We used male wild-type (WT) and Rad6B knockout (KO) mice throughout the experiments and were obtained from COMM (MGI: 102944). The embryonic stem cells of Rad6B knockout C57BL/6 were generated from the knockout mouse project (KOMP) library (UC Davis), embryonic stem cells were implanted into the uterus of mice to breed transgenic mice (Guo et al., 2018; Guo et al., 2019). These two mice strains were divided into 1-, 3-, and 12-month-old groups. All animals were housed at the Lanzhou University Animal Center. The mice were kept in standard animal facilities and given free access to food and water, and efforts were made to alleviate the suffering of the mice.

Murine skin fibroblasts were isolated from dorsal skin tissues, and the 0.5 × 1 cm full-thickness dorsal skin tissues were obtained. Briefly, skin tissues were steeped with 75% alcohol for about 30 s and subsequently washed three times in phosphate-buffered saline (PBS) with antibiotics (penicillin and streptomycin). Afterwards, the skin tissues were cut into small pieces and incubated with Type I collagenase (BIOSHARP) at 37°C for 2–3 h. After being centrifuged and washed repeatedly, the skin fibroblasts isolated were grown in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS) in an incubator at 37°C with 5% CO₂.

Adenosine triphosphate (ATP) contents of mouse skin tissues were detected using a commercial ATP Content Assay Kit (Solarbio, BC0300). Briefly, skin tissues (0.1 g) were lysed with 1 mL extraction buffer and centrifuged at 8000 × g for 10 min, the supernatant was collected, and 500 mL of chloroform were added, after which the samples were centrifuged at 10000 × g for 3 min. The absorbance of the supernatant was measured immediately using the Automatic Enzyme Marker System (TECAN, SPARK, 1710002409) at 340 nm. Relative ATP contents were normalized against the control samples.

For this, the dorsal skins of the mice were fixed in 4% buffered formalin, embedded in paraffin, and sectioned into 5 μm thick slices. After dewaxing, the sections were stained with hematoxylin and eosin (H&E). For immunohistochemistry analyses, the paraffin sections were incubated with anti-TFAM (Abcam, ab272885), anti-PGC-1α (Proteintech, 66369-1-AP) primary antibodies, subsequently with biotin-conjugated, affinity-purified goat anti-rabbit IgG (H&L) and HRP-conjugated streptavidin at 37°C, with 3,3-diaminobenzidine serving as the chromogenic substrate. For Masson’s trichrome staining, the sections were used sequentially with hematoxylin and ferric oxide, ponceau, glacial acetic acid, phosphomolybdic acid, and aniline blue, and then the sections were mounted with neutral gum.

Skin tissues were ground and lysed with radioimmunoprecipitation assay (RIPA) lysis buffer (Solarbio, 20180929) containing 1% phenylmethylsulfonyl fluoride (PMSF, Solarbio, 2877K). After being centrifuged, the supernatant was collected, and the protein concentration was determined using the bicinchoninic acid (BCA) Protein Assay Kit (Solarbio, PC0020). Afterwards, the samples were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene fluoride (PVDF) membranes. The membranes were incubated with primary antibodies for 8–16 h at 4°C and incubated with appropriate peroxidase-conjugated secondary antibodies for 1.5 h at 28°C, and the signals were visualized by enhanced chemiluminescence. The following primary antibodies were used in this study at the dilutions indicated: anti-Rad6B (Proteintech, 10733-1-AP, 1:700), anti-PGC-1α (Proteintech, 66369-1-AP, 1:1000), anti-Nrf2 (Immunoway, YT3189, 1:2000), anti-TFAM (Abcam, ab272885, 1:800), anti-Actin (Proteintech, 20536-1-AP, 1:5000), anti-ND1 (Proteintech, 19703-1-AP, 1:1000), anti-UQCRC2 (Proteintech, 14742-1-AP, 1:1000), anti-SDHB (Proteintech, 10620-1-AP, 1:20000), anti-COX4 (Proteintech, 11242-1-AP, 1:10000), anti-ATP5A (Proteintech, 14676-1-AP, 1:1000), anti-Mfn1 (Proteintech, 13798-1-AP, 1:700), anti-Mfn2 (Proteintech, 12186-1-AP, 1:4000), anti-Drp1 (Proteintech, 12957-1-AP, 1:1500), anti-p16 (Abcam, ab51243, 1:4000), anti-p21 (Abcam, ab109199, 1:1000), anti-P62 (Proteintech, 18420-1-AP, 1:2000), anti-LC3 (Proteintech, 14600-1-AP, 1:1000).

For OCR measurements, primary mouse skin fibroblasts were seeded on Seahorse cell culture plates at 2 × 10⁴ cells/well and incubated for 10–16 h. Before the examination, the medium was exchanged for 500 µL of assay medium (pH 7.4), which was composed of 10 mM glucose, 1 mM pyruvate, and 2 mM glutamine in XF Base Medium. Inhibitors of electron transport chain (ETC) complexes were injected into different ports of the Seahorse cartridge, including oligomycin A (oligo, 1 µM), carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP, 1 µM), antimycin A (AA, 0.5 µM), rotenone (rot, 0.5 mM), after which the corresponding OCR values were measured with the XF24 Seahorse Biosciences Extracellular Flux Analyzer (Seahorse Bioscience, 102238-100). Five replicates were analyzed for each experimental group.

For ECAR measurements, primary skin fibroblasts were plated on Seahorse cell culture plates at 2 × 10⁴ cells/well and incubated as described above. Experiments were performed in assay medium (pH 7.4) with 1 mM glutamine. Additional reagents required, i.e., glucose (10 mM), oligo (1 µM), and 2-deoxyglucose (2-DG, 50 mM), were injected into different ports of the Seahorse cartridge, after which the ECAR values were measured. Five replicates were performed for each experimental group.

To evaluate intracellular ROS levels, primary cells were incubated with 10 μM 2’,7’-dichlorodihydrofluorescein diacetate (DCFH-DA, Solarbio, CA1410) for 20 min at 37°C, subsequently washed three times with serum-free medium, and imaged using an Olympus fluorescence microscope (BX53).

For mitochondrial membrane potential, primary skin fibroblasts were loaded with MitoTracker Red (Solarbio, M9940) in serum-free medium for 15 min at 37°C in the dark, washed three times with PBS, and counterstained with DAPI before being imaged by laser confocal microscopy (Leica, TCS-SP8).

Primary skin fibroblasts were fixed in 4% paraformaldehyde, subsequently washed twice with PBS, and permeabilized using 0.5% Triton X-100. After being blocked with 3% bovine serum albumin in PBS, the cells were incubated with the primary antibodies against Vimentin (Proteintech, 10366-1-AP, 1:2000), COX4, and ND1 at 4°C for 8–16 h. For detection, goat anti-rabbit IgG-FITC (Abcam, ab6717) and anti-rabbit IgG-Cy3 (Abcam, ab6939) secondary antibodies were used according to standard immunostaining procedures. The nuclei were counterstained with 4’,6-diamidino-2-phenylindole (DAPI).

Cell viability was measured using the Cell Counting Kit-8 (CCK-8, Dojindo). To that end, primary fibroblasts were plated at a density of 3 × 10³ cells/well on 96-well plates and were incubated at 37°C for 24, 48, and 72 h, respectively, after which 100 µL of the assay reagent were added per well, and the plates were incubated for another 3 h. Absorbance was recorded at 450 nm using enzymatic activity as a marker, and relative cell viability was calculated.

The Real-Time Cellular Analysis System (ACEA Biosciences, xCELLigence RTCA S16) was used for measuring cell proliferation. After having determined the background impedance value of the assay plate (E-Plate), primary skin fibroblasts (3 × 10³ cells/well) were added, and the assay plate was placed on the test bench and incubated for 72 h. Growth curves were then recorded and analyzed.

Primary skin fibroblasts were seeded on 24-well plates at 2 × 104 cells/well and incubated for 48 h. Afterwards, the cells were prepared using the commercial Senescence β-gal Staining Kit (Beyotime) according to the manufacturer’s instruction and imaged using an Olympus fluorescence microscope.

Primary skin fibroblast clusters were fixed with 2.5% glutaraldehyde in 0.1 M PBS (pH 7.4), washed three times with PBS, and stained with 1% osmium tetroxide. Subsequently, the samples were dehydrated and embedded, and ultrathin sections (70 nm thick) were obtained using an ultramicrotome. Then, the sections were stained with 2% uranyl acetate and lead citrate and viewed under a transmission electron microscope (TEM, HITACHI HT7700).

Total DNA was isolated from skin tissues using Wizard^® SV Genomic DNA Purification System (Promega), and 1 µL DNA solution was added to PCR mixture for amplification. Subsequently, the 1% agarose gel was prepared, heated, and mixed, then poured into the gel mold to solidify for 30 min. Electrophoresis with 6 µL amplification product was added into each well (constant voltage 100 V, 40 min). After electrophoresis, the agarose gel was imaged with a multifunctional molecular imaging analysis system (Azure Biosystems, C500), and the genotype was identified. The primer sequences were used as following: Rad6B (5’- CCCCTGTGTATGTGTTATACCATCC-3’, 5’-TGAATTCAGGACTCAGGGATGCCTG-3’).

The data were analyzed using SPSS17.0 software. Figures were generated using Origin 8.5 software. Student’s t-test was used to assess differences between two groups. A P-value < 0.05 was considered statistically significant.

The results of the genotyping of WT and Rad6B KO C57 mice are shown in Suppl. Fig. S1. H&E staining of the skin showed that its thickness was reduced in KO groups, especially in 1-month-old KO mice compared with the WT group (Figs. 1A–1B). Quantitative histomorphometry revealed that the number of hair follicles in anagen were increased in 1-month-old WT groups, and there was evidence of a raised number of hair follicles in telogen in 1-month-old KO groups compared with other groups. Moreover, the number of hair follicles in the catagen was enhanced in 3-, and 12-month-old groups, especially in the 12-month-old KO group (Figs. 1C–1E). Masson’s trichrome staining showed that the collagen fiber content was also decreased in KO groups but significantly reduced in the 12-month-old KO group (Figs. 1F–1G). Taken together, the phenotypic skin-related differences between WT and KO mice indicated that Rad6B KO might induce alterations in skin physiological structure with age.

Mice with this genotype are known to feature decreased protein degradation and DNA damage repair capacities. In this study, the metabolism states of cells in the skin were determined. Immunohistochemical staining of samples from each group showed that cells positive for proliferator-activated receptor-γ co-activator 1α (PGC-1α), a regulator of mitochondrial synthesis, were less frequently detected in the skin of both 1- and 12-month-old KO mice, but there was no striking difference in the 3-month-old group (Figs. 2A, 2B). The number of mitochondrial transcription factor A (TFAM) positive cells in the skin was also decreased in both 1- and 12-month-old KO mice (Figs. 2C, 2D). Consistently, the expression of PGC-1α and TFAM proteins in skin tissues was decreased, especially in the 1- and 12-month-old KO groups (Figs. 2E–2G). Nrf2 is involved in the regulation of oxidative stress in the case of mitochondrial dysfunction (Sabouny et al., 2017; Hu et al., 2018). In this study, the expression of nuclear factor (erythroid-derived 2)-like 2 protein (Nrf2) was down-regulated in the KO group (Figs. 2E and 2H), which might have been indicative of different levels of ROS in WT and KO groups. These results suggested that Rad6B KO interfered with the metabolism of cells resident in mouse skin, and this phenotype was particularly prominent in both the 1- and 12-month-old groups.

The cells of the skin are highly proliferative relative to those in other organs; therefore, the mitochondria are presumed to be highly active and of prime importance. To investigate whether mitochondrial function was changed due to Rad6B KO, the expression of five mitochondrial complex proteins was analyzed. As shown in Figs. 3A, 3B and 3E, loss of Rad6B caused evident alternations in ND1 and COX4 expression profiles, which correspond to complexes I and IV, respectively. Protein expression analyses revealed that ND1 was markedly increased in the 1-month-old KO group but decreased in the 12-month-old KO group. Interestingly, COX4 expression was depressed in mice of the 1-month-old KO group and enhanced in those of the 12-month-old KO group. However, expression of SDHB, URCRC2, and ATP5A, subunits of complexes II, III, and V, respectively, was unchanged in each group (Figs. 3C, 3D and 3F). Subsequently, we examined the ATP content of skin tissues in each group, and the results showed that the ATP content was increased in 1-month-old KO mice but diminished significantly in 12-month-old KO mice compared with WT mice (Fig. 3G). Evidence has emerged that mitochondrial fusion and fission play important roles in cell metabolism and growth (Abate et al., 2019). Therefore, in the present study, we further investigated mitochondrial fusion (mitofusin 1 [MFN1] and mitofusin 2 [MFN2]) and fission (dynamin-related protein 1 [DRP1]) proteins in mouse skins. As shown in Figs. 3H–3K, expression of MFN1, MFN2, and DRP1 was down-regulated in the 12-month-old KO group compared with the WT group, but no differences were observed between other age groups. Hence, we speculated that the whole mitochondrial mass and status in the skin were degenerating with age in the case of Rad6B KO mice. Taken together, these data suggested that Rad6B KO reduced the activity of mitochondrial complexes I and IV, thus impairing mitochondrial function. Moreover, mitochondrial dynamics were declining with old age.

We extracted primary fibroblasts from mouse skins for the following experiments. First, the mitochondrial membrane potential of cells from each group was analyzed with MitoTracker Red (Figs. 4A, 4B), and the results revealed that the fluorescence intensity was markedly decreased in the 12-month-old KO group. As oxidative stress plays an important role in aging, we then clarified whether differences in ROS levels were related to Rad6B KO and mitochondrial complex dysfunction. As shown in Figs. 4C–4D, compared with WT cells, the ROS level was significantly increased in the KO groups. In addition, electron microscopic analysis showed that there was no striking difference in mitochondrial morphology in the 1- and 3-month-old groups; however, diminished mitochondrial cristae, elevated swollen and degenerated mitochondria were observed in cells of the 12-month-old KO group (Fig. 4E). To evaluate the viability and proliferation ability of the cells, CCK-8 and RTCA assays were performed, respectively. Rad6B KO resulted in a significant decrease in cell viability and proliferation ability over time (Figs. 4F–4I), and the impact on cell proliferation was particularly pronounced in the 12-month-old KO group. These results suggested that loss of Rad6B led to the dysfunction and destruction of the mitochondrial network in primary skin fibroblasts.

In fact, it has been shown that autophagy is a hallmark of the senescence process, and the mitophagy markers P62 and LC3-II/LC3-I also seem to be changed in the senescence process (Moreira et al., 2017; Bakula and Scheibye-Knudsen, 2020). Based on this, we decided to investigate the mitophagy proteins in primary skin fibroblasts. As shown in Figs. 5A–5C, the rate between microtubule-associated protein 1 light chain 3 (LC3)-II and LC3-I was significantly highest in the 12-month-old KO group. Consistently, expression of the Sequestosome 1 (P62) was also increased dramatically in the 12-month-old KO group compared had increased significantly in primary skin fibroblasts of the 12-month-old KO group (Figs. 5D and 5E). In line with this, p16 and p21 expression increased with age with the WT groups. To test whether Rad6B KO was associated with accelerated aging, we evaluated senescence-associated SA-β-gal activity, as well as the expression of p16 and p21 proteins, which are known biomarkers of aging, for each group. SA-β-gal activity and was hence highest in the 12-month-old KO group (Figs. 5F–5H). These data indicated that Rad6B KO caused the elimination of damaged mitochondria through mitophagy was disturbed, leading to accumulation of damaged mitochondria and accelerated aging.

In a previous study, the ATP content was changed in skin tissues from Rad6B KO mice (Fig. 3G), allowing us to speculate that OXPHOS in mitochondria of skin cells was affected by Rad6B KO. Moreover, considering that glycolysis was probably coupled with this cellular response, the capacity of OXPHOS and glycolysis of primary skin fibroblasts was examined using the Seahorse XF24 Extracellular Flux Analyzer. As shown in Fig. 6, the mitochondrial OCR was elevated in the 1-month-old KO group, and the ECAR was reduced (Figs. 6A and 6D). Dramatically decreased sOCR and increased EACR values were observed in the 12-month-old KO group compared with the WT group (Figs. 6C and 6F). It was considered that glycolysis might have been redeployed to compensate for the reduction in mitochondrial OXPHOS in the case of Rad6B KO. The OCR and ECAR values measured in the 3-month-old groups showed no differences (Figs. 6B and 6E). Together, these results clearly indicated that Rad6B KO caused a switch from OXPHOS to glycolysis with age, which was probably realized by reprogramming the metabolic network, thus compensating the lack of ATP production in old primary skin fibroblasts featuring a KO of Rad6B.