Fig 1: Expression of nuclear receptors for vitamin D3 hydroxyderivatives and CYP enzymes involved in vitamin D metabolism in murine ASZ001, human A431 and human SCC13 carcinoma lines. (A) Immunofluorescence detection (left panel) was performed using antibodies against VDR (MA1-710) and RORa and ROR? (54). The antigen is in green, while cell nuclei are red-counterstained with propidium iodide; scale bar=100 µm. Right panel shows detection of VDR and RORa and ROR? by western blotting (arrows) using specific anti-receptor antibodies (see Materials and methods) for isolated cytoplasmic or nuclear fractions (VDR) from the cells or whole extracts (CYP27B1, RORa and ROR?). Human hepatoma and human melanoma SKMEL-188 cells were used as positive controls for RORa and ROR?. Loading was evaluated using antibodies against lamin C and a-tubulin for nuclear or cytoplasmic fractions, respectively, while with anti-ß-actin for whole extracts. (B) shows expression at the mRNA level of Cyp27b1, Cyp24a1, Cyp11a1, Vdr, Rora and Rorc, respectively in ASZ001 murine carcinoma line treated with 10-7 M of the secosteroids listed on the x-axis. Data represent fold change, using ß-actin as a housekeeping gene and are shown as means ± SEM (n=4) with *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 by Student's t-test. ROR, retinoid-related orphan receptor; VDR, vitamin D receptor.
Fig 2: Dominant-negative Rora suppresses neutrophil motility and chemotaxis. (A) Schematic of the construct for neutrophil-specific expression of vector control or rora dominant-negative (DN). (B, C) Representative images and quantification of total neutrophil numbers in vector or rora DN zebrafish line. Scale bar: 500 µm. (D, E) Representative images and velocity of random neutrophil migration in vector or rora DN zebrafish line. Scale bar, 100 µm. Three embryos each from three different founders were imaged, and quantification of neutrophils in one representative video is shown, Kruskal–Wallis test. (F, G) Representative images and quantification of neutrophils recruited to the infected ear in vector or rora DN zebrafish line. Scale bar, 100 µm. (H, I) Representative images and quantification of neutrophil recruitment to the tailfin transection sites in vector or rora DN zebrafish line. Scale bar, 200 µm. The result is presented as mean, Mann–Whitney test. (J) Simultaneous imaging of utrophin-GFP distribution in neutrophils expressing either mCherry alone or with Rora DN. Data are representative of more than three separate time-lapse videos. Scale Bar, 50 µm. (B, C, F–I) Representative results for three individual trials are shown. (K) Schematics of the plasmid constructs for neutrophil-specific Cas9 expression (upper construct) and for ubiquitous expression of sgRNAs with a neutrophil-specific expression of GFP (lower construct). (L, M) Representative images (L) and quantification (M) of neutrophil motility at head region of 3dpf larvae from Tg(LyzC: Cas9, Cry: GRFP, U6a/c: rora guides, LyzC: GFP) fish. Student’s t-test. Scale bar, 100 µm. (N) Deep sequencing of the rora loci targeted by guide RNAs in (K). The sequences on the top are wild-type sequences, and the five most frequent types of mutations are shown. Point mutations, deletions, and insertions are all observed.
Fig 3: Pharmacological inhibition of Rora reduces neutrophil motility and chemotaxis in zebrafish and humans. (A, B) Tracks and quantification of neutrophil motility in zebrafish larva treated with SR3335 (100µM), SR2211 (100µM), or VPR66 (25µM). Scale bar, 200 µm. Three embryos, each from three different founders, were imaged. Quantification of neutrophils in one representative video is shown, Kruskal–Wallis test. (C, D) Representative images and quantification of neutrophils recruited to the infected ear in zebrafish larva treated with RORa specific inhibitor (SR3335, 100µM), ROR? specific inhibitor (SR2211, 100µM) or pan-ROR family inhibitor (VPR66, 25µM). Scale bar, 100 µm. (E, F) Representative images and quantification of neutrophils recruited to tail fin transection sites in zebrafish larva treated with SR3335 (100µM), SR2211 (100µM), or VPR66 (25µM). Scale bar: 500 µm. (C–F) The result from one representative experiment is shown as mean, Mann–Whitney test. (G, H) Representative tracks and mean velocity of primary human neutrophils treated with SR3335 (50µM), SR2211 (50µM), or SR1001 (pan-ROR family inhibitor) (50 µM) migrating towards fMLP in 3D matrigel. Scale bar, 100 µm. Representative results for three individual trials are shown. The result is presented as mean, Mann–Whitney test. (I) Neutrophil recruitments after zebrafish tail wounding at different dosages of SR3335 treatment compared to 1% DMSO treatment, Kruskal–Wallis test. (J) Transwell migration of primary human neutrophils treated with DMSO (0.1%) or SR3335 at 10, 30, or 100 µM toward 100 nM fMLP. Results are presented as mean ± s.d., from three independent experiments and normalized to DMSO (0.1%), Kruskal–Wallis test.
Fig 4: Quantitative analysis of nuclear receptor expression in SCC and BCC. (A) Quantitative comparison of immunostaining of VDR, RORa, ROR? and megalin in SCC (n=14), SCC in situ (n=22), BCC (n=12), normal (n=35) and peritumoral skin samples. Data are presented as mean values ± SD. Statistically significant differences were determined with ANOVA followed by Dunn's multiple comparisons test with *P<0.05, **P<0.01, with ***P<0.001, ****P<0.0001. The immunohistochemical stained archival formalin-fixed paraffin-embedded sections were used for quantifications as described in the Materials and methods. (B) Quantitative comparison of mRNA expression of RORA (probe 226682_at) and RORC (probe 228806_at) in SCC (n=11), BCC (n=15) and normal skin (n=4) was performed using data from the genomic repository (Gene Expression Omnibus, accession number GSE7553 http://www.ncbi.nlm.nih.gov/geo). Data are shown as means ± SD. Statistically significant differences were determined with the ANOVA followed by Dunn's multiple comparisons test with *P<0.05, **P<0.01. SCC, squamous cell carcinomas; BCC, basal cell carcinomas; ROR, retinoid-related orphan receptor; VDR, vitamin D receptor.
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