Additional file 1: of Physical activity and FTO genotype by physical activity interactive influences on obesity

Intake questionnaire. (DOC 51 kb

MOESM1 of Complete genome sequence of Clostridium perfringens CBA7123 isolated from a faecal sample from Korea

Additional file 1: Figure S1. A photomicrograph of Clostridium perfringens strain CBA7123 using Variable Pressure Field Emission Scanning Electron Microscope (VP-FE-SEM). Figure S2. Comparison of genomic structure between Clostridium perfringens CBA7123 and strains FORC 003, FORC 025, JP55, and JP838, using a progressive alignment algorithm in Mauve. The locally collinear blocks with identical colors represent highly homologous regions. The genomes were figured based on scale of the genome of strain CBA7123

Additional file 1: Table S1. of Next-generation sequencing methylation profiling of subjects with obesity identifies novel gene changes

The 13,130 differentially methylated cytosines (DMCs; P < 0.05) that were located within a promoter or untranslated regions (3' and 5'). (XLSX 2880 kb

Additional file 3: Table S3. of Next-generation sequencing methylation profiling of subjects with obesity identifies novel gene changes

The 170 differentially methylated regions (DMRs; P < 0.05) that were located within a promoter or untranslated regions (3' and 5'). (XLSX 35580 kb

Characterization of HSPB1 intramolecular cleavage.

<p><b>A</b> HSPB1 cleavage sites and a schematic representation of the resulting fragments. The amino acid sequence of the HSPB1 fragment from a Coomassie blue stained gel was analyzed by MALDI-TOF and LC mass spectrometry. The N-terminus or C-terminus HSPB1 fragments were detected by western blot analysis using N-terminal or C-terminal HSPB1 antibodies, respectively. <b>B</b> A schematic representation of the soluble vector for HSPB1. HSPB1 was tagged with an N-terminal 3XFlag and a C-terminal Myc tag. For secretion of HSPB1, the preprotrypsin leader sequence (PPTLS) precedes the Flag sequence; these vectors contain an additional C-terminal His tag. COS-7 cell lines stably transfected with control vector; PPTLS, secretory wild-type HSPB1; sHSPB1WT, N-terminal-deleted secretory HSPB1 (59–203); sHSPB1 (59–203), or possible cleavage sequence-deleted secretory HSPB1 (Δ54–63); sHSPB1 (Δ54–63) were cultured in OPTI-MEM media (1% FBS). Secretion of N-terminal Flag or C-terminal Myc tagged protein was detected by western blot analysis of the lysates and culture supernatants (bottom). <b>C</b> Recombinant protein (wild-type or mutant HSPB1) was incubated for 30 min with MMP9 (2 µg/mL) and HSPB1 digestion was examined by western blotting using the Flag antibody. <b>D</b> Recombinant protein of sHSPB1 (Δ54–63) was incubated for 30 min with MMP2, 3 and 9 (2 µg/mL) and HSPB1 digestion was examined by western blotting using the Flag antibody.</p

Effect of HSPB1 cleavage on tumor progression.

<p><b>A</b> B16F10 melanoma cell lines were stably transfected with PPTLS, sHSPB1WT, sHSPB1 (59–203), and HSPB1WT. Cell lysates and conditioned media of two stable lines (1,2) were repectively subjected to western blotting (top). <b>B</b> Effects of conditioned media of B16F10 stable cell lines on VEGF₁₆₅-induced VEGFR2 activation and proliferation activity in HUVECs. <b>C</b> Mean number of metastases at 14 days after intravenous injection of B6F10 stable cells into wild-type mice. The graph shows the mean ± SEM from three independent experiments of six mice per group f (*<i>P</i> <0.05 and **<i>P</i> <0.01 vs PPTLS). Right panel shows representative images. <b>D</b> Intratumoral microvessel density (MVD) per high-powered field (h.p.f.) was quantified by CD31 immunofluorescence. (*<i>P</i> <0.05 and **<i>P</i> <0.01 vs PPTLS). <b>E</b> Mean weight of liver with tumor at 14 days after intrasplenic injection of B6F10 stable cells into wild-type mice.</p

HSPB1 is cleaved by MMPs.

<p><b>A</b> HUVECs were treated for 24(2 µM). Cleaved HSPB1 levels in conditioned media (C.M.) were detected by western blot analysis using an HSPB1 antibody to amino acids 158-205. <b>B</b> Recombinant HSPB1 (2 µg/mL) was incubated with ADAMTS1, MMP3, or MMP9 (50 ng/mL) for the indicated times. HSP70 (2 µg/mL) was incubated with MMP9 as a negative control. HSPB1 cleavage was detected by immunoblot using the HSPB1 antibody to amino acids 158-205 as well as Coomassie staining. <b>C</b> Recombinant HSPB1 (2 µg/mL) was incubated with MMP9 (50 ng/mL) for the times indicated and analyzed by western blot using antibodies against the C- (158-205) and N-termini (10–21) of HSPB1.</p

Soluble HSPB1 is secreted by tumor endothelial cells.

<p><b>A</b> Co-localization of HSBP1 and CD31 on tumor vessels in soft tissue sarcomas and lung adenocarcinomas derived from Kras<i>^LSL-G12D/WT</i>; p53<i>^Flox/Flox</i> mice was detected by immunofluorescence. <b>B</b> Tumor cells and tumor endothelial cells (ECs) were isolated from sarcomas and lung adenocarcinomas of Kras<i>^LSL-G12D/WT</i>; p53<i>^Flox/Flox</i> mice and cell lysates were analyzed by western blot. Secreted HSPB1 was detected in conditioned media (C.M.) from these cells. arrows, full length HSPB1; open arrows, cleaved HSPB1 <b>C</b> Serum HSPB1 levels in Kras<i>^LSL-G12D/WT</i>; p53<i>^Flox/Flox</i> mice with or without tumors were measured by ELISA assays for detecting intact HSPB1 as described in Methods; blue bars, anti-HSPB1 (10-21)+anti-HSPB1 (158-205) antibody. Red and green bars obtained from general ELISA assays using antibodies to the N- (10-21) or C-termini (158-201) of HSPB1 (*<i>P</i><0.05 and **<i>P</i><0.01 vs intact HSPB1).</p

Effect of MMP9-induced HSPB1 cleavage on VEGF-mediated endothelial cell activation.

<p><b>A</b> HUVECs infected with MMP9 shRNA lentiviral and control vectors were pretreated for 30(1ug/ml) or rHSPB1 fragments (59–203; 1 µg/ml) and then incubated with VEGF (30 ng/mL). After 10 min, cell lysates and conditioned media were subjected to western blotting using indicated antibodies. Bar graph shows ± SEM of band intensities of P-VEGFR2 signals normalized to P-VEGFR2 signals of control for 3 independent experiments. <b>B</b> After 48 hr, proliferation of HUVECs was analyzed by MTT assay. <b>C</b> Transendothelial migration of HOS cells in the presence of rHSPB1 WT (1ug/ml) or rHSPB1 fragments (59–203; 1 µg/ml). After HOS cells were marked with CytoTracker, they were allowed to attach and migrate to the HUVEC monolayer. Fluorescence graph of migratory cells labeled with CytoTracker shows ± SEM for three independent experiments (*<i>P</i> <0.05 and **<i>P</i> <0.01, upper graph). HUVECs infected with MMP9 shRNA lentiviral and control vectors were subjected to tumor transendothelial migration assay in the presence of rHSPB1 WT (1ug/ml) (bottom graph). <b>D</b> HUVECs infected with MMP9 shRNA lentiviral and control vectors were transfected with sHSPB1WT or sHSPB1 fragments (1–58, 59–203) and then activated with VEGF (30 ng/mL). After 10 min, cell lysates and conditioned media were subjected to western blotting. Bar graph shows ± SEM of band intensities of P-VEGFR2 signals normalized to P-VEGFR2 signals of control for 3 independent experiments. <b>E</b> After 48 hr, proliferation of HUVECs was analyzed by MTT assay.</p