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. Author manuscript; available in PMC: 2016 May 1.

Published in final edited form as: Mol Cancer Res. 2015 Mar 2;13(5):944–953. doi: 10.1158/1541-7786.MCR-14-0412

(A) ChIP assay. CLL cell protein extract was incubated without or with anti-STAT3 antibodies, and DNA was extracted from chromatin fragments. As shown in the left panel, anti-STAT3 antibodies co-immunoprecipitated DNA of the STAT3 target genes c-Myc, p21, STAT3, VEGF-c, and ROR1 but not of the ribosomal RPL30 gene (used as negative control). As in Figure 4C, “Input” denotes DNA extracted from non-immunoprecipitated CLL cell chromatin fragments (negative control) IgG is the isotype of the anti-STAT3 antibodies. The right panel upper depicts ChIP of CLL cells. As shown, STAT3 co-immunoprecipitated DNA that was amplified with primers designed to amplify site 2 but not primers designed to amplify site 1 or site 3 of the LPL-promoter regions. The right lower panel depicts results of two separate experiments analyzed using qRT-PCR. Similar to the results depicted in the right upper panel, STAT3 co-immunoprecipitated DNA was significantly amplified with primer 2. (B) CLL cells were transfected with STAT3-shRNA or with an empty vector. Compared with cells transfected with empty vector (CTRL), the cells that were transfected with STAT3-siRNA expressed significantly lower levels of STAT3-regulated genes including LPL. (C) Western blot analysis of CLL cells transfected with STAT3 shRNA or empty vector showed that compared with an empty vector, STAT3 shRNA downregulated STAT3 and LPL protein levels by 80%.

(A) ChIP assay. CLL cell protein extract was incubated without or with anti-STAT3 antibodies, and DNA was extracted from chromatin fragments. As shown in the left panel, anti-STAT3 antibodies co-immunoprecipitated DNA of the STAT3 target genes c-Myc, p21, STAT3, VEGF-c, and ROR1 but not of the ribosomal RPL30 gene (used as negative control). As in Figure 4C, “Input” denotes DNA extracted from non-immunoprecipitated CLL cell chromatin fragments (negative control) IgG is the isotype of the anti-STAT3 antibodies. The right panel upper depicts ChIP of CLL cells. As shown, STAT3 co-immunoprecipitated DNA that was amplified with primers designed to amplify site 2 but not primers designed to amplify site 1 or site 3 of the LPL-promoter regions. The right lower panel depicts results of two separate experiments analyzed using qRT-PCR. Similar to the results depicted in the right upper panel, STAT3 co-immunoprecipitated DNA was significantly amplified with primer 2. (B) CLL cells were transfected with STAT3-shRNA or with an empty vector. Compared with cells transfected with empty vector (CTRL), the cells that were transfected with STAT3-siRNA expressed significantly lower levels of STAT3-regulated genes including LPL. (C) Western blot analysis of CLL cells transfected with STAT3 shRNA or empty vector showed that compared with an empty vector, STAT3 shRNA downregulated STAT3 and LPL protein levels by 80%.