SUPPLEMENTAL FIGURE LEGENDS

Transcription

1 SUPPLEMENTAL FIGURE LEGENDS Suppl. Fig. 1. Notch and myostatin expression is unaffected in the absence of p65 during postnatal development. A & B. Myostatin and Notch-1 expression levels were determined by semi-quantitative PCR analysis by isolating mrna from the skeletal muscles of P8 p65 +/+ and mice. Suppl. Fig. 2. IκBα-SR expression in muscles inhibits NF-κB activity. A. Whole cell lysates were prepared from P8 Ad-GFP and Ad-IκBα-SR skeletal muscle in homogenizing buffer followed by Western blot analysis for IκBα. Expression of the IκBα-SR protein is denoted by an asterisk. B. Whole cell extracts were isolated from the skeletal muscles of P8 Ad-GFP and Ad-IκBα-SR infected 3xκB-Luc- Tg mice and NF-κB activity was measured by luciferase assays and normalized to total protein. Suppl. Fig. 3. NF-κB activity in P8 postnatal muscle is localized to non muscle cells. A & B. Immunofluorescent staining for p-p65 (green, A & B), Pax7 (red, A), and MyoD (red, B) was performed on hind limb muscle cross-sections from P8 wild type mice. Dotted outlines denote the relative positions of the myofibers as assessed from overexposed fluorescent images and phase contrast microscopy. Scale bar = 5µm. C. Mononuclear cells were isolated from P8 NF-κB +/EGFP neonates and FACS sorting was performed. The GFP + and GFP - gates are indicative of cells which were collected and used for further analysis. Suppl. Fig. 4. NF-κB activity derived from fibroblasts is dispensable for C2C12 myotube formation. Primary p65 +/+ and fibroblasts were co-cultured for 24 hours with C2C12 myoblasts, and subsequently replaced with differentiation medium. After 3 days cells were fixed and stained with MyHC. Myotube numbers per field were determined by taking the average number of MyHC positive myotubes from 10 random fields from two independent experiments. Suppl. Fig. 5. expression can be efficiently silenced or induced by the transfection of sirna or a CMV expression plasmid, respectively in MEFs. A & B. Wild type MEFs were transfected with either control or sirna and probed for expression by semi-quantitative PCR (A) or Western blot analysis (B). C & D. Similar analysis was performed as above with respect to transfections of wild type MEFs with control or CMV driven expression plasmids. E &F. MEFs were transfected with either a control or an CMV driven expression plasmid and cells were subsequently probed for by semi-quantitative PCR (E) or Western blot analysis (F). Suppl. Fig. 6. Loss of expression has no effect on total myofiber count in early postnatal muscle. Hind limb cross-sections from P8 +/+ and -/- mice (n=3) were stained with H&E and subsequently analyzed for total fiber numbers as described in the Materials and Methods section. Suppl. Fig. 7. Alternative NF-κB signaling is activated late during postnatal muscle development. Whole cell lysates were prepared from the skeletal muscles of wild type P5 to P21 mice. Western blot analysis was performed probing for p100 processing to p52. 13

2 Supplemental Figure 1 A P8 B P8 p65 +/+ p65 +/+ myostatin Notch-1

3 Supplemental Figure 2 A IκBα Ad-GFP Ad-IκBα-SR * B Relative Light Units p<0.05 * Ad-GFP Ad-IκBα-SR

4 Supplemental Figure 3 A B C p-p65/myod/dapi p-p65/pax7/dapi P8 P8

5 Supplemental Figure 4 Myotubes Per Field p65 +/+ C2C12

6 Supplemental Figure 5 A -sirna Control-siRNA B -sirna Control-siRNA RNA Protein C CMV-Control CMV- D CMV-Control CMV- RNA Protein E F CMV-Control CMV- CMV-Control CMV- RNA Protein

7 Supplemental Figure 6 Fiber Number /+ -/-

8 Supplemental Figure 7 p100 p5 p8 p11 p15 p21 p52