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: Molecules

Article Title: Preferred Endocytosis of Amyloid Precursor Protein from Cholesterol-Enriched Lipid Raft Microdomains

doi: 10.3390/molecules25235490

Figure Lengend Snippet: Cellular cholesterol levels determined the localization of surface APP in lipid raft microdomains. To examine APP localization at the plasma membrane, cells were incubated with EZ-Link NHS-biotin at 4 °C to label all surface proteins. Then, biotin-labeled cell lysates were pooled for discontinuous sucrose density gradient to separate lipid raft and nonlipid raft fractions. Fractions 4 to 6 (lipid rafts, R) or 8 to 12 (nonlipid rafts, NR) were combined, and the same amount of protein was used for capturing biotin-labeled proteins with streptavidin beads. The biotin labeled proteins were run on Western blot, and detected with APP, β-actin, and caveolin (lipid raft marker) antibodies. ( a ) CHO PS1 WT cells were pretreated with 150 μM β-cholesterol before biotin-labeling. A typical result demonstrates the only surface APP levels from raft and nonraft fractions ( n = 6). ( b ) Quantitative analysis of surface APP from PS1 WT control and cholesterol-treated PS1 WT cells was shown ( n = 6). ( c ) CHO PS1 ΔE9 cells were pretreated with 5 mM MβCD to decrease cholesterol levels before labeling surface proteins. Representative Western blot result indicates surface APP levels from raft and nonraft fractions ( n = 5). ( d ) Quantitative analysis of surface APP from PS1 ΔE9 control and MβCD-treated PS1 ΔE9 cells was shown ( n = 5). Statistical analysis was carried out by one-way ANOVA: * p < 0.05, ** p < 0.01.

Article Snippet: After washing blocked membrane with PBS four times for 10 min, the membrane was incubated with the following primary antibodies at 4 °C for 16 h: APP (6E10; BioLegend, monoclonal, #803002), caveolin (cav-1; BD Transduction Laboratories, polyclonal, #610059), flotillin-1 (BD Transduction Laboratories, monoclonal, #610880), GAPDH (Cell Signaling Technology, Danvers, MA, USA, monoclonal, #14C10), β-actin (EnoGene, New York, NY, USA, monoclonal, #E12-041).

Techniques: Clinical Proteomics, Membrane, Incubation, Labeling, Western Blot, Marker, Control

Journal: Molecules

Article Title: Preferred Endocytosis of Amyloid Precursor Protein from Cholesterol-Enriched Lipid Raft Microdomains

doi: 10.3390/molecules25235490

Figure Lengend Snippet: Cellular cholesterol levels determined raft-dependent endocytosis of APP. To verify the contribution of lipid rafts to APP endocytosis, cells were incubated with EZ-Link NHS-SS-biotin at 4 °C to label surface proteins. Then, cells were incubated at 37 °C for 10 min to allow internalization of biotin-labeled surface proteins. After internalization, the remaining surface-bound biotin was removed and biotin-labeled cell lysates were loaded for discontinuous sucrose density gradient centrifugation to separate lipid raft and nonlipid raft fractions. Fractions 4–6 (lipid rafts, R) or 8–12 (nonlipid rafts, NR) were pooled, and biotin-labeled proteins were captured with streptavidin beads. Internalized biotin-labeled proteins were loaded on Western blots and detected with APP, β-actin, and caveolin (lipid raft marker) antibodies. ( a ) CHO PS1 WT cells were pretreated with 150 μM β-cholesterol before labeling surface proteins. Internalized biotin-bound APP levels from raft and nonraft fractions were detected by Western blotting ( n = 5). ( b ) Analysis of band densitometry shows the levels of internalized labeled APP from PS1 WT control and cholesterol-treated PS1 WT cells ( n = 5). ( c ) CHO PS1 ΔE9 cells were pretreated with 5 mM MβCD to reduce cholesterol levels before biotin-labeling. Internalized labeled APP from lipid raft and nonlipid raft fractions were monitored by Western blot ( n = 6). ( d ) The band densitometry of internalized labeled APP from lipid raft and nonlipid raft fractions was analyzed from PS1 ΔE9 control and MβCD-treated PS1 ΔE9 cells ( n = 6). Statistical analysis was carried out by one-way ANOVA: * p < 0.05, ** p < 0.01.

Article Snippet: After washing blocked membrane with PBS four times for 10 min, the membrane was incubated with the following primary antibodies at 4 °C for 16 h: APP (6E10; BioLegend, monoclonal, #803002), caveolin (cav-1; BD Transduction Laboratories, polyclonal, #610059), flotillin-1 (BD Transduction Laboratories, monoclonal, #610880), GAPDH (Cell Signaling Technology, Danvers, MA, USA, monoclonal, #14C10), β-actin (EnoGene, New York, NY, USA, monoclonal, #E12-041).

Techniques: Incubation, Labeling, Gradient Centrifugation, Western Blot, Marker, Control