TRiC binds Stat3 co-translationally and is required for its synthesis in RRLs.

<p>In panel (A), TRiC was immunoprecipitated from RRLs with a combination of antibodies to CCT2 and CCT5 (Anti-TRiC) or with a nonspecific control antibody (Control) following translation of the indicated proteins in the presence of ³⁵S-methionine. Immunoprecipitates were separated by SDS-PAGE and autoradiographed (left panel); the position of the 90-kDa MW marker is indicated. Half of each IP reaction prior to precipitation was run separately on SDS-PAGE and autoradiographed (right panel). In panel (B), RRLs were immunoblotted with CCT1 antibody following TRiC depletion using Protein-A agarose plus CCT1 antibody (TRiC-depleted) or control antibody (Mock-depleted). In panel (C), the indicated protein was translated in mock-depleted or TRiC-depleted RRLs in the presence of ³⁵S-methionine followed by SDS-PAGE and autoradiography. In panel (D), Stat3 was translated in TRiC-depleted RRLs following the addition of purified bovine TRiC in increasing amounts in the presence of ³⁵S-methionine followed by SDS-PAGE and autoradiography. The results shown are representative of three experiments.</p

TRiC knockdown reduces levels of activated Stat3 protein within cancer cells.

<p>HS-578T cells (A) or HepG2 cells (E) stably transfected with lentivirus containing CCT2 shRNA (knockdown) or control shRNA (control) were immunoblotted with antibody to CCT2 or β-actin. Levels of activated Stat3 (Y705-phosphorylated Stat3, pStat3), total (t)Stat3, and GAPDH within cells lysates were measured using Luminex beads. Levels of pStat3 in HS-578T and HepG2 CCT2 or control knockdown cells are presented normalized to GAPDH (B and F) or tStat3 (D and H); levels of tStat3 in HS-578T and HepG2 CCT2- or control-knockdown cells are presented normalized to GAPDH (C and G). Results in CCT2 knockdown cells indicated with an asterisk (*) differ from control knockdown cells (<i>p</i><0.02). The results shown are representative of two experiments.</p

Stat3 binds and cross-links to TRiC subunit CCT3 in vitro; CCT3 co-localizes with activated Stat3 within the cell nucleus.

<p>In panel (A), RRLs expressing the indicated ³⁵S-methionine-labeled TRiC subunit were mixed with RRLs expressing an unlabeled Flag-tagged Stat3 construct (Flag-Stat3) or no expression construct (Mock), incubated with DSP, immunoprecipitated with M2 anti-Flag antibody-bound agarose beads, incubated with β-mercaptoethanol, and analyzed by SDS-PAGE and autography. Autoradiographs are shown. In panel (B), transformed MEFs deficient in Stat3 and stably expressing GFP-Stat3α were transiently transfected with mCherry-CCT3 or mCherry-CCT7 and incubated without (<i>−</i>) or with (+) IL-6/sIL-6R (∼250 ng/ml), as indicated (±IL-6), before DAPI staining, fixation, and confocal fluorescent microscopic examination. Photomicrographs shown are representative images filtered to reveal the DAPI signal within the nucleus (column 1), the GFP signal within the cytoplasm and nucleus (column 2), the mCherry signal within the cytoplasm and nucleus (column 3), and the GFP plus mCherry merged signal (column 4). Bar, 10 µM.</p

TRiC knockdown reduces sensitivity of cancer cells to IL-6–mediated Stat3 activation.

<p>CCT2-knockdown (+) or control-knockdown (−) HepG2 cells were immunoblotted for pStat3 and total Stat3 (tStat3) 30 min after incubation in media alone or in media containing IL-6 at the indicated concentrations. Representative immunoblots are shown in the top panel. Densitometry readings of the immunoblot bands were normalized to the IL-6 concentration yielding maximum pStat3 levels (1 ng/ml) in two experiments. The ratios of densitometry values (pStat3/tStat3) for these experiments is shown in the bottom panel; results in CCT2 knockdown cells that are indicated with an asterisk (*) differ from control knockdown cells (<i>p</i><0.05).</p

vTBD-Stat3 demonstrates increased binding to TRiC; increased binding is mediated by residues within the vTBD Box 1 and 2 and is accompanied by improved SH2 domain function.

<p>The top portion of panel (A) delineates the vTBD including the location of Box 1 and Box 2 and critical residues therein including those shown in red, which were mutated to alanine. Following translation of the indicated proteins in the presence of ³⁵S-methionine, TRiC was immunoprecipitated from RRLs with a combination of antibodies to CCT2 and CCT5 (TRiC-IP). Immunoprecipitates were separated by SDS-PAGE and autoradiographed (middle portion). Autoradiography of an equivalent volume of each immunoprecipitation reaction pair (Input) is shown in the lower portion. In panel (B), equivalent amounts of ³⁵S-methionine labeled Flag-Stat3 or Flag-vTBD-Stat3 (Input) were incubated with biotinylated tyrosine phosphorylated dodecapeptide 1068 (p1068) or unphosphorylated dodecapeptide (1068) bound to streptavidin agarose beads. Bead-bound proteins (pull down) were separated by SDS-PAGE and autoradiographed. Autoradiographs of Input (left panel) and Pull Down (middle panel) proteins of a representative of three experiments are shown. Densitometry of the underlined bands from all experiments were normalized to the Stat3 in experiment 1 and the mean ± SEM plotted (right panel). The asterisk (*) indicates increased binding to p1068 by vTBD-Stat3 versus Stat3 (<i>p</i> = 0.0028).</p

Stat3 interacts with TRiC within MEF cells.

<p>In panel (A), lysates of MEF-Stat3^Δ/Δ cells transiently expressing Flag-Stat3α were immunoprecipitated (IP) with a mixture of rabbit antibodies to CCT2 and CCT5 (+) or control rabbit antibody to human IgG (Mock) and Western blotted (WB) with antibodies to Flag tag or CCT1. In panel (B), TRiC lysates of MEF-Stat3^Δ/Δ cells transfected with Flag-tagged Stat3α were prepared in the presence or absence of ATP and MgCl₂, and analyzed by immunoprecipitation and Western blotting. The input is shown to the right.</p

Formation of TRiC-Stat3 complexes and refolding of guanidine hydrochloride-denatured Stat3 within RRLs containing ATP.

<p>In panel (A), Stat3-containing complexes were detected within RRLs following native-PAGE analysis of <i>in vitro</i> translation and refolding reaction products by Western blot using an anti-Stat3 antibody. Lane 1 shows RRL reaction in the absence of addition of exogenous plasmid. Lane 2 shows RRL reaction in the presence of addition of exogenous plasmid encoding Stat3. Stat3 previously unfolded using 6 M guanidine hydrochloride was added to RRLs without ATP (lane 3) or RRLs containing ATP (lane 4). Arrows indicate the position of Stat3 aggregate, TRiC-Stat3 complex, and native endogenous and refolded Stat3. In panel (B), the blot was stripped and re-probed with anti-CCT1 antibody.</p