Fig 2: BTG3 suppresses EMT by antagonizing the AKT-GSK3ß–ß-catenin signaling axis. (a and b) Overexpression of BTG3 led to diminished GSK3ß phosphorylation (a), whereas BTG3 depletion with siRNA enhanced it in U2OS osteosarcoma cells. (b) Cells were starved for 30 h and collected at the indicated times after serum stimulation. Lysates were analyzed by western blotting using the indicated antibodies. siBTG3-1 and -2 are siRNAs targeting two different sequences in BTG3. (c) Biochemical fractionation showing increased levels of ß-catenin in the nuclear fractions of BTG3-downregulated U2OS cells. (d) The transcriptional activity of ß-catenin/TCF was increased in BTG3-knockdown cells. U2OS cells were transfected with wild-type (8 × TOP) or mutant (8 × FOP) ß-catenin/TCF reporters, serum starved for 24 h, and collected at the indicated times after the addition of serum. All luciferase activities were normalized to the co-transfected internal control and expressed as fold induction relative to the wild-type reporter in serum-starved cells (0 h). The mean±S.D. of three independent experiments is shown. (e) Increased expression of markers indicative of EMT in Tet-On shBTG3 U2OS cells after the induction of BTG3 shRNA expression by doxycycline. Immunoblot analysis was performed 72 h after the addition of doxycycline. Expression was quantified and mean±S.D. from three independent experiments is shown. *P<0.05, ***P<0.001, ****P<0.0001. (f) Enhanced migration of Tet-On shBTG3 U2OS cells after the induction of BTG3 shRNA expression by doxycycline (doxy). The mean±S.D. of three independent duplicated experiments is shown. (g) Inhibition of ß-catenin/TCF activity by BTG3 requires GSK3ß activity. The 293 T cells were transfected with the ß-catenin/TCF reporters together with BTG3 or the control vector. Cells were treated with DMSO (solvent) or 2 µM CT99021 (a GSK3-specific inhibitor) for 3 h before collection (n=3). NS, nonsignificant

Fig 3: PTEN affects ß-catenin nuclear localization.(a) Confocal microscopy revealed cells (labelled with arrows) with a heavily laden ß-catenin (bcat) nuclear staining (b), in contrast to nearby PTEN-GFP-positive cells, where ß-catenin staining could seldom be observed within the nucleus, arrowheads. Cells were counterstained with 4,6-diamidino-2-phenylindole (DAPI) (c). Merged is shown (d). Scale bar, 10 µm. Immunofluorescence experiments for the Hs944T cells were performed three times with similar results. (e–h) Human melanoma Lyse cells mutated for NRAS (Q61K), which produce PTEN, were transfected with siScr (e,f) and siPTEN (g,h). Cells were labelled for ß-catenin (e,g) and counter stained with DAPI (f,h). Scale bar, 10 µm. Immunofluorescence experiments for the Lyse cells were performed two times with similar results. (i,j) Confocal microscopy showing the localization of ß-catenin in Tyr::Cre/°;PTENf/+=PTENf/+ (i) and Tyr::Cre/°;PTENf/f=PTENf/f (j) melanocytes. Note the increase of nuclear ß-catenin staining in PTENf/f cells. Scale bar, 10 µm. Immunofluorescence experiments for the murine PTENf/+ and PTENf/f cells were performed four times with similar results. (k) Immunoblot analysis of PTEN, AKT (total and phosphorylated form Ser473), GSK3ß (total and phosphorylated form Ser9), ß-catenin (total, phosphorylated form Thr41/Ser45 and Ser675) and ß-actin proteins in Hs944T transfected with either expression vector encoding GFP (CMV::GFP) or PTEN (CMV::PTEN-GFP). Cells expressing either exogenous GFP or PTEN treated with LY294002 or LiCl for 1 h. It is noteworthy that a higher concentration of GSK3ß antibody reveals a second upper band. Western blot analyses were performed two times for all antibodies with similar outputs. (l) Immunoblot blot analysis for GFP, PTEN, AKT (total and pSer473), ß-catenin pSer675 and ß-actin of Hs944T lysates co-transfected with either empty vector GFP or PTEN and WT or constitutively active p110 E545K mutant. Cells were starved for 2 h with 0.1% serum before lysis. Western blot analyses were performed two times for all antibodies with similar outputs. (m) Immunoblot blot analysis for PTEN, AKT (total and pSer473), ß-catenin pSer675 and ß-actin of Hs944T lysates co-transfected with a constitutive active form of p110 (p110 CAAX) or a kinase-dead form (p110 KD) in the presence of GFP or PTEN. Western blot analyses were performed three to six times, depending on the antibody with similar outputs.

Fig 4: Following acute exercise Haptoglobin deficiency results in altered mitochondrial signaling in skeletal muscle.A) mRNA analysis for mitochondrial biogenesis (names of genes as indicated): PGC1a transcript is induced by exercise in WT mice and not in Hp-/-; Tfam and Nrf1 are downregulated by exercise in Hp-/- skeletal muscle. B) Exercise does not affect Atrogin-1 and MuRF1 mRNA levels in any of the two genotypes. C) mRNA amount of autophagy related genes (names as indicated) in control and RUN WT and Hp-/- skeletal muscle. (D-I) Quantification of Western blots analysis on protein isolated from skeletal muscle of control and RUN WT and Hp-/- mice; data are expressed as percentage of “control WT”, where control WT is considered 100%. D) Beclin-1 protein levels, E) LC3 lipidation (ANOVA treatment effect P<0.05), F) p62 protein levels, G) Akt phosphorylation (ratio between signal on Ser 473 and total Akt), H) S6 phosphorylation (ratio between signal on Ser 235/236 and total p70S6), I) 4EBP1 phosphorylation (ratio between signal on Thr 37/46 and total 4EBP1). J) 4EBP1 mRNA level. Real Time PCR and western blot analysis were respectively performed on Tibialis anterior cDNA and Gastrocnemius muscles protein lysates. GAPDH is used as control of equal loading in both cases. Muscles were dissected from 5 months old mice. Data are expressed as mean ± SEM. *P<0.05; **P<0.01; §§P<0.01; §§§P<0.001.

Fig 5: The effects of obesity on skeletal muscle protein balance in the absence of Haptoglobin.Each panel shows WT (white bars) and Hp-/- (black bars) skeletal muscle under regular Chow Food Diet Feeding (the same as figures 1, 2 and 3, referred to as “CFD”, n = 6–8) and in the obesity condition following 12 weeks of High Fat Diet (referred to as “HFD”, n = 6). A) Obesity results in Atrogin-1 transcript upregulation in HFD WT mice and in MuRF1 transcript upregulation both in HFD WT and HFD Hp-/- mice. B) mRNA levels of autophagy related genes (names as indicated) in CFD and HFD WT and Hp-/- skeletal muscle. (C-H) Quantification of Western blot analysis on skeletal muscle of CFD and HFD WT and Hp-/- mice; data are expressed as percentage of “CFD WT”, where CFD WT is considered 100%. C) LC3 lipidation, D) p62 protein levels, E) Beclin-1 protein levels, F) Akt phosphorylation (ratio between signal on Ser 473 and total AKT), G) S6 phosphorylation (ratio between signal on Ser 240/244 and total p70S6), H) 4EBP1 phosphorylation (ratio between signal on Thr 37/46 and total 4EBP1). I) 4EBP1 mRNA level is increased in both obese WT and HFD Hp-/- skeletal muscle with respect to relative CFD. Real Time PCR and western blot analysis were respectively performed on Tibialis anterior cDNA and Gastrocnemius muscles protein lysates. GAPDH is used as control of equal loading in both cases. Muscles were dissected from 5 months old mice. Data are expressed as mean ± SEM. *P<0.05; **P<0.01; §P<0.05; §§P<0.01; §§§P<0.001.