Additional file 1: Figure S1. of Che-1 sustains hypoxic response of colorectal cancer cells by affecting Hif-1Îą stabilization

Che-1 is involved in the metabolic switch in response to hypoxia. A- and B- HT29 (A) and A549 (B) cells were transiently transfected with stealth siRNA negative control (siControl) or siRNA Che-1 (siChe-1) and exposed to hypoxia for 16 h where indicated and pH was measured. C- and D- Score plots indicating metabolic differences between hypoxic and normoxic samples from HT29 (C) and A549 (D) cells transiently transfected as in A. E- HT29 cells were transfected as in A and the medium lactate content was evaluated. (TIF 5841 kb

Additional file 2: Figure S2. of Che-1 sustains hypoxic response of colorectal cancer cells by affecting Hif-1α stabilization

Che-1 regulates genes transcription in response to hypoxia. A- Quantitative RT–PCR (qRT–PCR) for metabolic genes expression was performed in HT29 cells transiently transfected with Stealth siRNA negative control (siControl) or siRNA Che-1 (siChe-1) and exposed to hypoxia for 4 h. Values were normalized to RPL19 mRNA expression. Error bars represent the standard error of three different experiments. *P = 0,0010, **P ≤ 0,0003, ***P ≤ 0,004, n.s., not significant. (TIF 1835 kb

Muscle specific expression of Vp16-Jazz in transgenic mice.

<p>A: Western Blot analysis of total proteins extracted from the skeletal muscle of wild type (wt) and transgenic mice derived from two different founders (tg9 and tg41). The expression of Vp16-Jazz transgene was monitored by the anti-myc monoclonal 9E10 antibody. Detection of α−tubulin was used to normalize the amount of proteins. B: Western Blot analysis of total proteins extracted from the skeletal muscle, heart and brain of transgenic mice from families tg9 and tg41.</p

Vp16-Jazz and its DNA target sequence.

<p>A: Schematic representation of the Vp16-Jazz gene in the pMex-vector, used to generate transgenic mice. The 9 base pair long Vp16-Jazz DNA target sequence is indicated. B: The nucleotide sequence of the mouse utrophin promoter A. The Vp16-Jazz DNA target sequence is indicated in bold characters and underlined. The main transcription factor binding sites present in this promoter region are indicated.</p

Vp16-Jazz and utrophin up-regulation.

<p>A: Vp16-Jazz chromatin immunoprecipitation, performed in skeletal muscle derived from wt mice and transgenic mice (family tg9) using myc monoclonal antibody/protein G-agarose beads or protein G-agarose beads as a control (no-Ab). Immunoprecipitates from each sample were analyzed for the presence of utrophin promoter by PCR. A sample representing linear amplification of the total input chromatin (input) was included (lane 1). As control, samples from transgenic mice were also tested for the presence of dystrophin promoter sequence. B: Real-time PCR analysis of the utrophin gene expression rate in Vp16-Jazz transgenic mice (tg9 and tg41) and control wt mice. The gene expression ratio between utrophin and β-glucoronidase (GUS) and β2-microglobulin (β2M) is shown as means±S.D. from three independent experiments performed in triplicate. C: Western blot of total protein extracts derived from skeletal muscle and heart from wt mice and Vp16-Jazz transgenic mice (tg9 and tg41) incubated with monoclonal antibody against utrophin. The same membrane was incubated with anti-α-tubulin monoclonal antibody for loading normalization. D: Relative utrophin expression of wt and transgenic mice (tg9 and tg41) was determined by densitometric analysis. E: Total protein extracts from skeletal muscle of wt and transgenic mice (tg9 and tg41) were subjected to immunoblotting to detect the expression levels of the dystrophin and α-sarcoglycan proteins. The anti-α-tubulin and anti-myc antibodies were used to normalize the protein content and to test the Vp16-Jazz transgene expression respectively.</p

Effects of Vp16-Jazz in transgenic mice.

<p>Immunohistochemistry of Tibialis Anterior (TA) muscle derived from wt (panel A) and transgenic mice tg9 (panel B) stained with anti-utrophin antibody. Nuclei are counterstained with Hoechst 33258. C: TA from wt and transgenic mice tg9 were co-stained with anti-utrophin antibody and the α-bungarotoxin-Alexa Fluor to visualize the acetylcholine receptor (AChR) at the neuromuscular junctions. The anti-utrophin monoclonal antibody reveals an extra-synaptic distribution of utrophin only in transgenic mice. D: Relationship between contractile response (g) and stimulation frequency (Volts) in diaphragm and EDL muscle preparations obtained from wt, tg9 and tg41 mice. E: Scatter plots of the DEG with natural log transformed expression values averaged over the 4 replicates. The x–axis reports the mean of the replicates of transgenic mice, while the y-axis is the mean across the replicates of the control wt mice. Expression values are colour coded with red representing up-regulated genes in tg9/tg41 and green down-regulated genes compared to wild-type and lines on the graph set at 2-folds differential expression.</p