Fig 2: mTOR is primarily membrane-associated in osteoclasts. RAW264.7 cells and RAW-derived osteoclasts (RAW-OCs) were cultured and cytosolic and membrane fractions were prepared as described in “Materials and Methods”. Equal protein amounts were separated on 4–20% gradient gels, transferred to a nitrocellulose membrane and probed for mTOR, Raptor, LAMTOR1, LAMP2, a3, and V1A. Representative cropped blots of 4 independent experiments. M = membrane fraction; C = cytosolic fraction; CTRL = control, no treatment; STRV = starvation, HBSS, 1 hr.

Fig 3: Hepatic CCRK-mTORC1 signaling recruits MDSCs to form a tumor-prone liver microenvironment. a mTORC1 signaling was activated in the livers of CCRK TG mice. The downstream molecules of mTORC1 signaling were detected by Western blot. b mTORC1 signaling components were activated in the primary hepatocytes isolated from CCRK TG mice. c Schematic diagram of CCRK TG mouse model with intrahepatic injection of Ccrk KO Hepa1–6 cells by CRISPR/Cas9, and lentiviral-mediated Raptor knockdown. d CCRK TG mice (n = 8) developed significantly larger tumors compared to controls (n = 11), which was abolished by down-regulation of Raptor (shRaptor; n = 5) (scale bar = 1 cm). e PMN-MDSCs were induced in CCRK TG mice, but were reduced after Raptor knockdown, and f PMN-MDSC levels positively correlated with tumor weight. g PMN-MDSCs were induced by HFHC diet, but were reduced by Ccrk knockdown in NASH-HCC model (CD+shCtrl, n = 8; HFHC+shCtrl, n = 15; HFHC+shCcrk, n = 15), wherein h PMN-MDSC levels positively correlated with tumor multiplicity. i G-csf mRNA levels were induced by HFHC diet, but were reduced by Ccrk knockdown in NASH-HCC model. j G-CSF mRNA level was augmented by CCRK over-expression in LO2 cells, which could be attenuated by Raptor knockdown. k G-CSF mRNA expression was down-regulated in CCRK KO Huh7 cells. Data are presented as mean ± SD. *p < 0.05 and **p < 0.01 as calculated by one-way ANOVA followed by Bonferroni post hoc test (d, e, g, i, j), Pearson correlation (f, h), and unpaired two-tailed Student’s t-test (k)

Fig 4: Activation of Akt recovers muscle transcriptome and protein degradation. (A) Principal component analysis of RNA‐Seq data of transgenic mice (Raptor ko, Akt, and wt) in control (sham) or post‐C26 inoculation (n = 3 animals per group). (B) Subset of genes after two‐way ANOVA comparing wt and Akt animals. Genes with interaction P value <0.001 are shown using Euclidean distance and the complete method. See also Table S1 for a heat map representation in Excel format. (C) Line plot visualizing Cluster 2 identified in (B) and genes of the proteasomal and autophagy system. IQR identifies the cluster inter‐quartile range. (D) Protein expression of autophagy markers, LC3 and p62, is restored after Akt activation during cancer cachexia (n = 3 wt sham, n = 4 wt C26, Akt sham, and Akt C26). (E) Western blot analysis of Lys48 ubiquitinated proteins and related quantification (n = 3 wt sham, n = 4 wt C26, Akt sham, and Akt C26). Data presented as mean ± SEM. Two‐way ANOVA with Tukey's multiple comparison post hoc test was performed. *P < 0.05 and **P < 0.01.

Fig 5: HSP60 protected RPTOR from the glucocorticoide-phosphorylation, aggregation, and ubiquitination.a HSP60 restored the glucocorticoid-mediated loss of phosphorylated ERK, phosphorylated Ser473-Akt, and phosphorylated RPTOR abundances. b It attenuated glucocorticoid-enhanced Bax, cytochrome c, and cleaved caspase3 levels. c Forced HSP60 expression alleviated the glucocorticoid elevation of protein aggregation along with decreased RPTOR aggregation. Actin immunoblots indicate equal amount of cell lysates pipetted for protein aggregation assay. d, e HSP60 overexpression reduced the glucocorticoid-upregulated levels of ubiquitinated proteins and RPTOR. Knocking down HSP60 impaired RPTOR phosphorylation but increased RPTOR aggregation and ubiquitination. f MG132 attenuated the glucocorticoid-induced RPTOR ubiquitination and restored RPTOR and LC3-II levels and LC3-II/LC3-I ratio. g Images of TUNEL staining in osteoblasts. HSP60 and RPTOR attenuated glucocorticoid-mediated apoptosis. Scale bar, 85 μm. Experiments results are expressed as mean ± standard error. Asterisks (*) resemble a significant difference (P < 0.05) vs. scramble group, and hashtag (#) indicates a distinguishable difference (P < 0.05) vs. glucocorticoid-treated group