br Formoterol administration and autophagy flux br C bearing
Formoterol administration and autophagy flux
C26-bearing mice were subdivided in two groups, intraperitoneally treated daily either with saline or with formoterol at 1 μg/g of body weight, starting the day after C26 cell injection until day 14 of tumor growth. For the subsequent in vivo autophagy flux experiment, the mice from each group were further subdivided into two and administered either 0.4 μg/g/day of colchicine or saline for 2 days before sacrifice (from day 10 to day 12 of tumor growth in order to avoid animal death in end-stage cachectic mice).
Body composition and grip strength
Body composition was evaluated in living awake, conscious (restrained but not anesthetized) animals at given time points using a magnetic resonance whole-body composition analyzer (EchoMRI, ). Lean mass, fat mass and total water mass were determined based on radio pulse emission properties able to differentiate between distinct tissue types.
Mouse skeletal muscle strength was assessed by the grip-strength test using a Panlab/Harvard Apparatus device . At least three measurements were taken per mouse on both baseline and test days, and the results were averaged for analysis.
Human skeletal muscle biopsies
The cDNA used for TP53INP2 analysis in the rectus abdominis muscle from control and cancer (either cachectic or not) patients was obtained from a
previous study performed by our group . Patient characteristics are reported in the above-mentioned study; they were considered cachectic according to the international consensus definition on cancer cachexia (; body weight loss N 5% in the previous 6 months).
Protein extraction and Western blotting r> Total protein extracts were obtained by homoge-nization in RIPA buffer followed by clarification through centrifugation at 15,000g. Mitochondrial-enriched fractions were obtained by homogenization in 250 mM sucrose, 50 mM KCl, 5 mM EDTA, 5 mM MgCl2 and 1 mM sodium pyrophosphate (pH 7.4) followed by two centrifugation steps at 740g in order to remove the unsoluble fraction. The mitochondria were pelleted by two centrifugation steps at 10,000g, and the supernatant was collected as cytoplasmic fraction. Protein concentration was quantified using the bicinchoninic Methylpiperidino pyrazole (BCA) method (Thermo Scientific Pierce) with bovine serum albumin as standard. The samples were resolved in 10%, 12.5% or 15% acrylamide gels for SDS-PAGE and were then transferred onto PVDF Transfer Membranes (Millipore) at 350 mA for 1 h 30 min on ice. The following antibodies were used: Beclin-1, LC3B, p62 (Sigma) or SDHa (Santa Cruz). GAPDH or α-tubulin
(Sigma) antibodies were used as a loading control. Proteins were detected by the enhanced chemilu-minescence method and quantified by scanning densitometry.
Ten-micrometer-thick transverse frozen sections of the TA muscle were cut and stained with hematoxylin and eosin. Digital images were obtained for measuring myofiber CSA counting ∼300–400 fibers per muscle section using the Image J software (http://rsb.info.nih.gov/ij/). For immunofluorescence, transverse sections were fixed in 4% paraformalde-hyde and probed with the primary anti p62 (BD Bioscience), BNIP3 (Abcam) or SDHa (Santa Cruz) antibodies. Detection was performed using Alexa Fluor 555- or 488-conjugated secondary antibodies (Invitrogen). Nuclei were stained with the Hoechst 33342 fluorochrome and the images captured using an epiilluminated fluorescence microscope (Axiovert 35; Zeiss) or a confocal laser scanning microscope (LSM 800; Zeiss).
Gene expression analysis
Following the supplier's instruction, RNA from muscle was extracted using a protocol combining TRIzol reagent (Invitrogen) and RNAeasy® minikit columns (Qiagen), including DNAse I digestion to avoid potential contaminations of DNA. One
2684 Autophagy and mitochondria in cancer cachexia
micrograms of RNA was reverse-transcribed into cDNA with the SuperScript RTIII kit (Invitrogen). The expression of target genes of interest was quantified by quantitative real-time PCR (RT-PCR) and was per-formed using the ABI Prism 7900 HT real-time PCR device (Applied Biosystems) and the SYBR® Green PCR Master Mix or the Taqman Probes 20X (Applied Biosystems). Primers used for amplification are listed below. All measurements were normalized to the expression of housekeeping genes ARP, β- actin and GAPDH.
Symbol Forward Reverse
AAGCGCGTCCT CCGCAGGGGCA ARP GGCATTGTCT GCAGTGGT
GCAAACACTGCC CTTGAGGGGAAA Atrogin1 ACATTCTCTC GTGAGACG
β-Actin GGCAACGA GTCAGCAATGCCTGG
CACCTTTATCACTC GATTTTGTTTTTCA Bnip3 TGCTGAATTCTCT TTTCCAGTCTTTTAA
GCAGCAAGAACC GTTGTCCCGGTCTTT Cathepsin L TCGACCAT GGCTA
AGCCAATCCATCTC ATCGCCTACAGGTAC DRP-1 AAGGTTTTCTC TTTGGTCATTC