The 3T3-L1 cell was purchased from the Korean Cell Line Bank. Dulbecco's modified Eagle's medium (DMEM) and calf serum (CS) used for cell culture were purchased from HyClone (Cytiva) and penicillin/streptomycin was purchased from Corning, Inc. MDI used for differentiation was purchased from MilliporeSigma, fetal bovine serum (FBS) HyClone (Cytiva). Cyclosporine A (CsA) used as an inhibitor of CypB was purchased from Cell Signaling Technology, Inc. and Rapamycin used as an mTOR inhibitor was purchased from MilliporeSigma. RNA extraction was performed using TRIzol^® (Invitrogen; Thermo Fisher Scientific, Inc.) and cDNA synthesis was performed using a kit (Thermo Fisher Scientific, Inc.). Reverse transcription PCR was performed using a 7500 Reverse transcription PCR system (Applied Biosystems; Thermo Fisher Scientific, Inc.) and with Power SYBR^® Green PCR Master Mix (Applied Biosystems; Thermo Fisher Scientific, Inc.). All reagents used for Oil Red O Staining were purchased from MilliporeSigma. For cell cycle analysis, propidium iodide solution and RNase A solution were purchased from MilliporeSigma. Cell proliferation assay was performed using Chromo-CK cell viability assay reagent (Monobio). Antibodies used in the present study included mouse anti-β-actin; cat. no. sc-47778, mouse anti-vinculin; cat. no. sc-25336, mouse anti-C/EBPβ; cat. no. sc-7962, mouse anti-C/EBPα; cat. no. sc-166258, goat anti-FABP4; cat. no. sc-18661, rabbit anti-Adiponectin; cat. no. PA1-054 (Thermo Fisher Scientific, Inc.), mouse anti-hemagglutinin (HA); cat. no. sc-7392, mouse anti-CyclinD; cat. no. sc-8396, mouse anti-CyclineE; cat. no. sc247, mouse anti-CyclinA; cat. no. sc-271645, rabbit anti-AKT; cat. no. sc-8312 (Santa Cruz Biotechnology, Inc.), rabbit anti-PPAR; cat. no. 2443s, rabbit anti-CDK4; cat. no. 12790s, rabbit anti-p27; cat. no. 2552s, rabbit anti-pRB; cat. no. 8516s, rabbit anti-pAKT; cat. no. 9271s, rabbit anti-pmTOR; cat. no. 5536s, rabbit anti-mTOR; cat. no. 2983s, rabbit anti-phosphorylated (p-) p70S6K; cat. no. 9204L, rabbit anti-p70S6K; cat. no. 9202L (Cell Signaling Technology, Inc.), rabbit anti-CypB; cat. no. ab16045 (Abcam). Secondary antibodies used were goat anti-rabbit IgG; cat. no. 31460, goat anti-mouse IgG; cat. no. M32607 (Invitrogen; Thermo Fisher Scientific, Inc.) and rabbit anti goat IgG; cat. no. SA007-500 (GenDEPOT, LLC).

3T3-Ll cells were cultured in DMEM supplemented with 10% CS and 1% P/S at 37°C in a 5% CO₂ atmosphere. For 3T3-L1 cell differentiation, the cells when confluent were incubated for 2 more days (day 0) and then treated with MDI (500 µM IBMX, 0.25 µM dexamethasone, 10 µg/ml insulin) for 48 h at 37°C to induce differentiation. At 48 h after initiating 3T3-L1 cell differentiation, the DMEM medium (10% FBS, 1% P/S) was supplemented with 10 µg/ml insulin for 2 days (day 4). Day 4 after 3T3-L1 cell differentiation, DMEM medium (10% FBS, 1% P/S) was supplemented for 3 days (day 7). On day 7 of differentiation, differentiating cells were observed by capturing five different random fields of views for each well using a light microscopy at ×40 magnification (model IX73; Olympus Corporation). CsA (a CypB inhibitor) and mTOR-specific inhibitor rapamycin were dissolved in DMSO and added into the medium at 5 µg/ml (CsA) and 0.5 µM (Rapamycin), respectively at 37°C.

The 3T3-L1 cells were seeded at a density of 5.5×10⁵ cells/ml in a 60 mm dish or 12×10⁵ cells/ml in a 100 mm dish and then incubated for 24 h at 37°C in a 5% CO₂ atmosphere. Afterward, plasmid or siRNA transfection was performed using X-tremeGene^® HP DNA Transfection Reagent (MilliporeSigma) or X-tremeGENE siRNA Transfection Reagent (MilliporeSigma) according to the manufacturer's instructions and treated for 48 h at 37°C in a 5% CO₂ atmosphere. Subsequent experimentation was then performed 2 days later. CypB was HA-tagged at its 5' end and subcloned into a pcDNA vector (HA tag; 5′-TAC CCA TAC GAC GTC CCA GAC TAC GCT-3′). CypB-specific siRNA (5′GGA AAG ACU GUU CCA AAA A3′; D-001136-01-20) and scrambled siRNA (5′UAA GGC UAU GAA GAG AUA C3′) were purchased from Dharmacon.

Cells were lysed in RIPA buffer (50 mM Tris-HCl; pH 7.6; 150 mM NaCl; 1% Triton X-100; 1% Sodium deoxycholate). Protein concentrations was determined by BCA assay. Equal amounts of protein (20 µg) were separated with 6-15% SDS-PAGE gel and transferred to BioTrace NT nitro-cellulose membrane (Pall Life Sciences) and incubated with 5% blocking solution [(BSA; cat. no. A0100-010; GenDEPOT, LLC) or skimmed milk (cat. no. MB-S1667; MB cell)] for 1 h at room temperature. Afterward, the membrane was incubated with primary antibody overnight at 4°C. Next, the reaction was performed at room temperature for 1 h using a secondary antibody matching the primary antibody. The primary antibodies and secondary antibodies concentration used in the experiment were 1:1,000 and 1:10,000, respectively. The bands were detected with the enhanced chemiluminescence (Clarity™ Western ECL Substrate; cat. no. 170-5061; Bio-Rad Laboratories, Inc.), and the density and size of the bands were quantified using ImageJ 1.50i software (National Institutes of Health) by normalizing to β-actin and Vinculin.

The 3T3-L1 cells were seeded at a density of 5.5×10⁵ cells/ml in a 60 mm dish. Total RNAs were extracted by TRIzol^® (Invitrogen; Thermo Fisher Scientific, Inc.). cDNA was synthesized from 0.2 µg total RNA using RevertAid First Strand cDNA Synthesis kit (Invitrogen; Thermo Fisher Scientific, Inc.). Reverse transcription PCR was performed using 7500 Real Time PCR system with SYBR-Green PCR Master Mix according to the manufacturer's instructions. The Real Time PCR cycling conditions consisted of an initial activation step of 95°C for 15 min, denaturation of 95°C for 15 sec, annealing of 60°C for 30 sec, extension for 72°C for 30 sec; for 40 cycles. The primer sequences used are shown in Table I. The data were quantified using the 2^−ΔΔCq method and were normalized against the levels of GAPDH (37). These experiments were performed in triplicate and repeated five times with similar results.

After day 7, differentiated 3T3-L1 cells were washed three times with phosphate-buffered saline (PBS) and then fixed with 3.7% paraformaldehyde for 1 h at room temperature and washed thrice again with PBS. The cells were stained with 0.5% Oil Red O in isopropanol for 1 h at room temperature. Stained cells were washed three times with distilled water. Then, the stained cells were observed by capturing five different random fields of views for each well using a light microscopy at ×100 magnification (model IX73; Olympus Corporation) and lipid droplets were measured with a microplate reader (Molecular Devices, LLC.) at 510 nm.

The cells were harvested after MDI treatment for 48 h. Harvested cells were fixed 70% cold ethanol overnight at -20°C and washed with cold PBS. The fixed cells were centrifuged at 500 × g for 10 min at room temperature. The centrifuged cell pellet was resuspended in 10 µg/ml of RNase A at 37°C for 1 h. Then, 40 µg/ml propidium iodine solution was added at 37°C for 10 min in the dark. Cells were analyzed using a FACSCalibur system (BD Biosciences) according to the manufacturer's instructions. The results were averaged, and the percentage of cell cycle progression was calculated using the BD CellQuest™ Pro software (version 5.1; BD Biosciences).

Results were presented as the mean ± standard deviation. Error bars represent the mean ± standard deviation of three or five independent experiments performed in triplicate. Depending on the design of the experiment, data were analyzed one-way ANOVA with post hoc multicomparison analysis (Tukey's test) and comparisons between the two groups were performed using an unpaired two-tailed Student's t-test. P<0.05 was considered to indicate a statistically significant difference.

Post confluent 3T3-L1 pre-adipocytes were treated with MDI, an adipogenic inducer and were observed for 7 days to determine the expression level of CypB during adipocyte differentiation and adipogenesis. Fig. 1A shows the morphology image of 3T3-L1 cells during the differentiation period under a micro-scope and Fig. 1B shows that the lipid droplets were stained with Oil Red O staining assay for each day, confirming that the differentiation was normal. For reference, the expression levels of key adipogenic markers such as C/EBPβ, PPARγ, C/EBPα, FABP4 and adiponectin were monitored. The C/EBPβ gene is expressed in the early stage of adipogenesis and at the middle and later stages, PPARγ, C/EBPα, FABP4 and Adiponectin are expressed (38). As shown in Fig. 1C, CypB and C/EBPβ were transiently induced on day 2 and the protein expression levels of PPARγ, C/EBPα, FABP4 and Adiponectin were significantly increased after day 4. Notably, CypB protein was upregulated by 5-6 fold after day 2. mRNA levels of CypB were measured by RT-qPCR during adipocyte differentiation (Fig. 1D). Consistent with previous reports (16,38), the mRNA levels of adipogenic genes including C/EBPβ, PPARγ, C/EBPα and FABP4 were increased after treatment with MDI. The mRNA level of CypB was also significantly upregulated during MDI-induced adipocyte differentiation, consistent with the CypB protein expression data.

To investigate the functional roles of CypB in 3T3-L1 cell adipogenesis, the effects of CypB gene silencing were monitored using siRNA on adipocyte differentiation. Following CypB-knockdown cells were treated with MDI for 48 h to induce differentiation, Oil Red O staining assay was performed to evaluate the effects of CypB knockdown on lipid accumulation during adipocyte differentiation. The results showed that lipid accumulation was reduced by >50% in siCypB, compared to siControl (Fig. 2A). Next, western blot analysis was performed to monitor a key early-stage adipogenic marker, C/EBPβ. As shown in Fig. 2B, both CypB and C/EBPβ were significantly suppressed in CypB knockdown, compared with siControl. Other key adipogenic factors such as FABP4, C/EBPα and PPARγ were significantly downregulated in CypB knockdown, compared with siControl (Fig. 2C). Likewise, mRNA levels of key adipogenic markers such as FABP4, C/EBPα and PPARγ were significantly decreased in siCypB, compared to siControl (Fig. 2D). To confirm these results, CsA was used. Consistent with the CypB knockdown data, treatment with CsA inhibited lipid accumulation by >50%, compared to the DMSO (Fig. 2E). In addition, the CsA treatment led to a reduction the expression levels of key adipogenic markers such as FABP4, C/EBPα and PPARγ (Fig. 2F). These data suggested that CypB was required for adipogenesis in 3T3-L1 cells.

To investigate the functional role of CypB in adipogenesis, 3T3-L1 pre-adipocytes were transfected with a CypB expression vector. After the pre-adipocytes were treated with MDI for 2 days, Oil Red O staining assay was performed to evaluate the effects of overexpression of CypB on lipid accumulation during adipocyte differentiation. The results showed that overexpression of CypB increased lipid accumulation by almost two-fold, compared to pcDNA (Fig. 3A). Next, western blot analysis was performed to monitor the levels of a key early-stage adipogenic marker, C/EBPβ. As shown in Fig. 3B, C/EBPβ was significantly increased during CypB-overexpression, compared with pcDNA. Other key adipogenic factors such as FABP4, C/EBPα and PPARγ were significantly upregulated under the conditions of CypB-overexpression on day 7, compared with pcDNA (Fig. 3C). Likewise, CypB overexpression significantly increased mRNA levels of key adipogenic markers such as FABP4, C/EBPα and PPARγ, compared to pcDNA (Fig. 3D).

AKT/mTOR plays an important role in adipocyte differentiation, which affects lipid metabolism through the insulin pathway (39). To examine whether CypB affects the AKT/mTOR pathway in adipocyte differentiation, CypB-HA was overexpressed in 3T3-L1 preadipocytes before inducing the differentiation of 3T3-L1 cells with MDI, followed by western blotting. As shown in Fig. 4A, CypB overexpression activated the AKT/mTOR pathway. P70S6 kinase (P70S6K) is a mitogen-activated Ser/Thr protein kinase that is required for cell growth and G₁ cell cycle progression. CypB overexpression increased the expression of p-P70S6K. Next, the effects of CypB gene silencing were tested using siRNA on the AKT/mTOR pathway by western blotting (Fig. 4B). The expression levels of p-AKT, p-mTOR and p-P70S6K were significantly reduced in CypB knockdown. To confirm these results, rapamycin was used in the assay. Treatment with rapamycin negated the effects of overexpressed CypB on the AKT/mTOR pathway (Fig. 4C). These results suggested that CypB plays an important role in the AKT/mTOR pathway involved in adipocyte differentiation.

Next, how cell cycle events during mitotic clonal expansion are affected by CypB were examined. G₁-arrested 3T3-L1 cells synchronously re-enter the cell cycle and undergo mitotic clonal expansion during adipogenesis (11). As shown in Fig. 5A, CypB knockdown (siCypB) reduced the G₂/M population during adipocyte differentiation, compared to siControl. The DNA content of the cells in the G₁ phase in the siControl and siCypB groups was 37.6 and 54.0%, respectively, while that of the cells in the G₂/M phase in the two groups was 32.7 and 16.8%, respectively. Next, the expression of cell cycle-associated genes such as CyclinD, CylclinE, CyclinA, CDK4, p27 and pRB in CypB knockdown were monitored (Fig. 5B). Compared to siControl, expression levels of CyclinD, CylclinE, CyclinA, CDK4 and pRB were significantly reduced in siCypB, while p27 expression level was increased in siCypB by two-fold. These results suggested that the cell cycle transition from G₁ into the G₂/M phase was inhibited in CypB knockdown. As shown in Fig. 5C, overexpression of CypB increased the G₂/M population during adipocyte differentiation, compared to pcDNA control. The DNA content of the cells in the G₁ phase in pcDNA and CypB-HA groups was 36.6 and 23.8%, respectively, while that of the cells in the G₂/M phase in the two groups was 35.7 and 53.5%, respectively. Compared to pcDNA, expression levels of CyclinD, CylclinE, CyclinA, CDK4 and pRB were significantly increased by CypB overexpression, while p27 expression level was significantly decreased in CypB overexpression. These results suggested that the cell cycle transition from G₁ into the G₂/M phase was facilitated by CypB overexpression. Altogether, the data suggested that CypB regulates adipocyte differentiation by modulation of cell cycle progression.

Our previous study reported that CypB interacts with p300 to degrade CHOP in tumor cells (40). It has also been reported that CHOP inhibits adipogenesis and p300 is the transcriptional co-activator of C/EBPα (25,41). Therefore, whether CypB regulates the expression of CHOP in 3T3-L1 cells was tested by western blotting. CypB knockdown decreased the expression level of C/EBPβ and increased the expression level of CHOP (Fig. 6A). Compared with pc-DNA, overexpression of CypB increased the expression level of C/EBPβ and significantly decreased the expression level of CHOP (Fig. 6B). By contrast, CypB knockdown decreased the expression level of C/EBPβ and increased the expression level of CHOP (Fig. 6B). As expected, these data suggested that CypB regulates adipogenesis by regulating CHOP.

The present study showed that CypB regulated adipocyte differentiation through modulation of mitotic clonal expansion and AKT/mTOR signaling pathway (Fig. 7).