DOI: 10.18710/uglrk4
title: Extended data for: The proteoglycan decorin does not influence adiposity, glucose tolerance or aerobic exercise capacity in mice

description: Supplementary figures to manuscript: The proteoglycan decorin does not influence adiposity, glucose tolerance or aerobic exercise capacity in mice. 

Supplementary figure 1: Dcn+/+ mice and Dcn-/- littermates were placed on a HFD for 35 weeks (n=12). 

A; mRNA expression in quadriceps muscle. For reverse transcriptase quantitative PCR (RT-qPCR), tissues were homogenized in TRI Reagent solution (Thermo Fisher) and phase-separated by adding chloroform. RNA was then isolated with a Nucleospin RNA extraction kit (Macherey-Nagel) according to the manufacturer’s instructions. cDNA was synthesized with a Tetro cDNA synthesis kit (Bioline). qPCR was performed using TaqMan reagents and predeveloped assays from Applied Biosystems. Raw data is found in "Raw data SFig1a.csv"; CT values in duplicates, as quantified in CFX manager (Bio-Rad).

B; Protein expression in gastrocnemius muscle measured with western blotting. Muscle tissue was homogenized in RIPA buffer and subjected to SDS-PAGE with criterion™ TGX™ gels (Bio-Rad). Protein transfer was done using the Trans-Blot Turbo transfer system and RTA transfer kit (Bio-Rad). For total protein measurements the membranes were either stained with Ponceau S, or visualized with TGX stain-free technology (Bio-Rad) for fluorescent detection of proteins. The membranes were then blocked in Tris buffered saline containing 0.1% Tween-20 and 5% BSA, and incubated over night with primary antibodies in 2.5% BSA in TBS-T. The following antibodies were used: Total OXPHOS rodent antibody cocktail (MitoScience LLC Cat# MS604, RRID:AB_2629281), glycogen synthase (GS; Cell Signaling Technology Cat# 3886, RRID:AB_2116392), phospho-glycogen synthase (pGS; Cell Signaling Technology Cat# 3891, RRID:AB_2116390), Ppargc1a (PGC1α; Santa Cruz Biotechnology Cat# sc-13067, RRID:AB_2166218), PI3 Kinase p85 (Millipore Cat# 06-497, RRID:AB_310141), Stat-3 (Cell Signaling Technology Cat# 4904, RRID:AB_331269) and phopho-Stat3 ((Cell Signaling Technology Cat# 9167, RRID:AB_561284)), Decorin (LF-114 antisera from Fisher et al(19)). For detection we used HRP-conjugated secondary antibodies (Jackson Immunoresearch) and the ChemiDoc™ Touch Imaging System (Bio-Rad). Quantification of band intensities was done in ImageJ and were normalised to loading control (ponceau stain or TGX stain-free fluorescent detection of proteins). For the OXPHOS proteins, the band detected slightly above 50 kDa was assumed to be complex 5 (ATP5A), complex 4 (MTC01) was assumed to be the band at around 40 kDa and complex 2 (SDHB) was detected at around 30 kDa. Raw data is found in "Raw data SFig1b.csv"; band intensities and loading control intensities, as quantified in ImageJ. 

Data in Supplementary Figure 1a and 1b are presented as fold change, Dcn-/- relative to Dcn+/+. Statistical testing was done with a student one-sample t-test. *p&lt;0.05, significant difference from 1. 

Supplementary Figure 2. Cross sectional view of the tibialis anterior muscle from female (A) and male (B) Dcn+/+ and Dcn-/- mice. Stained with Hematoxylin and eosin stain. Muscle tissue was fixed in 10% phosphate-buffered formalin, embedded in paraffin, sectioned and stained with hematoxylin and eosin for routine histology at the Garvan Histopathology Core Facility. Cross sections from whole muscles were scanned with a 5x lens on a Leica DM 6000 Power Mosaic microscope with a stepping stage for mosaic image acquisition. Fiber cross sectional area and Feret diameter was measured by manual annotation of 50-150 fibers per section, using ImageJ software (RRID:SCR_003070).
description: ImageJ
description: Bio-Rad CFX Manager
description: GraphPad Prism, 10