Moving Through Adolescence: Developmental Trajectories of African American and European American Youth. II: Method

<p>Heat map of TLR4-dependent gene expression changes induced by cotreatment with palmitate and mmLDL (A). J774 cells were stimulated with palmitate for 16h and incubated with or without mmLDL and LPS. Profiles of mRNA were determined by RNA-seq analysis (B-top). To validate RNA-seq results, independent real time PCRs were used to assess the expression of <i>Ccr5</i>, <i>Il-6</i>, <i>Csf-3</i>, <i>Il-1β</i>, and β–actin (B-bottom). The combination of palmitate and mmLDL caused an increase of mRNA expression of <i>Il-6</i>, <i>Csf-3</i>, and <i>Il-1β</i> genes (normalized to that of <i>Actb</i>). The real time PCR was conducted with technical duplicates, and the data shown represent three independent replicate experiments. *p <0.05 and **p <0.01 compared to LPS treatment without palmitate or mmLDL.</p

Expression of <i>lcat</i>, but not <i>cetp</i>, is significantly decreased in <i>apoc2</i> mutant zebrafish.

<p>In situ hybridization (A) and qPCR (B) results showing <i>lcat</i> and <i>cetp</i> mRNA expression in 5.3 dpf zebrafish embryos. mRNA expression of <i>apoc2</i>, <i>lcat</i> and <i>cetp</i> (C) and <i>apoa1</i>, <i>apoe</i>, <i>apob</i> and <i>mtp</i> (D) in adult zebrafish liver. Results are mean±s.e.m.; numbers of biological replicates are indicated on the graphs; *P<0.05, **P<0.01 and ***P<0.001 (Student’s t-test).</p

Patients with familial chylomicronemia syndrome (FCS) have disproportional FC and CE levels in plasma.

<p>TG levels (A), cholesterol levels (B) and the FC/CE ratio (C) in plasma of healthy subjects (Ctrl), in FCS patients’ whole plasma (FCS-W), and in chylomicron-depleted FCS plasma (FCS-CD). (D) A linear regression analysis of the FC/CE ratio and plasma TG levels (n = 4 in control group and n = 5 in FCS-W and FCS-CD groups). (E) LCAT activity in healthy subjects’ and FCS-CD plasma (n = 5 in control group and n = 6 in FCS-CD group). Results are mean±s.e.m.; *P<0.05, **P<0.01 and ***P<0.001 (Student’s t-test).</p

Reducing hypertriglyceridemia does not correct the FC/CE ratio.

<p>Plasma TG levels (A), cholesterol levels (B) and the FC/CE ratio (C) in WT, <i>apoc2</i> mutants and the <i>apoc2</i> mutants fed a low fat diet (LFD). Results are mean±s.e.m.; n = 3 in each group; *P<0.05, ***P<0.001 (Student’s t-test).</p

Reduced Syk expression in macrophages from Syk^flox/flox/LysM-Cre mice.

<p>(<b>A</b>) Total RNA was isolated from BMDM of Syk^flox/flox/LysM-Cre(−) (WT) and Syk^flox/flox/LysM-Cre(+) (Syk^−/−) mice, and RT-PCR with Syk and GAPDH primers was performed. (<b>B</b>) Lysates of resident peritoneal macrophages (PM) and BMDM were run on SDS-PAGE, transferred to a membrane and immunoblotted with anti-Syk and anti-GAPDH antibodies. (<b>C</b>) WT and Syk^−/− BMDM were incubated with 50 µg/ml mmLDL (to induce macropinocytosis) and 200 µg/ml of native LDL (lipid carrier) for 40 hours. The cells were stained for neutral lipid with Oil Red O.</p

c-Jun and c-Fos DNA binding in WT and Syk^−/− macrophages stimulated with mmLDL.

<p>BMDM from WT or Syk^−/− mice (<b>A</b> and <b>B</b>) and J774 expressing control of Syk-specific shRNA (<b>C</b> and <b>D</b>) were incubated for 1 hour with media or 50 µg/ml mmLDL. Nuclear extracts were isolated and used in a transcription factor DNA binding plate-based assay. Mean ± SEM (n = 4). *, p<0.05; **, p<0.005 WT vs. Syk^−/−.</p

AP-1 activation in Syk-expressing CHO cells.

<p>(<b>A</b>) CHO cells were transfected with 1 µg Flag-Syk (or empty vector), 200 ng AP-1-Luc reporter, and 200 ng β-galactosidase plasmid. After 36 hours, cells were incubated with media or mmLDL for 6 hours and luciferase activity was measured and normalized to β-galactosidase activity. Mean±SEM (n = 3). *, p<0.05 vector vs. Syk, and media vs. mmLDL. (<b>B</b>) CHO Cells were transfected with 300 ng empty vector or Flag-Syk. After 24 hours, cells were incubated with mmLDL for 6 hours and cell lysates were tested for expression of Syk (Flag) and for p-JNK, p-c-Jun and GAPDH.</p

Secretion of CXCL2 (MIP-2) and IL-6 by WT and Syk^−/− macrophages stimulated with mmLDL.

<p>BMDM from WT or Syk^−/− mice (<b>A</b> and <b>B</b>) and resident peritoneal macrophages from Syk^flox/flox mice infected with adenovirus expressing GFP or Cre (for 48 hours at 500 MOI) (<b>C</b> and <b>D</b>) were incubated for 6 hours with media or 50 µg/ml mmLDL. Cell culture media were collected and CXCL2 and IL-6 protein levels were measured by ELISA. Mean ± SEM (n = 3). *, p<0.05; ****, p<0.00005 WT vs. Syk^−/−.</p

Mouse strain characteristics.

<p>Data are averages±standard error. 7–10 mice per group. * p<0.05 vs diet-matched WT, # p<0.05 vs strain-matched NC. ns - not significant.</p

Proinflammatory chemokine production by 15LO-expressing cells.

<p>A. Fold difference in OPN and MCP-1 mRNA expression in murine fibroblasts constitutively expressing human 15LO or LacZ. OPN and MCP-1 mRNA levels were measured by qPCR of total RNA and were normalized to GAPDH mRNA. B. Levels of MCP-1 protein in conditioned media from 15LO and LacZ cells, as assayed by ELISA. C. Autoradiograph of OPN protein in conditioned media from 15LO and LacZ cells, as determined by western blotting.</p