*2.7. Measurement of Mitochondrial Ca2*<sup>+</sup>

We used Rhod-2/AM (R1244, Thermo Fisher Scientific, USA), which demonstrates increased fluorescence upon Ca2<sup>+</sup> binding in the mitochondria, to determine mitochondrial free Ca2<sup>+</sup> [27]. Briefly, after washing samples twice with fresh PBS, dye (5 μM Rhod-2/AM) was slowly added along the sides of the single muscle fibers, followed by incubation in the dark at 37 ◦C for 30 min. After incubation, the glass slide-mounted Rhod-2/AM-loaded fibers were washed with fresh PBS three times (20 s/time, 1-min process). The slide was then quickly placed on the microscope stage, and the fibers were focused

in the bright field (20-s process) and scanned via laser confocal microscopy in combination with an Olympus FV10-ASW system (Japan) under 594-nm krypton/argon laser illumination, with fluorescence detected at 618 nm. Analysis and statistical methods were similar to those used for the measurement of cytoplasmic Ca2<sup>+</sup> mentioned above.

#### *2.8. Total RNA Extraction and Quantitative Real-Time Polymerase Chain Reaction (RT-PCR)*

As per Fu et al. (2016) [6] and in accordance with the manufacturer's protocols, we extracted total RNA from the muscle samples using an RNAiso Plus kit (TaKaRa Biotechnology, 9109, Dalian, China). RNA quality was characterized using the OD260/OD280 ratio, after which selected samples (those exhibiting OD260/OD280 > 1.8) were reverse transcribed into cDNA using an appropriate reagent (TaKaRa Biotechnology, RR036A China) and stored (−20 ◦C) for the following analyses. Here, qRT-PCR was undertaken using a SYBR Premix Ex Taq II kit (TaKaRa Biotechnology, RR820A, China), following the protocols stated by the manufacturer. The resultant dissolution and amplification curves were observed and selected, with the α-tubulin reference gene and 2−ΔΔct method then being applied to analyze the relative mRNA concentrations of STIM1, ORAI1, RyR1, LETM1, PMCA3, SERCA1, MCU, MICU1, MICU2, CALM, and α-tubulin. The primers used for the above genes (Sangon, Nanjing, China) are listed in Table 1.

**Table 1.** Primers used for quantitative real-time PCR experiments.


#### *2.9. Protein Extraction and Western Blotting Analysis*

Muscle samples (~0.1 g) were weighed and fully homogenized with 1 mM RIPA Lysis Buffer (Heart, WB053A, Xi'an, China), 1% protease inhibitor cocktail (Heart, WB053B, Xi'an, China), and 1% phenylmethylsulfonyl fluoride (PMSF, Heart, WB053C, Xi'an, China). After 15 min of centrifugation at 4 ◦C and 15,000 rpm, the supernatants were removed and placed into new tubes, with soluble protein concentrations then detected using a PierceTM BCA Protein Quantitation kit (Thermo Fisher Scientific, 23227, USA). The supernatants were mixed with 1 × SDS loading buffer (100 mM Tris, 5% glycerol, 5% 2-β-mercaptoethanol, 4% SDS, and bromophenol blue, pH 6.8) at a 1:4 *v*/*v* ratio, followed by boiling and then storage at −20 ◦C for further analysis.

Western blotting procedures were as described by Zhang et al. (2017) [28]. In brief, we first separated the muscle protein extracts using SDS-PAGE on 10% Laemmli gels (acrylamide/bisacrylamide ratio of 37.5:1 for STIM1, ORAI1, LETM1, PMCA3, SERCA1, MCU, MICU1, CALM) and on 6% Laemmli gels (acrylamide/bisacrylamide ratio of 37.5:1 for RyR1), respectively. Following electrophoresis (for 60 min at 120 V), the proteins were electrically transferred to 0.45-μm pore polyvinylidene difluoride (PVDF) membranes (Millipore, IPVH00010, Merck kGaA, Darmstadt, Germany) using the Bio-Rad (1703930) semidry transfer apparatus (Hercules, CA, USA) at 15 V for 30–40 min. We then blocked the membranes at room temperature for 2 h using 5% skim milk in TBST (containing 10 mM Tris-HCl, 150 mM NaCl, 0.05% Tween-20, pH 7.6), followed by overnight incubation at 4 ◦C with primary STIM1 (1:1000, CST, 5668S, Danvers, MA, USA), ORAI1 (1:1000, Thermo, MA5-15776, Eugene, OR, USA), RyR1 (1:1000, CST, 8153S, USA), LETM1 (1:1000, CST, 14997S, USA), PMCA3 (1:1000, Abcam, ab3530, Cambridge, UK), SERCA1 (1:1000, CST, 4219S, USA), MCU (1:1000, CST, 14997S, USA), MICU1 (1:750, CST, 12524S, USA), and CALM (1:1000, CST, 4830S, USA) antibodies in TBST containing 0.1% BSA. The membranes where then washed three times with TBST (10 min/time), followed by 1.5-h incubation at room temperature with horseradish peroxidase (HRP)-conjugated anti-rabbit or anti-mouse secondary antibodies (Thermo Fisher Scientific, A27014, USA). The membranes were again washed with TBST (four times × 10 min), with the resulting immunoblots being visualized using enhanced chemiluminescence reagents (Thermo Fisher Scientific, NCI5079, USA), in accordance with the manufacturer's instructions. Blot quantification was conducted using Image-Pro Plus 6.0 software. Total protein staining of the gel was used as the normalization control for all blots. In detail, as described previously, 0.5% 2,2,2-trichloroethanol (TCE) was first added to the gel [29–31]. After electrophoresis, the gel was irradiated on the UV platform of the electrophoresis gel imaging analysis system (G: box, GBOX Cambridge, UK) for 5 min, with the signal then being collected. As described previously [32,33], the original images captured with no gain were stored. After that, the fluorescence intensity of each lane (after removal of the background fluorescence intensity) was determined with Image-Pro Plus 6.0, with the internal reference being used to correct the fluorescence intensity of the target protein. Specificity detection of the complete SDS-PAGE lane for each antibody used in the present study is shown in Figure S1.

#### *2.10. Co-Localization Analysis of ORAI1*/*STIM1*

Briefly, 10-μm thick frozen cross-sections were cut from the muscle mid-belly at −20 ◦C with a CM1850 cryostat (Leica, Wetzlar, Germany). After fixing samples in 4% paraformaldehyde for 30 min, slices were permeabilized in 0.1% Triton X-100 for 30 min, blocked with 1% BSA in PBS at room temperature for 60 min, and then incubated at 4 ◦C overnight with an anti-ORAI1 antibody (1:50, Thermo, MA5-15776, USA). On the second day, after washing samples three times with PBS (10 min/time), the sections were incubated at 37 ◦C for 2 h with an Alexa Fluor FITC-conjugated secondary antibody (1:300, Thermo Fisher Scientific, Rockford, IL, USA). After again washing samples three times with PBS (10 min/time), the slices were incubated at 4 ◦C overnight with an anti-STIM1 antibody (1:300, CST, 5668S, USA). On the third day, after washing samples three times with PBS (10 min/time), the sections were incubated at 37 ◦C for 2 h with a 647-labeled IgG secondary antibody (1:200, Thermo Fisher Scientific, A-21235, USA). The slices were then washed three times with PBS (10 min/time) and dried and treated with anti-fade mounting medium (Life Technologies, 1427588, USA). Images were visualized and captured via confocal laser scanning microscopy (Olympus, FV1000, Japan) at a 40× objective magnification with krypton/argon laser illumination at 488 and 647 nm and captured at 526 and 665 nm. As previously described [34,35], the co-localization of ORAI1/STIM1 was calculated by Pearson's correlation coefficients using Image-Pro Plus 6.0.

#### *2.11. Statistical Analysis*

Data are presented as means ± SEM. SPSS Statistics 17.0 was used for all statistical tests. Group differences were determined via one-way analysis of variance (ANOVA) with Fisher's least significant difference (LSD) post hoc test. When no homogeneity was detected, ANOVA-Dunnett's T3 method was applied. A value of *p* < 0.05 was considered statistically significant.

#### **3. Results**

#### *3.1. Skeletal Muscle Mass and Single Muscle Fiber CSA of PL and AM during Di*ff*erent Hibernation Periods*

Changes in skeletal muscle morphology were observed by analyzing the muscle mass (MM) (Figure 2B) and muscle fiber CSA (Figure 2A,C) in different groups. The results showed that, compared with the SA group, slight decreases in muscle mass and single muscle fiber CSA (15–20%) were observed during hibernation.

**Figure 2.** Changes in muscle mass and single muscle fiber CSA in PL and AM muscles during different periods. (**A**) Representative fluorescence images of single muscle fiber CSA in PL and AM muscles. 400× magnification, scale bar = 100 μm. (**B**) Histogram depicting muscle mass of PL and AM muscles during different periods. (**C**) Histogram depicting single muscle fiber CSA in PL and AM muscles during different periods. CSA, cross-sectional area; PL, plantaris; AM, adductor magnus. SA, summer active group; PRE, pre-hibernation group; LT, late torpor group; IBA, inter-bout arousal group; ET, early torpor group; POST, post-hibernation group. Values are means ± SEM, n = 6–8. \* *p* < 0.05 compared with SA; # *p* < 0.05 compared with PRE.
