G) Representative Western blots for NPHS1, CFP, RXRa, and VDR in vit D- and calcipotriol-treated mouse glomeruli after 9 days and untreated glomeruli on day 0. (B,D,F,H) Quantification of Western blot lanes. The protein expression of NPHS1, CFP, and VDR is significantly increased after treatment with vit D and calcipotriol compared to DMSO. Treatment was done with 100 nM vit D, 1 µM calcipotriol, and 0.1% DMSO. Values are presented as medians + Min/Max. *p <0.05; **p <0.01; ***p <0.001. Vit D = vitamin D, calcip = calcipotriol, SFI = Stain Free Imager. (n = 5, 5, 3, 5).

Fig 3: (a) Normalized expression of human nhr-2 orthologs RXRA, RXRB, RXRG, and PPARG in samples from 38 patients with ET and mutations in CALR (26 with type 1 mutations and 12 with type 2 mutations), 21 patients with ET and mutations in JAK2, and 10 healthy donors. Individual values, means, and standard deviations are shown. When no expression was detected in any of the samples analyzed, no statistic is shown (ND, not detected). (b) Individual heatmaps with RXRA, RXRB, RXRG, and PPARG expression data in the samples. Some of the samples were extracted from bone marrow (BM) and others from peripheral blood (PB). Additionally, the time of sample collection is indicated, either at the time of diagnosis or during follow-up of the disease. In all cases, average fold change values are represented from shades of green (negative fold change) to shades of red (positive fold change). (c) The percentage of viable cells quantified by the MTS assay after exposing MARIMO and SET-2 cell lines to increasing concentrations (0, 0.1, 1, and 10 μM) of bexarotene, HX531, and ATRA without ruxolitinib or in combination with ruxolitinib at 1 μM (n = 3). * p < 0.05, ** p < 0.01, and *** p < 0.001.

Fig 4: Expression of RARA, RXRA, and VDR mRNA in osteosarcoma cell lines after treatment with selected differentiation inducers. Changes in the expression of the VDR (A), RARA (B), and RXRA (C) genes in osteosarcoma cell lines after 24 h of incubation with 1 µM ATRA, 10 nM calcitriol, or 100 nM calcidiol were measured using RT-qPCR. GAPDH served as a loading control. The results are presented as fold changes compared to the mRNA levels detected in untreated control cells. In addition, mRNA levels in untreated patient-derived osteosarcoma cell lines were compared with mRNA levels in the Saos-2 reference cell line, which served as an arbitrary calibrator. Experiments were performed once in technical triplicate. The data represent the mean ± SD.

Fig 5: Insulin stimulates phosphorylation of RXRα at S22 in vitro and in vivo. Representative immunoblots with quantified pRXRα (56 kDa and 65 kDa, separately) and RXRα (56 kDa and 65 kDa together) band intensities in brown pre-adipocytes and mouse BAT presented as mean ± SE. Immunoprecipitation (IP) was performed using control IgG or RXRα antibody (D6H10). A different RXRα antibody (D-20) was used for immunoblotting. Lysate input or immunoprecipitated RXRα were prepared from (a) WT or Rxra−/- brown pre-adipocytes treated with insulin or vehicle; n = 3; or (b) BAT from mice injected with insulin or saline; n = 5; two-tailed t-test or two-way ANOVA with Šídák’s multiple comparison tests. (c) BAT from mice that were fasted or refed for 2 or 6 h; n = 6; one-way ANOVA and Dunnett’s multiple comparison test. (d) BAT from mice that were fed with CD or HFD and were injected with insulin or saline; n = 5–6; two-way ANOVA and Šídák’s multiple comparison test. All phosphoprotein band intensities are normalized to the total amount of each respective protein, except in immunoprecipitation experiments where pRXRα and RXRα are quantified separately. Asterisk (*) represents a significant difference (p < 0.05) from vehicle-treated control cells, fasted mice or saline-injected control mice. Hash (#) represents a significant difference (p < 0.05) from WT cells, BAT RXRα IP or insulin-injected CD mice