Fig 1: CC-induced CD36 expression, oxLDL uptake and foam cell formation require STAT1 interaction with PPARγ. A. Quiescent cells were treated with vehicle or CC (40 μg/ml) in the presence and absence of GW9662 (5 μM) and analyzed for CD36 expression, oxLDL uptake or foam cell formation as described in Fig. 1, panel C. B. Upper panel: Quiescent cells were treated with and without CC in the presence and absence of GW9662 for 1 h and analyzed for STAT1 acetylation as described in Fig. 1, panel F. Middle and bottom panels: Equal amounts of protein from control and the indicated time periods of CC-treated cells were immunoprecipitated with anti-STAT1 or anti-PPARγ antibodies or IgG and the immunocomplexes were analyzed by Western blotting for the indicated proteins using their specific antibodies and normalized for STAT1 or PPARγ. C. Cells were transfected with vector, PFS1YF or K410R/K413R, quiesced, treated with and without CC for 1 h and equal amounts of protein from control and each treatment were immunoprecipitated with anti-STAT1 antibodies and the immunocomplexes were analyzed by Western blotting for PPARγ. The blots were reprobed for STAT1 over expression. The bar graphs represent Mean±S.D. of three experiments *p<0.01 vs vehicle control or vector control; **p<0.01 vs CC or vector control+CC.
Fig 2: STAT1, PPARγ and p300 mediate CC-induced CD36 promoter activity. A. Cells were transfected with vector, pGL3-hCD36 or pGL3-hCD36m (STAT-binding site at −107 nt was mutated), growth-arrested, treated with and without CC (40 μg/ml) for 6 h and the luciferase activity was measured. B, C and E. After transfection with vector or pGL3-hCD36, cells were quiesced, treated with vehicle or CC in the presence and absence of Apocyanin (100 μM), DPI (10 μM), Allopurinol (100 μM), PCI32765 (10 μM) or GW9662 (5 μM) for 6 h and the luciferase activity was measured. D and F. Cells were co-transfected with vector or pGL3-hCD36 in combination with pcDNA3.1, PFS1YF, K410R/K413R, p300WT or p300ΔHAT, quiesced, treated with vehicle or CC for 6 h and the luciferase activity was measured. *p<0.01 vs vector; **p<0.01 vs vector+CC or STAT1+CC or p300WT+CC. RLU, relative luciferase units.
Fig 3: CC-induced CD36 expression, oxLDL uptake and foam cell formation require STAT1 acetylation. A. Quiescent cells were treated with vehicle or CC (40 µg/ml) for the indicated time periods and either protein extracts were prepared or RNA was isolated. The protein extracts and RNA were analyzed by Western blotting and RT-PCR for the indicated scavenger receptors expression and normalized to β-tubulin protein and β-actin mRNA levels, respectively. B. Cells were treated with vehicle or CC (40 µg/ml) for 6 h and tested for their cytotoxicity and proliferation by LDH release and MTT assays, respectively. C. Upper panel: Cells were transfected with the indicated ASO, quiesced, treated with vehicle or CC for 4 h and analyzed by Western blotting for CD36 levels and the blot was reprobed for β-tubulin to show the effects of the ASO on its target and off target molecules levels. Middle and bottom panels: All the conditions were the same as in the upper panel except that cells were subjected to CC-induced oxLDL uptake (middle panel) or foam cell formation (bottom panel) assays. D. Equal amounts of protein from control and various time periods of CC-treated cells were analyzed by Western blotting for pSTAT1, pSTAT2, pSTAT3, pSTAT4, pSTAT5 and pSTAT6 levels and normalized to their total levels. E. Upper panel: Cells were transfected with vector or PFS1YF, quiesced, treated with and without CC for 1 h or 4 h, cell extracts were prepared and analyzed by Western blotting for pSTAT1 (1 h samples) and CD36 levels (4 h samples) and the blots were reprobed for STAT1 over expression and β-tubulin normalization. Middle and bottom panels: All the conditions were the same as in the upper panel except that after quiescence cells were subjected to CC-induced oxLDL uptake (middle panel) and foam cell formation (bottom panel) assays. F. Cells were transfected with control or STAT1 ASO, quiesced, treated with and without CC and analyzed for CD36 expression, oxLDL uptake or foam cell formation as described in panel C. The CD36 blot was reprobed for STAT1 and β-tubulin to show the effect of the ASO on its target and off target molecules levels. G. Upper panel: Equal amounts of proteins from control and the indicated time periods of CC-treated cells were immunoprecipitated with anti-STAT1 antibodies or IgG and the immunocomplexes were analyzed by Western blotting using anti-acetyl lysine (Ac-Lys) antibodies followed by normalization to STAT1. Lower panels: Cells were transfected with vector, K410R/K413R or PFS1YF, quiesced, treated with and without CC for 1 h and analyzed for STAT1 acetylation and phosphorylation as described in the upper panel and panel C, respectively and the blots were reprobed for STAT1 levels. H. Cells were transfected with vector or K410R/K413R, quiesced, treated with and without CC and analyzed for CD36 expression, oxLDL uptake or foam cell formation as described in panel C. The bar graphs represent Mean±S.D. values of three experiments. *p<0.01 vs vehicle control or vector control or control ASO; **p<0.01 vs CC or control ASO+CC or vector control+CC.
Fig 4: CC induces ROS production, BTK activation, p300-STAT1-PPARγ interactions, CD36 expression and foam cell formation in mouse primary peritoneal macrophages. A–C. Mouse primary peritoneal macrophages were isolated from WT mice, quiesced overnight, treated with and without CC (40 μg/ml) for the indicated time periods and either ROS production was measured or cell extracts were prepared and analyzed for BTK and p300 tyrosine phosphorylation, p300 association with STAT1, STAT1 acetylation and its association with PPARγ and CD36 expression as described in Figure legends 5A, 4B, 4A, 3A, 1F, 2B and IA, respectively. D and E. Mouse primary peritoneal macrophages after overnight growth arrest were treated with and without CC (40 μg/ml) for 6 h and analyzed for oxLDL uptake and foam cell formation as described in Figure legend 1 C. *p<0.01 vs control.
Fig 5: Gliadin-induced phopho-STAT3 protein (P-STAT3) expression in the PBMCs of healthy controls and patients with NCGS. The figure shows representative figures of (a) total protein staining (fluorescence) of PBMC lysates separated on 4–12% TGX Stain-Free™ protein gel after transfer to nitrocellulose, and (b) Western blot for P-STAT3 in the PBMC of patients with NCGS and healthy controls before and after 24 h stimulation with gliadins. Two biological replicates were performed for n = 5 out of 8 healthy controls and n = 3 out of 5 patients with NCGS, and one analysis each for the other subjects. Image Lab 6.1 (BioRad) was used to analyze the expression of P-STAT3 protein. Normalization was performed on proteins of 40 kD, i.e., ß-actin (Supplementary Figure S1). (c) The figure shows the x-fold enhancement of STAT3 and P-STAT3 protein in the PBMCs from healthy controls and patients with NCGS after stimulation of PBMCs with gliadins compared to a medium without cereal stimulants. Results are shown as median with interquartile range. In the healthy controls, the one-sample t-test revealed a significant upregulation of P-STAT3 by gliadins compared to medium control. (a) p = 0.021. The two-tailed t-test with Welch’s correction revealed significant differences in upregulation of P-STAT3 in PBMCs induced by gliadins between healthy controls and NCGS (p = 0.029). p < 0.5 is considered to be statistically significant.
from Cell Signaling Technology for Stat1/2/3/5 Control Cell Extracts