amplify the conditional allele (2-lox) and recombined allele (1-lox) following tamoxifen

Transcription

1 Supplementary data Supplementary Table 1. Real-time PCR primer sequences Supplementary Fig. 1. (A) Genomic map of the Vhl (2-lox) conditional allele. Numbered boxes represent exon 1, which is targeted for excision. The locations of loxp sites and primers used to amplify the conditional allele (2-lox) and recombined allele (1-lox) following tamoxifen treatment are shown with red triangles and black arrows, respectively. (B) Genomic PCR analysis of DNA isolated from the kidney (K), Lung (Lu), and Liver (Li) of control (Cre - ) and Ubc-Vhl -/- mice following tamoxifen treatment. (C) Immunoblot analysis was performed for Hif-1α and Hif-2α using nuclear extracts isolated from Cre - and Ubc-Vhl -/- kidneys according to published protocols (15). (D) Immunohistochemical analysis of Hif-1α expression in Cre - and Ubc-Vhl -/- kidneys (NB ; Novus, Littleton, CO). Black arrows point to epithelial cells, red arrows indicate glomerular cells, and double-headed arrows point to interstitial cells with positive nuclear staining. Asterisks depict dilated tubules. (E) qrt-pcr analysis of Pgk-1, Ldh, Epo, and Ngal mrna levels in CTL and UUO kidneys 8 days after ligation. Shown are relative expression values normalized to 18S rrna. Data points represent individual mice. Error bars represent mean ± SEM; p<0.05, p<0.01. (F) Shown are representative images of paraffinembedded tissue sections from wild type kidneys treated either with or without tamoxifen, and stained for collagen with Sirius red (original magnification x200) and for macrophages with F4/80 immunohistochemsitry (original magnification x200). Supplementary Fig. 2. (A) Genomic map of the Hif-1α and Hif-2α (2-lox) conditional allele. Numbered boxes represent exon 2, which is targeted for excision. The locations of loxp sites and primers used to amplify the conditional allele (2-lox) and recombined allele (1-lox) following

2 Kobayashi et al. tamoxifen treatment are shown with red triangles and black arrows, respectively. (B) Genomic PCR analysis of DNA isolated from the kidney (K), Lung (Lu), and Liver (Li) of control (Cre - ) and Ubc-Hif -/- mice following tamoxifen treatment. (C) qrt-pcr analysis of Pgk-1 and Epo in CTL and 8-day post UUO kidneys from control (Cre - ) and Ubc-Hif -/- mice. Shown are relative expression values normalized to 18S rrna. Data points represent individual kidneys. Error bars represent mean ± SEM; p<0.01. Supplementary Fig. 3. (A) qrt-pcr for Hif-1a exon 2/exon 8 (Hif exon 2 is flanked by loxpsites and excised upon Cre-mediated recombination) was performed in CD11b + cells isolated from Cre - littermate control and LysM-Hif -/- UUO kidneys to determine recombination efficiency. Shown are representative figure. (B) qrt-pcr analysis of Vegfa in M/M isolated from LysM-Vhl -/- or LysM-Hif -/-. M/M isolated from Cre - littermates served as control. Shown are relative expression values normalized to 18S rrna. Error bars represent mean ± SEM; p<0.05, p<

3 Kobayashi et al., Supplementary Table 1 Supplementary Table 1: Real-time PCR primer sequences. Gene Primer Sequence (5 3 ) Kim-1 (F) Kim-1 (R) Ngal (F) Ngal (R) Ldh (F) Ldh (R) Ccl2 (F) Ccl2 (R) Ccl5 (F) Ccl5 (R) Ccr2 (F) Ccr2 (R) Ccr5 (F) Ccr5 (R) AAACCAGAGATTCCCACACG GTCGTGGGTCTTCCTGTAGC CAAGCAATACTTCAAAATTACCCTGTA GCAAAGCGGGTGAAACGTT CGCCCCCCCATCGT GTTGCCATCTTGGACTTTGAATC CCCAATGAGTAGGCTGGAGA TCTGGACCCATTCCTTCTTG CCTCACCATCATCCTCACTG GAGGGAGAGGTAGGCAAAGC ATGAGTAACTGTGTGATTGACAAGCA GCAGCAGTGTGTCATTCCAAGA CAGGGCTGTGAGGCTCATCT GGCAGCAGTGTGTCATTCCA

4 Kobayashi et al., Fig. S1 A. N N 1 N Vhl 2-lox Vhl 1-lox Cre-mediated recombination B. Cre- Vhl-/- K Lu Li K Lu Li Vhl 2-lox 1-lox C. Cre- Vhl-/- Cre- Vhl-/- Hif-1α Hif-2α n.s. Ponceau Ponceau D. Vhl-/- Cre- Vhl-/- Hif-1α E. Pgk-1 Ldh Epo Ngal mrna/18s F. Sirius Red Vehicle TAM F4/80 Vehicle TAM

5 Kobayashi et al., Fig. S2 A. B. E P 2 P Hif1a 2-lox Cre-mediated recombination Hif1a 1-lox Cre- Hif-/- K Lu Li K Lu Li Hif1a 1-lox 2-lox E 2 Hif2a 2-lox Cre- Cre+ K Lu Li K Lu Li Hif2a Cre-mediated recombination Hif2a 1-lox 2-lox 1-lox C. Pgk-1 Epo mrna/18s

6 Kobayashi et al., Fig. S3 A. Hif1a Ex2/Ex8 Cre - LysM Hif-/- B. Vegfa Vegfa mrna/18s Cre - LysM Vhl-/- Normoxia Cre - LysM Hif-/- Cre - LysM Hif-/- Normoxia Hypoxia