Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Dexras1 plays a crucial role in glucocorticoid-induced osteonecrosis of the femoral head by mediating imbalance between osteogenesis and adipogenesis

doi: 10.1007/s00018-026-06170-9

Figure Lengend Snippet: Dexras1 KO restores the osteogenic-adipogenic balance in the femoral head of GIONFH rats. ( A ) IF staining of the femoral head for OCN and PPARγ in each group. ( B ) Fluorescence intensity of OCN and PPARγ relative to that in the WT group by quantification analysis. n = 4 samples per group. ( C ) Relative mRNA expression of Runx2, OCN, PPARγ and C/EBPα of the femoral head in each group analyzed by RT-PCR. n = 4 samples per group. * p < 0.05, ** p < 0.01

Article Snippet: C/EBPα , Cell Signaling Technology (#2295) , 1:1000 (WB).

Techniques: Staining, Fluorescence, Expressing, Reverse Transcription Polymerase Chain Reaction

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Dexras1 plays a crucial role in glucocorticoid-induced osteonecrosis of the femoral head by mediating imbalance between osteogenesis and adipogenesis

doi: 10.1007/s00018-026-06170-9

Figure Lengend Snippet: Dexras1 KO restores the osteogenic-adipogenic balance of BMSCs isolated from GIONFH rats. ( A , B ) Relative mRNA expression of Dexras1, Runx2, ALP, PPARγ and C/EBPα of BMSCs in each group analyzed by RT-PCR. n = 3 independent cell cultures. ( C , D ) Expression levels of Dexras1, Runx2, ALP, PPARγ and C/EBPα of BMSCs in each group analyzed by western blot. n = 3 independent cell cultures. ( E , F ) ALP and Oil Red O staining of BMSCs from different groups. n = 3 independent cell cultures. * p < 0.05, ** p < 0.01

Article Snippet: C/EBPα , Cell Signaling Technology (#2295) , 1:1000 (WB).

Techniques: Isolation, Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Staining

Journal: Journal of Diabetes Investigation

Article Title: Kbtbd11 gene expression in adipose tissue increases in response to feeding and affects adipocyte differentiation

doi: 10.1111/jdi.12995

Figure Lengend Snippet: Kbtbd11 expression levels in epididymal white adipose tissue. (a) The expression of Kbtbd11 in epididymal white adipose tissue of C57BL/6J mice; mice were fasted for 24 h or fasted for 24 h/re‐fed for 12 h; n = 3 per group, * P < 0.01 versus fasted. (b) The expression of Kbtbd11 in epididymal white adipose tissue of diet‐induced obesity mice; diet‐induced obesity mice were fed a high‐fat diet for 1 month. The mice were fasted for 24 h; n = 3 per group, * P < 0.01 versus chow.

Article Snippet: Furthermore, Kbtbd11 knockdown inhibited adipogenesis, but only before MCE (not mature 3T3‐L1 adipocytes), and Kbtbd11 overexpression induced MCE, leading to the expression of Cebpa and Pparg (Figure c,f).

Techniques: Expressing

Journal: Journal of Diabetes Investigation

Article Title: Kbtbd11 gene expression in adipose tissue increases in response to feeding and affects adipocyte differentiation

doi: 10.1111/jdi.12995

Figure Lengend Snippet: Kbtbd11 messenger ribonucleic acid (mRNA) expression in differentiating 3T3‐L1 cells. Total RNA was extracted from 3T3‐L1 cells at day 0, 2, 4, 8 and 10 after the induction of differentiation by treatment with an adipogenic cocktail; n = 3 per group, * P < 0.01 versus day 0.

Article Snippet: Furthermore, Kbtbd11 knockdown inhibited adipogenesis, but only before MCE (not mature 3T3‐L1 adipocytes), and Kbtbd11 overexpression induced MCE, leading to the expression of Cebpa and Pparg (Figure c,f).

Techniques: Expressing

Journal: Journal of Diabetes Investigation

Article Title: Kbtbd11 gene expression in adipose tissue increases in response to feeding and affects adipocyte differentiation

doi: 10.1111/jdi.12995

Figure Lengend Snippet: Kbtbd11 messenger ribonucleic acid (mRNA) expression in response to adipogenic stimuli at early time points. 3T3‐L1 preadipocytes were cultured to confluence, serum‐starved for 12 h and treated with (a) 5 μg/mL insulin, (b) 2.5 μmol/L dexamethasone (DEX) or (c) 200 μmol/L 3‐isobutyl‐1‐methylxanthine (IBMX) for the indicated times; n = 3 per group, * P < 0.01 versus 0 h.

Article Snippet: Furthermore, Kbtbd11 knockdown inhibited adipogenesis, but only before MCE (not mature 3T3‐L1 adipocytes), and Kbtbd11 overexpression induced MCE, leading to the expression of Cebpa and Pparg (Figure c,f).

Techniques: Expressing, Cell Culture

Journal: Journal of Diabetes Investigation

Article Title: Kbtbd11 gene expression in adipose tissue increases in response to feeding and affects adipocyte differentiation

doi: 10.1111/jdi.12995

Figure Lengend Snippet: The effects of knockdown and overexpression of Kbtbd11 on 3T3‐L1 cellular differentiation. (a) The expression of Kbtbd11 messenger ribonucleic acid (mRNA) in Kbtbd11 knockdown 3T3‐L1 cells at day 8. Kbtbd11 knockdown adenovirus particles were used with either of the two independent Kbtbd11 short hairpin ribonucleic acid (shRNA) constructs (shRNA#1 and shRNA#2); n = 3 per group, * P < 0.01 versus LacZ‐specific short hairpin ribonucleic acid (shLacZ); (b) triglyceride accumulation in 3T3‐L1 cells on day 8, visualized using Oil Red O staining; (c) the mRNA levels in 3T3‐L1 cells expressing each shRNA at various time points after inducing differentiation; n = 3 per group, * P < 0.01 versus shLacZ; (d) the expression of Kbtbd11 mRNA in Kbtbd11 ‐overexpressing 3T3‐L1 cells at day 8. Cells were infected with adenoviral vectors for expressing green fluorescent protein (GFP) or mouse Kbtbd11 ; n = 3 per group, * P < 0.01 versus GFP; (e) triglyceride accumulation in 3T3‐L1 cells on day 6 was detected using Oil Red O staining. (f) Relative mRNA levels in each group of 3T3‐L1 cells at various time points after inducing differentiation; n = 3 per group, * P < 0.01 versus GFP.

Article Snippet: Furthermore, Kbtbd11 knockdown inhibited adipogenesis, but only before MCE (not mature 3T3‐L1 adipocytes), and Kbtbd11 overexpression induced MCE, leading to the expression of Cebpa and Pparg (Figure c,f).

Techniques: Knockdown, Over Expression, Cell Differentiation, Expressing, shRNA, Construct, Staining, Infection

Journal: Journal of Diabetes Investigation

Article Title: Kbtbd11 gene expression in adipose tissue increases in response to feeding and affects adipocyte differentiation

doi: 10.1111/jdi.12995

Figure Lengend Snippet: Effects of knockdown of Kbtbd11 on mature 3T3‐L1 adipocytes. (a) Triglyceride accumulation in Kbtbd11 ‐knockdown mature 3T3‐L1 adipocytes at 48 h after either of the two independent Kbtbd11 short hairpin ribonucleic acid (shRNA) adenoviral infections (shRNA#1 and shRNA#2) visualized using Oil Red O staining; (b) RNA was harvested at 48 h after adenoviral infection, and expression levels of Kbtbd11 , adipocytes ( Pparg and aP2 ) and inflammation markers ( Tnfa and Il6 ), and lipogenic ( Fasn ) and proapoptotic genes ( Bax and Bcl2 ) were measured using quantitative polymerase chain reaction; n = 3 per group, * P < 0.01 versus shLacZ.

Article Snippet: Furthermore, Kbtbd11 knockdown inhibited adipogenesis, but only before MCE (not mature 3T3‐L1 adipocytes), and Kbtbd11 overexpression induced MCE, leading to the expression of Cebpa and Pparg (Figure c,f).

Techniques: Knockdown, shRNA, Staining, Infection, Expressing, Real-time Polymerase Chain Reaction

Journal: Experimental and Therapeutic Medicine

Article Title: Cigarette smoke extract promotes proliferation of airway smooth muscle cells through suppressing C/EBP-α expression

doi: 10.3892/etm.2017.4126

Figure Lengend Snippet: Knockdown of calreticulin suppresses proliferation of human ASMCs. Human ASMCs were transfected with calreticulin siRNA or siRNA NC for 24 h and then stimulated with 10% of CSE for 24 h prior to analysis. (A) Reverse transcription -quantitative polymerase chain reaction and (B) western blot analyses were performed to determine the mRNA and protein levels of calreticulin and C/EBP-α in the ASMCs. (C) Immunostaining of human ASMCs was performed using antibody against C/EBP-α (red); nuclei were counter-stained with DAPI (blue). Magnification, ×200. (D) Cell proliferation was assessed by MTT assay. (E) Cell apoptosis was analyzed using the FITC Annexin V/Dead Cell Apoptosis kit. (F) Bar chart showing the percentage of apoptotic cells after 10% CSE treatment for 24 h. Values are expressed as the mean ± standard deviation. *P<0.05 vs. control. AMSC, airway smooth muscle cell; C/EBP-α, CCAAT/enhancer-binding protein alpha; CSE, cigarette smoke extract; siRNA, small interfering RNA; siRNA NC, scrambled control siRNA; FITC, fluorescein isothiocyanate; PI, propidium iodide.

Article Snippet: ASMCs were seeded into 6-well plates and incubated for 24 h prior to transfection with 50 nM control small interfering siRNA vector, siRNA calreticulin (5′-GGAGGAUGAUGAGGACAAATT-3′) or siRNA C/EBP-α (5′-GACAAGAACAGCAACGAGUTT-3′; Santa Cruz Biotechnology, Inc., Dallas, TX, USA).

Techniques: Knockdown, Transfection, Reverse Transcription, Real-time Polymerase Chain Reaction, Western Blot, Immunostaining, Staining, MTT Assay, Standard Deviation, Control, Binding Assay, Small Interfering RNA

Journal: Experimental and Therapeutic Medicine

Article Title: Cigarette smoke extract promotes proliferation of airway smooth muscle cells through suppressing C/EBP-α expression

doi: 10.3892/etm.2017.4126

Figure Lengend Snippet: Knockdown of C/EBP-α and calmodulin enhances ASMC proliferation. Human ASMCs were transfected with calreticulin siRNA together with either C/EBP-α siRNA or control siRNA for 24 h and then stimulated with 10% CSE for 24 h prior to the following analyses. (A) Reverse transcription-quantitative polymerase chain reaction and (B) western blot analyses were performed to determine mRNA and protein levels of C/EBP-α in the ASMCs. (C) Immunostaining of human ASMCs was performed using antibody against C/EBP-α (red); nuclei were counter-stained with DAPI (blue). Magnification, ×200. (D) Cell proliferation was analyzed by MTT assay. (E) Cell apoptosis was analyzed using the FITC Annexin V/Dead Cell Apoptosis kit. (F) Bar chart showing the percentage of apoptotic cells after 10% CSE treatment for 24 h. *P<0.05 vs. control. AMSC, airway smooth muscle cell; C/EBPα, CCAAT/enhancer-binding protein α; CSE, cigarette smoke extract; siRNA, small interfering RNA; siRNA NC, scrambled control siRNA; FITC, fluorescein isothiocyanate; PI, propidium iodide.

Article Snippet: ASMCs were seeded into 6-well plates and incubated for 24 h prior to transfection with 50 nM control small interfering siRNA vector, siRNA calreticulin (5′-GGAGGAUGAUGAGGACAAATT-3′) or siRNA C/EBP-α (5′-GACAAGAACAGCAACGAGUTT-3′; Santa Cruz Biotechnology, Inc., Dallas, TX, USA).

Techniques: Knockdown, Transfection, Control, Reverse Transcription, Real-time Polymerase Chain Reaction, Western Blot, Immunostaining, Staining, MTT Assay, Binding Assay, Small Interfering RNA

Journal: Cell Death and Differentiation

Article Title: MORC2 regulates C/EBPα-mediated cell differentiation via sumoylation

doi: 10.1038/s41418-018-0259-4

Figure Lengend Snippet: MORC2 regulates C/EBPα-mediated C2C12 muscle cell differentiation. a In differentiation medium MORC2 overexpressing C2C12 cells keep proliferating and do not start differentiation. Phase-contrast microphoto of C2C12 and LV-MORC2 differentiation cells at 0, 4, and 7 days of differentiation. Original magnification 10×. To induce skeletal muscle differentiation, when reaching 70–80% confluence, cells of infection with Lentivirus Flag-control vector (LV-NC) and Lentivirus Flag-MORC2 (LV-MORC2) were cultured in DM. DM was changed every 2 days and differentiation was completed in 7 days. b The ratios of myotube formation in LV-MORC2 cells (13.0% ± 1.5%) on day 6 of differentiation showed significant less than LV-control vector cells (36.4% ± 2.2%) P < 0.0001. c With the process from C2C12 myoblast to mature myocytes triggered by 2% horse serum, muscle-related protein molecules expression changed prior to simulation. The differentiating C2C12 myoblast cells was investigated by subjecting confluent C2C12 cells from GM (growth medium) to DM (differentiation medium) to induce differentiation for 7 days, and isolating mRNA extracts at the indicated time point. Real-time PCR detected the mRNA levels of myoD, myogenin, myosin and C/EBPα. The mRNA levels of MyoD (myogenic differentiation antigen), myogenin and myosin increased gradually in LV-control C2C12 cells (“3 group” and “5 group” compared to “0” group in upper panel) compared to “0 day” of differentiation (the mRNA levels of LV-control C2C12 in “0 day” of the differentiation were set to 1 and used as a control), but their expression in LV-MORC2 C2C12 cells showed no significant difference (“3 group” and “5 group” compared to “0” group in lower panel) compared to “0 day” (the mRNA levels of LV-MORC2 C2C12 were the relative ratio to the control vector in “0 day” of the differentiation, “0” group in lower panel). C/EBPα mRNA was decreased in the MORC2 overexpressing C2C12 compared to control C2C12 cells in “0 day” (Fig. 4c, lower panel compared to upper panel of “0 group”), but the C/EBPα mRNA expressed approximately equivalent levels in “3 days” and “5 days” compared to “0 day” (Fig. 4c, lower panel). d The expression levels of C/EBPα and MORC were detected with LV-MORC2 and control vector C2C12 cells during induction differentiation by western blotting. The differentiating C2C12 myoblast cells was investigated by subjecting confluent C2C12 cells to induce differentiation for 6 days, and isolating protein extracts at the indicated time point. “0 day” shown cells were cultured with growth medium. Total lysate from C2C12 cells undergoing differentiation was prepared on the days indicated and analyzed by immunoblotting with anti-MORC2, anti-C/EBPα and anti-GAPDH. The C/EBPα protein and its sumoylation levels are gradually increased in LV-control vector C2C12 cells (left panel, “5 days” and “3 days” compared to “1 day”), its protein and sumoylation levels were gradually decreased in LV-MORC2-C2C12 cells (right panel, “5 days” and “3 days” compared to “1 day”). e The C/EBPα-KO C2C12 cells were identified the knockout endogenous C/EBPα expression by western blot. C2C12 cells were transfected with the human C/EBPα double nickase plasmid (from Santa Cruz Company) and Selected with puromycin (2 g/mL) to product stable expressing C/EBPα-KO C2C12 cells. f MORC2 overexpression promotes the sumoylation of wild-type C/EBPα not mutant K161R in stable co-expressing C2C12 cells. These stable expressing of C2C12 cell lines including LV-control vector, LV-MORC2, LV-C/EBPα-WT, LV-C/EBPα-K161R, LV-MORC2-C/EBPα-WT and LV-MORC2-C/EBPα-K161R were transfected into T7-sumo1, western blot detected the sumoylation levels of C/EBPα with C/EBPα antibody. g MORC2 promotes the sumoylation of C/EBPα and its subsequent degradation. These stable expressing of C2C12 cell lines as showed in Fig. 4g were transfected into T7-sumo1 and HA-ub, western blot detected the sumoylation level of C/EBPα and its degradation. h MORC2 promotes C/EBPα-mediated C2C12 cell cycle transition from G1 to S. Flow cytometry analyzed the cell cycle transition with these stable expressing of C2C12 cell lines including LV-control vector, LV-MORC2, LV-C/EBPα-WT LV-C/EBPα-K161R, LV-MORC2-C/EBPα-WT and LV-MORC2-C/EBPα-K161R. i The died cell number of LV-NC and LV-MORC2 cells, cell viability was determined by Trypan blue dye exclusion assay. After differentiation induction, we counted dead cell number—collecting cell medium after 1 up to 4 days of differentiation—and expressed it as a percentage of dead cells, floating in medium, on total cells (sum of dead and viable cells, in suspension and adhering to plate). j MORC2 inhibits C/EBPα-mediated C2C12 cell differentiation via maintaining cell cycle progression. Flow cytometry analyzed cell cycle of LV-C/EBPα-WT, LV-MORC2 and LV-MORC2-C/EBPα-WT cells with the increasing differentiation induction days

Article Snippet: C2C12 cells were transfected with the human C/EBPα double nickase plasmid (from Santa Cruz Company) and Selected with puromycin (2 g/mL) to product stable expressing C/EBPα-KO C2C12 cells. f MORC2 overexpression promotes the sumoylation of wild-type C/EBPα not mutant K161R in stable co-expressing C2C12 cells.

Techniques: Cell Differentiation, Infection, Control, Plasmid Preparation, Cell Culture, Expressing, Real-time Polymerase Chain Reaction, Western Blot, Knock-Out, Transfection, Over Expression, Mutagenesis, Flow Cytometry, Exclusion Assay, Suspension