Journal: Journal of Lipid Research

Article Title: CXCL3 positively regulates adipogenic differentiation

doi: 10.1194/jlr.M067207

Figure Lengend Snippet: CXCL3 treatment promotes adipogenic differentiation of 3T3-L1 cells. A: 3T3-L1 cells were induced to differentiate by the addition of DEX, insulin, and IBMX with the treatment of 10 ng/ml of recombinant CXCL2, CXCL3, or CXCL13 protein for 8 days. Cells were stained with Oil Red O to determine lipid droplet appearances. After the staining, extracted dye was monitored spectrophotometrically at 540 nm. Each staining assay with three biological replicates was performed at three times. Error bars represent SD. B: After the treatment as in A, total RNAs were isolated and reverse transcribed. The gene expression of adipogenic markers was analyzed by real-time PCR. Each experiment was performed at least three times producing consistent results. Relative mRNA expression levels compared with RPL13a are shown. Error bars represent SD. Statistical significance was determined by Student’s t-test (*P < 0.01). C: After the treatment as in A, cells were lysed in RIPA lysis buffer at the indicated day. Cell lysates were separated by SDS-PAGE, and Western blotting was performed with the indicated antibodies.

Article Snippet: Reagents and antibodies Recombinant mouse CXCL2 and CXCL3 proteins were purchased from Prospec (Rehovot, Israel).

Techniques: Recombinant, Staining, Isolation, Reverse Transcription, Gene Expression, Real-time Polymerase Chain Reaction, Expressing, Lysis, SDS Page, Western Blot

Journal: Journal of Lipid Research

Article Title: CXCL3 positively regulates adipogenic differentiation

doi: 10.1194/jlr.M067207

Figure Lengend Snippet: CXCL3 treatment facilitates adipogenesis, but not osteogenesis, in ST2 cells. A: ST2 cells were cultured in adipogenic differentiation medium with the treatment of 10 ng/ml CXCL2, CXCL3, or CXCL13 for 4 or 10 days. Total RNAs were isolated and reverse transcribed. Adipogenic marker gene expression was analyzed by real-time PCR. Each experiment was performed at least three times producing consistent results. Relative mRNA expression levels compared with RPL13a are shown. Error bars represent SD. Statistical significance was determined by Student’s t-test (* P < 0.01). B: ST2 cells were cultured in osteogenic differentiation medium with 10 ng/ml of CXCL2 or CXCL3. Osteogenic marker gene expression was examined as in A. C: ST2 cells were cultured in adipogenic differentiation medium with the treatment of 10 ng/ml CXCL3 for the indicated days. Adipogenic marker gene expression was analyzed as in A.

Article Snippet: Reagents and antibodies Recombinant mouse CXCL2 and CXCL3 proteins were purchased from Prospec (Rehovot, Israel).

Techniques: Cell Culture, Isolation, Reverse Transcription, Marker, Gene Expression, Real-time Polymerase Chain Reaction, Expressing

Journal: Journal of Lipid Research

Article Title: CXCL3 positively regulates adipogenic differentiation

doi: 10.1194/jlr.M067207

Figure Lengend Snippet: Transfection with CXCL3- or CXCR2-specific siRNA inhibits adipogenic differentiation of 3T3-L1 cells. A: 3T3-L1 cells were incubated in adipogenic differentiation with CXCL3-specific siRNA. The supernatants of culture medium were collected from undifferentiated (day 0) and differentiated (day 8) cells. CXCL3 ELISA was performed at least three times in three different cells. B: 3T3-L1 cells were cultured in adipogenic differentiation medium with the transient transfection of CXCL1-, CXCL2-, CXCL3-, or CXCR2-specific siRNA for 8 days. Cells were stained with Oil Red O to determine lipid droplet appearances. After the staining, extracted dye was monitored spectrophotometrically at 540 nm. Each staining assay with three biological replicates was performed three times. Error bars represent SD. C: After the treatment as in B, total RNAs were isolated and reverse transcribed. The indicated gene expression was analyzed by real-time PCR. Each experiment was performed at least three times producing consistent results. Relative mRNA expression levels compared with RPL13a are shown. Error bars represent SD. Statistical significance was determined by Student’s t-test (* P < 0.01). D: After the treatment as in B, cells were lysed in RIPA lysis buffer at the indicated day. Cell lysates were separated by SDS-PAGE, and Western blotting was performed with the indicated antibodies. E: 3T3-L1 cells were cultured in adipogenic differentiation medium with the transient transfection of CXCL3-specific siRNA and CXCL3 recombinant proteins for 8 days. Cells were lysed in RIPA lysis buffer at the indicated day. Cell lysates were separated by SDS-PAGE, and Western blotting was performed with the indicated antibodies.

Article Snippet: Reagents and antibodies Recombinant mouse CXCL2 and CXCL3 proteins were purchased from Prospec (Rehovot, Israel).

Techniques: Transfection, Incubation, Enzyme-linked Immunosorbent Assay, Cell Culture, Staining, Isolation, Reverse Transcription, Gene Expression, Real-time Polymerase Chain Reaction, Expressing, Lysis, SDS Page, Western Blot, Recombinant

Journal: Journal of Lipid Research

Article Title: CXCL3 positively regulates adipogenic differentiation

doi: 10.1194/jlr.M067207

Figure Lengend Snippet: CXCL3 promotes c/ebpb and c/ebpd expression through ERK and JNK activation. A: 3T3-L1 cells were stimulated by 10 ng/ml of recombinant CXCL2, CXCL3, or CXCL13. Cells were lysed in RIPA lysis buffer at the indicated time. Cell lysates were separated by SDS-PAGE and Western blotting was performed with the indicated antibodies. B: 3T3-L1 cells were pretreated with CXCR2 siRNA for 24 h. After the siRNA transfection, cells were stimulated as in A. ERK and JNK phosphorylation was analyzed by Western blotting. C: 3T3-L1 cells were pretreated with 2.5 μM of U0126, 5 μM of SB203580, or 2.5 μM of SP600125 for 1 h. After the treatment, cells were stimulated with 10 ng/ml of CXCL3 for 3 h. Total RNAs were isolated and reverse transcribed. The gene expression of cebpb and cebpd was analyzed by real-time PCR. Each experiment was performed at least three times producing consistent results. Relative mRNA expression levels compared with RPL13a are shown. Error bars represent SD. Statistical significance was determined by Student’s t-test (* P < 0.01). D: After the treatment as in B, CXCL3-induced cebpb and cebpd expression was analyzed by real-time PCR.

Article Snippet: Reagents and antibodies Recombinant mouse CXCL2 and CXCL3 proteins were purchased from Prospec (Rehovot, Israel).

Techniques: Expressing, Activation Assay, Recombinant, Lysis, SDS Page, Western Blot, Transfection, Phospho-proteomics, Isolation, Reverse Transcription, Gene Expression, Real-time Polymerase Chain Reaction