Fig 2: TBP is required for myoblast differentiation into myotubes.(A) Representative images of C2C12 myoblasts differentiation into myotubes upon TBP downregulation. TBP was downregulated using Tbp-targetting siRNA in C2C12 myoblasts. C2C12 myoblasts were differentiated into myotubes for two days, and immunolabeled with anti-Myosin Heavy Chain (MHC) antibody (red) to monitor myotybes formation. The nuclei were counterstained with Hoechst 33,258 (blue). Images were taken by Olympus IX71 microscope with 20x objective. Size bar 20 µm. (B) Quantification of nuclei residing within differentiated MHC-positive myotubes in A, represented as differentiation index. Error bars represent a standard deviation among three biological replicates. Student’s t test was used for statistical analyses (***p<0.001). (C) Immunoblot analysis of the whole cell lysate of C2C12 to assess the TBP protein levels after siRNA-mediated silencing of TBP. (D) mRNA isolated from C2C12s grown in differentiation medium after siRNA-mediated downregulation of TBP was analyzed by RT-PCR for expression of skeletal muscle specific genes Myog, Ckm and Acta1. Relative expression of indicated genes is presented as a fraction of Gapdh expression. Error bars represent a standard deviation among three biological replicates. Student’s t test was used for statistical analyses (***p < 0.001).DOI: http://dx.doi.org/10.7554/eLife.12534.006

Fig 3: VCP protein expression in canine lymphoma. a Immunoblotting analysis was done for VCP. Representative blots are shown, each lane represents a single lymph node or tumor sample from one patient. Quantitative analyses of were done using n = 4–9 tumor samples per grade and type and normalized to ACTB (ß actin) as a loading control. Data are presented as mean (cross bar) ± SEM (error bars). Asterisk (*) indicates a statistically significant difference (*P < 0.05) compared to low grade. b VCP immunohistochemistry for B-cell lymphoma compared to normal lymph node. c Immunoblotting analysis of VCP in PBMCs (signal intensity = 1.01 arbitrary units) and lymphoma cell lines (17–71, CL-1 and CLBL-1) (average signal intensity = 3.33 arbitrary units). High, Inter (Intermediate) and Low refer to grade, T and B refer to lymphoma cellular subtype, LN = normal lymph node

Fig 4: Signal transduction, metabolic activity, and apoptosis after treatment of Ba/F3 cell lines expressing four distinct FLT3-ITD variants with 17-AAG after pre-incubation with tunicamycin. Immunoblotting analysis of signaling pathways (left) after sequential incubation of four Ba/F3 cell lines stably expressing different FLT3-ITD variants with 1 µg/mL tunicamycin and 17-AAG. (A) Ba/F3 cells were incubated with 0.1% (v/v) DMSO for 24 h followed by treatment with 17-AAG at the indicated concentrations for 24 h. (B) the incubation was started with 1 µg/mL tunicamycin for 24 h prior to treatment with 17-AAG at indicated concentrations for a further 24 h. Whole cell protein lysates were subjected to SDS-PAGE and immunoblotting to assess the expression and phosphorylation status of the signaling proteins. For loading control, the levels of beta-Actin were detected. Representative blots of the three independent experiments are shown. Corresponding assays indicating metabolic activity (MTS) and apoptosis (Annexin V) are illustrated in the right section of all Ba/F3 FLT3-ITD cell lines. The data for MTS assays are shown as relative absorption compared to untreated controls. Apoptosis is demonstrated by Annexin V-negative cells indicating viable, non-apoptotic Ba/F3 cells. Graphs are shown as the mean ± SD that were obtained from three independent experiments. Statistical significance between each Ba/F3 cell lines comparing DMSO vs. tunicamycin group was calculated using paired t-tests. p values of Student’s t-test, raw data, as well as IC50 values are shown in Table S1 in the Supplement.

Fig 5: Effect of SL on expression of osteoclast-associated genes. (A) Inhibitory effects of SL on expression of osteoclast-associated mRNA was verified by reverse transcription-quantitative PCR and (B) normalized to Actb. The results are expressed as the mean ± SEM (n=3). #P<0.05, ##P<0.01 vs. untreated; *P<0.05 and **P<0.01 vs. RANKL-alone. SL, Solanum nigrum Line; RANKL, receptor activator of NF-?B ligand; NFATc1, nuclear factor-activated T cells c1; Tnfrsf11a, receptor activator of NF-?B; Acp5, tartrate-resistant acid phosphatase; Ctsfk, cathepsin K; Mmp9, matrix metallopeptidase 9; Oscar, osteoclast-associated immunoglobulin-like receptor; Atp6v0ds, ATPase H+ transporting V0 subunit d2; Ca2, carbonic anhydrase II; Actb, ß-actin.