Fig 2: Pip5k1c regulates Runx2 expression through CaMK2 in BMSC.A and B, Cytosol Ca2+ assay. Primary BMSCs isolated from 4-month-old male mice were plated in confocal dish and knockdown (KD) of Pip5k1c by RNA interference for 48 h. Cells were then applied for Fluo-4/AM staining and signals were collected as described in Methods (A). Scale bar, 20 µm. The total fluorescence intensity was used as the cytoplasmic Ca2+ concentrations (B). Results were expressed as mean ± SD; N = 20 cells per group were analyzed. **p < 0.01 versus si-NC; unpaired two-tailed t test. C–E, primary BMSCs isolated from 4-months-old male mice were seeded in 6-well plate at 4 × 105 cells per well. Pip5k1c was knocked down by RNA interference for 48 h. Protein extracts isolated from the cells were subjected to western blotting with the indicated antibodies (C and D) or RT-qPCR analysis for expression of Pip5k1c and Runx2 mRNAs (E). Gapdh protein and mRNA were used as internal controls for Western blotting and RT-qPCR analysis, respectively. Results were expressed as mean ± SD; N = 3 biologically independent experiments. **p < 0.01 versus si-NC; unpaired two-tailed t test. F and G, primary BMSCs isolated from 4-months-old male mice were seeded in 6-well plate at 4 × 105 cells per well. Pip5k1c was knocked down by RNA interference for 48 h. Protein extracts isolated from the cells were subjected to Western blotting with the indicated antibodies (F). Gapdh protein were used as internal controls for western blotting analysis. Quantification results (G) were expressed as mean ± SD; N = 3 biologically independent experiments. **p < 0.01 versus si NC; unpaired two-tailed t test. H, IHC staining. Tibial sections of control and cKO mice were subjected to IHC staining for expression of Runx2. Scale bar, 50 µm. I, quantification of (H). Results were expressed as mean ± SD; N = 6 mice per group were analyzed. **p < 0.01 versus control; unpaired two-tailed t test. J and K, tibial sections of control and cKO mice were subjected to IF staining for expression of Osx (J). Scale bar, 50 µm. (K) quantification of (J). Results were expressed as mean ± SD; N = 6 mice per group were analyzed. **p < 0.01 versus control; unpaired two-tailed t test. L–O, Flag-Runx2 plasmid (1 µg) was transfected into COS-7 cells together with increasing amounts of plasmids expressing a kinase-dead CaMK2a-V5 (KD-CaMK2a-V5) (L and M) or constitutively active CaMK2a-V5 (CA-CaMK2a-V5) (N and O) plasmid. Runx2 expression was determined by Western blotting with anti-Flag antibody at 24 h after transfection. Results were expressed as mean ± SD; N = 3 biologically independent experiments. **p < 0.01 versus 2 µg; unpaired two-tailed t test. P and Q, cycloheximide experiments. Flad-Runx2 plasmid was transfected into COS-7 cells together with KD-CaMK2a-V5 or CA-CaMK2a-V5 plasmid. At 24 h after transfection, cells were treated with CHX at 100 µg/ml for the indicated times. Quantification of levels of Runx2 protein was performed (Q). Results were expressed as mean ± SD; N = 3 biologically independent experiments. **p < 0.01 versus control; unpaired two-tailed t test. si-NC, control siRNA; CA-CaMK2a, constitutively active form of CaMK2a; CHX, cycloheximide; BMSCs, bone marrow stromal cells; IHC, immunohistochemical.

Fig 3: EOFAZ inhibited PPAR-? downregulation and the high level of p-CREB and p- CaMKII in vivo and in vitro. Western blot analysis was used to analyze PPAR-?, p-CREB (ser133), p-CaMK II (ser-286), total CREB and total CaMK II expression in rats (a, b). Western blots analysis was used to detect the expression of PPAR-?, p-CREB (ser133), and p-CaMK II (ser-286), total CREB and total CaMKII levels in RMCs (c, d), mRNA level of PPAR-? in RMCs detected by qRT-PCR (e). Data are expressed as mean ± SEM. n = 3. **P < 0.01 vs. non-diabetic rats or control group of RMCs, #P < 0.05, ##P < 0.01 vs. Diabetic rats or HG group

Fig 4: Skeletal muscle intracellular CaMKII, AMPKa and p62 signalling in response to incremental exercise to exhaustion in normoxia and severe hypoxia and the application of immediate ischaemic or non-ischaemic recovery. Protein expression levels of pThr287 CaMKII (A), pThr172 AMPKa (B), AMPKa total (C), pThr172 AMPKa/AMPKa total ratio (D), pSer349 p62 (E), p62 total (F), pSer349 p62/p62 total ratio (G), and association between the ratio of pThr172 AMPKa/AMPKa total and the ratio of pSer349 p62/p62 total (H). Nx; test performed in normoxia (FIO2 = 0.21, PIO2: 143 mmHg), Hyp; test performed in severe acute normobaric hypoxia (FIO2 = 0.104, PIO2: 73 mmHg); Pre, before exercise; Post, 10 s after the end of exercise with ischaemic recovery; Oc1m, 60 s after the end of exercise with ischaemic recovery; FC1m, 60 s after the end of exercise in the leg recovering without occlusion (free circulation); p62/SQSTM1 (shortened to p62). n = 11 in all conditions except for Oc1m Nx (n = 9), Post Hyp (n = 10), and Oc1m Hyp (n = 10). In (H), large symbols represent the mean of the subjects studied in each condition. The correlation coefficient and regression line have been calculated using the individual values (small white circles, n = 73). A detailed description of the experimental phases is explained in Fig. 1. The statistical analysis was performed with logarithmically transformed data for the pThr172 AMPKa/AMPKa total ratio and p62 total. The values shown are means ± standard errors and expressed in arbitrary units (a.u.). †p < 0.05 vs Pre Nx; *p < 0.05 vs Pre Hyp; #p < 0.05 vs Post Hyp; §p < 0.05 vs Oc1m Hyp.

Fig 5: Schematic model of the regulation of Nrf2 and Keap1 signalling in human skeletal muscle immediately after incremental exercise to exhaustion in normoxia and severe hypoxia. Under basal conditions, Keap1 continuously targets Nrf2 for ubiquitination and degradation, allowing for minimal levels of Nrf2. The production of RONS during exhaustive exercise stimulates the activation of AMPK and CaMKII. Concomitantly, CaMKII acts indirectly as an upstream AMPK activator (by an unknown mechanism). AMPK promotes the increase of Nrf2 levels by two main mechanisms. Firstly, by phosphorylating p62 at Ser349, which stimulates the p62-mediated degradation of Keap1 via autophagy; secondly, by phosphorylating and blocking GSK3-ß, which activates ß-TrCP (an E3 ubiquitin-protein ligase) which tags Nrf2 for proteasomal degradation (not measured here). RONS may also activate PKCd which phosphorylates Nrf2 at its Serine 40 promoting its nuclear translocation and genes transactivation. The lowered levels of Keap1 and the reduced amount of p62 observed here are suggestive of co-degradation following exercise. Overall, the augmented Nrf2 total and phosphorylated protein expression together with the rise in the Nrf2-to-Keap1 ratio elicited by exhausting exercise should be sufficient to enhance the Nrf2-mediated antioxidant response. A central role of Catalase is manifested by a remarkable increase in its protein content following exercise, which was exacerbated during exercise in severe acute hypoxia, likely as a response to increased H2O2 production, by superoxide dismutases. This process is facilitated in hypoxia due to the upregulation of SOD1. No acute changes in SOD2 protein expression were observed. Most changes evoked by the exhaustive exercise bout were almost entirely reverted to baseline in less than 60 s by an O2-dependent mechanism. Activating/inhibiting actions are represented by blue/red connecting lines (dashed if the effect is indirect). Changes on cellular locations are presented with black dashed lines. The arrows and symbols depicted inside dashed grey boxes and located beside the specific markers illustrate the overall protein expression changes in this investigation, as follows: Thin arrows in green (phosphorylated form) and black (total form) depict the overall direction of the outcomes (increase/decrease) for the particular muscle protein; thick arrows in darker green represent the overall effect on stimulation/inhibition of Nrf2 signalling; the symbol § indicates a significant difference between the biopsies taken 60 s after the end of the exercise, i.e., between the legs recovering with and without ischaemia. A differential modulation due to FIO2 is illustrated by the presence of arrows in red (normoxia) and blue (severe hypoxia). The size of each arrow is commensurate with the magnitude of the change. Abbreviations not defined in the text: ARE, antioxidant response element; OS: oxidative stress. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)