Figure 1 from Oncogenic Cell Tagging and Single-Cell Transcriptomics Reveal Cell Type–Specific and Time-Resolved Responses to Vhl Inactivation in the Kidney

A novel reporter model for Vhl inactivation in the mouse kidney. A, Design and recombination of the cell marking conditional Vhlpjr allele. Double and single arrows indicate reversible and irreversible processes, respectively. Vhlpjr.fl, Vhlpjr.inrec, and Vhlpjr.KO refer to “floxed,” “incompletely recombined,” and “knockout” forms of the Vhlpjr allele. P, Vhl promoter; U, untranslated region; E, Vhl exon; I, Vhl intron; pA, polyadenylation site; P2A, porcine teschovirus 2A peptide; SA, splice acceptor. Dashed lines, spliced and translated regions; lightning symbols, excitation and emission wavelengths for tdTomato fluorescence. Red stop sign indicates no interaction between VHL exon 1 fragment and HIFA-1/2 or Elongin B/C. B, Representative tdTomato IHC counterstained with hematoxylin in kidney sections and tdTomato fluorescence-based flow cytometry on renal cells from Vhlwt/pjr.fl; Pax8-CreERT2 mice untreated (top) or given 5 × 2 mg tamoxifen (TMX; bottom) and harvested at the early time point. Scale bar, 250 μm. Magnification, ×20. FACS gates are shown. C, Gel electrophoresis of genomic PCR for Vhlwt, Vhlpjr.fl, and Vhlpjr.KO alleles performed on FAC-sorted tdTomato-positive (left) or tdTomato-negative (right) cells from kidneys of Vhlwt/pjr.fl; Pax8-CreERT2 mice given tamoxifen and harvested at the early time point. D, Representative immunoblots (IB) for HIF1A, HIF2A, or tdTomato protein in tdTomato-negative (−) or tdTomato-positive (+) cells sorted by flow cytometry from dissociated kidneys of Vhljae.KO/pjr.fl or Vhlwt/pjr.fl Pax8-CreERT2 mice given 5 × 2 mg tamoxifen and harvested at the early time point (n = 3 per genotype).