Fig 2: Total CaMKII isoforms in human skeletal muscle in response to incremental exercise to exhaustion in normoxia and severe hypoxia, and post-exercise ischaemia. Levels of protein expression of (A) Total CaMKII ßM, (B) Total CaMKII dA, and (C) Total CaMKII ?. Total CaMKII dD is included in Fig. 2E. Nx: normoxia session (FIO2 = 0.21, PIO2 = 143 mmHg); Hyp: severe normobaric hypoxia session (FIO2 = 0.104, PIO2 = 73 mmHg); Pre: before exercise; Post: 10 s after exercise cessation during ischaemic recovery; Oc1m: 60 s after exercise cessation during ischaemic recovery; FC1m: 60 s after exercise cessation during recovery with free circulation. n = 11 for all conditions except for Oc1m Nx (n = 9). See Fig. 1 for a detailed description of the experimental phases. Values presented are means ± standard errors and expressed in arbitrary units (a.u.). No statistically significant effects were detected.

Fig 3: WNT5a mediated decrease in active-CTNNB1 is dependent on Ca2+/calmodulin-dependent protein kinase II activity. (A) OSE cells were treated with a CAMKII inhibitor (KN-93) for 1 h before challenge with WNT5a for 2 h. Western blot analyses were performed to detect active and total CTNNB1 and phosphorylated and total CAMKII. Representative immunoblots show 3 samples per treatment from three independent experiments. Quantitative analyses of active-CTNNB1/CTNNB1 and p-CAMKII/CAMKII protein levels for each time point in OSE are presented below the blots. Western blot for each protein is normalized to ACTB of their own blot (n = 3) and the representative image of ACTB belongs to active-CTNNB1 and p-CAMKII blots. Data are means ± SEM of three independent replicates. Different letters above histograms indicate significant differences among treatments. ANOVA with Tukey’s post-test, P < 0.05. (B) Representative images of immunofluorescence staining for active-CTNNB1 on OSE treated with an inhibitor of CAMKII (KN-93) for 1 h before challenge with WNT5a for 2 h. Mean relative fluorescence units for each group are indicated at the bottom. ANOVA with Dunnett’s post-test, ***P < 0.001. Scale bar = 50 µm.

Fig 4: WNT5a modulated the activity of multiple signaling pathways in OSE cells. (A) OSE cells were cultured and challenged with WNT5a for the times shown. Total cell protein was used to measure active and total CTNNB1 and phosphorylated and total CAMKII by western blot. Representative immunoblots show 3 samples per time point from three independent experiments. Quantitative analyses of active-CTNNB1/CTNNB1 and p-CAMKII/CAMKII protein levels for each time point in OSE are presented below the blots. Western blot for each protein is normalized to ACTB of their own blot (n = 3) and the representative image of ACTB belongs to active-CTNNB1 and p-CAMKII blots. Data are means ± SEM of three independent replicates. ANOVA with Dunnett’s post-test, **P < 0.01, indicates significant differences from control vs. WNT5a treatment. (B) Representative images of immunofluorescence staining for active-CTNNB1on OSE cells treated with WNT5a for 2 h (n = 3). Scale bar = 50 µm.

Fig 5: UVR-induced phosphorylation of CaMKII and keratinocyte phagocytosis was regulated by TRPA1 in HaCaT cells. TRPA1 channels promoted phosphorylation of CaMKII (A and B), (A) the protein levels of CaMKII and phosphorylated CaMKII (pCaMKII) were measured by Western blot in HaCaT cells, which were treated with UVA or UVB exposure, JT010 (1 µM, TRPA1 agonist), HC-030031 (10 µM, TRPA1 antagonist). (B) Relative protein levels of pCaMKII to CaMKII. Values are mean value of the ratio relative to control from three independent tests ± SD. (C) Quantification of fluorescence intensity indicating the uptake of fluorescent microspheres by HaCaT cells detected by Flow cytometry. Values are expressed as mean ±SD from three independent tests. (D) Fluorescent microspheres uptake was observed by fluorescence confocal microscopy, with red fluorescence indicating microspheres (0.5 µm microspheres), and green fluorescence indicating cell membrane stained with DiO. HaCaT cells were treated with UVA or UVB exposure, or JT010 (1 µM), HC-030031 (10 µM). Scale bar: 50 µm. (E) Quantification of fluorescent microspheres per HaCaT cell. Values are expressed as mean ± SD from three independent tests. (**p < 0.01; ***p < 0.001).