Fig 2: PABPC4 knockdown increases PPARβ activity.A, either shGFP or shPABPC4 myotubes were seeded in 60 mm Petri dishes and maintained for 12 h in high (25 mM) or low (5 mM). Cells were then trypsinized, counted, and loaded on Oroboros chambers, and the oxygen consumption rate (OCR) was recorded (n = 2). B, lactate released into the media was measured after the cells were maintained in high (25 mM) or low (5 mM) glucose-containing media. C, C2C12 were seeded in 60 mm Petri dishes and differentiated into myotubes and maintained for 12 h in high (25 mM), low (5 mM), or low plus GW (100 mM GW501516, a PPARD agonist) medium (n = 4). Cells were then lysed, and nuclear and cytosolic fractions were isolated and then subjected to Western blotting to detect NCoR1 content (n = 1). D, MEF cells were seeded in 48-well plates, and when the confluency reached 50%, the cells were transiently transfected with non-target siRNA (siSCR) or siRNA specific to PABPC4 (siPABPC4). Twenty-four hours after siRNA transfection, cells were transiently transfected with PPAR response element (PPRE) and non inducible Renilla luciferase–expressing plasmids. Twenty-four hours after plasmid transfection, the medium was changed, and cells were maintained in a high (25 mM), low (5 mM), or low + GW (5 mM glucose + 100 mM GW501516) medium for 12 h, and the luminescence was recorded. Firefly luminescence was normalized by Renilla luminescence, and values expressed as fold change related to siSCR (n = 6). E, MEF cells were seeded in 48-well plates and transiently transfected with siRNA as described before. Twenty-four hours after siRNA transfection, cells were transiently transfected with PPRE and non inducible Renilla luciferase–expressing plasmids. Additionally, cells were also transiently transfected with pcDNA-GFP-FLAG (EV)– or pCMX-NCoR1-FLAG (NCoR1 OE)–expressing plasmids. Forty-eight hours after plasmid transfection, cells were lysed and both the Firefly and Renilla luminescences were recorded. Firefly luminescence was normalized by Renilla luminescence, and values expressed as fold change related to siSCR EV (n = 4). F, Western blotting for proteins of mitochondrial electron transport chain in differentiated C2C12 transiently transfected with siSCR or siPABPC4. CV: ATP5A; CIII: UQCRC2; CII: SDHB. G, MEF cells were transiently transfected with pcDNA-GFP-FLAG (EV)– or pCMV6-PABPC4-FLAG (PABPC4 OE)–expressing plasmids, and 48 h after transfections, OCR was evaluated (n = 2). H, either control (shGFP) or PABPC4 knockdown cells (shPABPC4) were transiently transfected with pcDNA-GFP-FLAG (EV)– or pCMX-NCoR1-FLAG (NCoR1 OE)–expressing plasmids and 48 h after transfections, subjected to OCR (n = 2). Bars represent the mean ± SD. ∗p < 0.05 and ∗∗∗p < 0.001 versus control. MEF, mouse embryonic fibroblastic; NCoR1, nuclear receptor corepressor 1; PPAR, peroxisome proliferator-activated receptor; PABPC4, poly(A)–binding protein cytoplasmic 4.

Fig 3: Pull-down of PABPC4 and different NCoR1 constructions. Hek293T cells were transiently transfected with pCMX-NCoR1-Flag (NCoR1 full-length), pCMV6–NCoR1 N-terminal-FLAG (NCoR1 N-terminal), or pCMV6–NCoR1 C-terminal-FLAG (NCoR1 C-terminal) plasmids. PABPC4-GST expression was performed in Escherichia coli and the lysate used in the pull-down assays. The glutathione agarose resin was functionalized by first binding 0.668 mg of PABPC4-GST. A, either bacteria expressing PABBPC4-GST or Hek293T cells expressing NCoR1 full-length were lysates. After that, different amounts of NCoR1 full-length were loaded into the column. Bacterial PABPC4-GST lysate was used to demonstrate that NCoR1 or any other bacterial protein interacts with PABPC4 and could be detected by the FLAG antibody. Different amounts of NCoR1 full-length (1, 2, or 4 mg) were loaded in the column containing PABPC4-GST and incubated as described in the Experimental procedures section. After that, the column was washed, and proteins were eluted and assayed by Western blotting. NCoR1 band was identified using an anti-FLAG antibody. B, the same experiment described in (A) but with different mass ratios between PABPC4-GST and NCoR1 full-length; 1: 0.668 mg of PABPC4-GST and 1 mg of NCoR1 full-length cell lysate; 2: 0.668 mg of PABPC4-GST and 2 mg NCoR1 full-length cell lysate; 3: 1 mg of PABPC4-GST and 1 mg of NCoR1 full-length cell lysate; 4: 1 mg of PABPC4-GST to 3 mg of NCoR1 full-length cell lysate. C, Hek293T cell expressing either NCoR1 full-length or NCoR1 C-terminal constructs were lysate, loaded into the column, washed, eluted, and then identified by Western blotting. NCoR1 band was identified using an anti-FLAG antibody. D, either bacteria expressing PABBPC4-GST or Hek293T cells expressing NCoR1 full-length or NCoR1 C-terminal were lysate. The lysates form both NCoR1 full-length and NCoR1 C-terminal were used in a pull-down assay. The NCoR1 band was identified using an anti-FLAG antibody. E, schematic representation depicting NCoR1 structure in mice and its binding partners. At the N-terminus (1-1622), it is shown the corepressor domains (RD1, RD2, and RD3) and also some of the proteins known to make up the core NCoR1 complex, such as HADH3, GPS2, TBL1 e TBL1XR1. At the C-terminus (1629-2453), it is shown the nuclear receptor–interaction domains (ID1 and ID2). Our results show that PABPC4 interacts with NCoR1 at its N-terminus region. FT, Flow-through (fraction not bound to column); NCoR1, nuclear receptor corepressor 1; PABPC4, poly(A)–binding protein cytoplasmic 4; WB, western blotting.

Fig 4: PABPC4 is required for NCoR1 stability.A, Siah2 gene expression analysis was performed by RT-qPCR in either shGFP or shPABPC4 myotubes (n = 4). B, NCoR1 gene expression analysis was performed by RT-qPCR in shGFP or shPABPC4 myotubes maintained in high (25 mM) or low (5 mM) glucose DMEM, for 12 h (n = 3). C, NCoR1 protein half-life was analyzed in myotubes transiently transfected with siSCR or siPABPC4 by treating the cells with cycloheximide (CHX) followed by Western blotting (n = 1). CHX (50 or 100 μg/ml) was added at times indicated in the Figure. D1, trypsin-resistant tandem ubiquitin-binding entities (TR-TUBE) allow low-expressed and rapid-degraded proteins to be immunoprecipitated since it binds to ubiquitin molecules, stabilizing them and avoiding their degradation by the proteasome. Since the sequence is fused with a FLAG peptide, it allows proteins to be easily immunoprecipitated using an anti-FLAG antibody. D2, MEF cells were transiently transfected with WT TR-TUBE-FLAG (WT TR-TUBE) or mutated TR-TUBE-FLAG (Mut TR-TUBE). Cells were treated with 10 μM MG132 for 4 h and then subjected to immunoprecipitation using a FLAG antibody-bound resin as described in the Experimental procedures section (n = 1). Exposure time varied from short to long for capturing ubiquitinated proteins in the eluate and the input, respectively. E, MEFs stable GFP (shGFP) or PABPC4 (shPABPC4) knockdown were transiently transfected with pmCherry-NCoR1–expressing plasmid. Forty-eight hours after transfection, cells were fixed and observed in a fluorescence microscope. Nuclei were stained with Hoechst 33342. Magnification = 200 ×. F, gene expression performed by RT-qPCR in control (shGFP) or PABPC4 knockdown (shPABPC4) myotubes (n = 4). G, MEF cells were transiently transfected with pcDNA-GFP-FLAG (EV), pCMX-NCoR1-FLAG (NCoR1 OE), or pCMV6-PABPC4-FLAG–expressing plasmids, and 48 h after transfections, the assay of cellular viability was performed using propidium iodide (PI) and Hoechst 33342 (HO). Twelve hours before PI and HO addition, cells were maintained in DMEM containing either high glucose (25 mM) or galactose (10 mM). The fluorescence was read in 535/617 nm (Ex/Em) for PI and 350/461 nm (Ex/Em) for HO (n = 8). PI fluorescence was normalized by HO fluorescence, and values were expressed as a percentage related to EV high. H, Western blotting in WT or NCoR1 KO myotubes (n = 2). Bars represent the mean ± SD. ∗p < 0.05, ∗∗p < 0.01 and ∗∗∗p < 0.001 versus control. Atp5a1, ATP synthase F1 subunit alpha; Cpt1b, carnitine O-palmitoyltransferase 1; Creb1, cyclic AMP-responsive element-binding protein 1; CS, citrate synthase; Cycs, cytochrome c; DMEM, Dulbecco’s modified Eagle’s medium; ETC, electron transport chain; MEF, mouse embryonic fibroblastic; Nrf1, nuclear respiratory factor 1; Ndufs5, NADH:ubiquinone oxidoreductase subunit s5; NCoR1, nuclear receptor corepressor 1; Pdk4, pyruvate dehydrogenase (acetyl-transferring) kinase isozyme 4; Ppard, peroxisome proliferator-activated receptor delta; PABPC4, poly(A)–binding protein cytoplasmic 4; Sdha, succinate dehydrogenase (ubiquinone) flavoprotein subunit; Sod2, superoxide dismutase (Mn); Tfam, transcription factor A; TF/NR, transcription factor/nuclear receptor; TR, thyroid hormone receptor; TUBE, Tandem Ubiquitin Binding Entity; Vegfa, vascular endothelial growth factor A; Uqcrc1, ubiquinol-cytochrome c reductase core protein 1.

Fig 5: PABPC1 do not play a role in mitochondria function in myotubes.A, PABPC1 and PABPC4 mRNA levels were assessed by RT-qPCR in both myoblast (MB) and myotubes (MT) (n = 3). Expression was calculated as fold change related to PABPC1 mRNA. B, C2C12 cells knockdown for either PABPC4 or PABPC1 were differentiated, lysed, and samples were subjected to Western blotting (n = 2). C and D, bar graph representing the PABPC1 or PABPC4, respectively, quantitation from (B). E, C2C12 cells knockdown for either PABPC4 or PABPC1 were seeded in 6-well plates and treated with 10 μg/ml of puromycin for 30 min before the analysis of protein synthesis. Prior addition of puromycin, 30 μg/ml cycloheximide (CHX) was added, for 30 min, to inhibit the protein synthesis process. Upper panel: Western blotting using anti-puromycin antibody; middle panel: Ponceau red–stained membrane from the same membrane probed with anti-puromycin antibody in the upper panel; lower panel: a Coomassie Brilliant Blue (CBB)-stained gel run in parallel and with the same samples as in upper panel (n = 2 for puromycin-treated cells; n = 1 for CHX pretreated cells). F, quantitation from (E). Quantitation was performed by normalizing the optical density from puromycin-labeled membrane divided by the optical density from the ponceau-stained membrane. G, oxygen consumption rate (OCR) in PABPC1 knockdown cells (n = 2). H, lactate production in PABPC1 or PABPC4 knockdown cells in high glucose (25 mM) or galactose (10 mM, no glucose), for 12 h (n = 4). I, mitochondrial content was analyzed using MitoTracker Deep Red (MTDR) staining in cells exposed to high glucose (25 mM) or galactose (10 mM, no glucose), for 12 h (n = 8). The MTDR fluorescence intensity was normalized by the Hoechst 33342 (HO), and the results were expressed as a percentage related to shGFP. Bars represent the mean ± SD. ∗p < 0.05, ∗∗p < 0.01 and ∗∗∗p < 0.001 versus control condition. PABPC4, poly(A)–binding protein cytoplasmic 4.