Journal: Aging (Albany NY)

Article Title: PDCD10 promotes the aggressive behaviors of pituitary adenomas by up-regulating CXCR2 and activating downstream AKT/ERK signaling

doi: 10.18632/aging.204206

Figure Lengend Snippet: Activation of CXCR2 rescues the inactivation of AKT/ERK signaling and the tumor-suppressive effects induced by PDCD10 silencing. ( A , B ) Western blotting was performed to examine the phosphorylation levels of AKT and ERK1/2 after recombinant CXCL2 administration (200ng/ml) in Att-20 and TtT/GF cells with PDCD10 silencing. ( C , D ) CCK-8 assay was used to evaluate cell proliferation potential after CXCL2 administration in Att-20 and TtT/GF cells with PDCD10 silencing. ( E , F ) Relative migration rate of ATT-20 and TtT/GF cells with PDCD10 silencing was analyzed by scratch assay after CXCL2 administration (magnification: 100x). ( G , H ) Transwell invasion assay was employed to assess the invasion capacity of Att-20 and TtT/GF cells with PDCD10 silencing after CXCL2 administration (magnification: 200x). * P < 0.05.

Article Snippet: Recombinant mouse CXCL2 protein (R&D System) was added into the culture medium to study the signaling pathways in a subset of experiments as indicated.

Techniques: Activation Assay, Western Blot, Phospho-proteomics, Recombinant, CCK-8 Assay, Migration, Wound Healing Assay, Transwell Invasion Assay

Journal: Journal of Lipid Research

Article Title: CXCL3 positively regulates adipogenic differentiation

doi: 10.1194/jlr.M067207

Figure Lengend Snippet: mRNA inductions of chemokine and chemokine receptor genes during adipogenic or osteogenic differentiation of 3T3-L1 and ST2 cells. A, B: 3T3-L1 cells were induced to differentiate by a combination of DEX, insulin, and IBMX for 4 or 8 days. Total RNAs were isolated and reverse transcribed. The gene expression of fabp4, chemokine (A), and chemokine receptor (B) genes was analyzed by real-time PCR. Each experiment was performed at least three times producing consistent results. Relative mRNA expression levels in comparison with ribosomal protein L 13a are shown. Error bars represent SD. C, D: ST2 cells were cultured in adipogenic differentiation media as in (A) or osteogenic differentiation media (280 μM l-ascorbic acid 2-phosphate trisodium and 5 mM β-glycerophosphate) for 10 days. Gene expression levels of adipogenic (fabp4) and osteogenic (bglap) markers, and chemokines and chemokine receptors were analyzed as in A. E: The culture supernatants were collected from undifferentiated (day 0) and differentiated (days 4 and 8) 3T3-L1 cells. CXCL3 ELISA was performed at least three times in three different cells.

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

Techniques: Isolation, Reverse Transcription, Gene Expression, Real-time Polymerase Chain Reaction, Expressing, Comparison, Cell Culture, Enzyme-linked Immunosorbent Assay

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: 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: 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: 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: 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

Journal: Journal of Lipid Research

Article Title: CXCL3 positively regulates adipogenic differentiation

doi: 10.1194/jlr.M067207

Figure Lengend Snippet: CXCL3 is an accessorial factor for adipogenic differentiation. A: 3T3-L1 cells were cultured with the addition of either adipogenesis-inducing factors (DEX, insulin, and IBMX) or recombinant CXCL3 protein (10 ng/ml) for 6 days. Total RNAs were isolated and reverse transcribed. The expression of adipogenic marker genes 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: 3T3-L1 cells were cultured with the combination of adipogenic cocktail including DEX, IBMX, insulin, and CXCL3 for 5 days. Adipogenic marker expression was analyzed as in A.

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

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

Journal: Journal of Lipid Research

Article Title: CXCL3 positively regulates adipogenic differentiation

doi: 10.1194/jlr.M067207

Figure Lengend Snippet: CXCL3 expression is highly induced in the inflammation states of adipocytes. 3T3-L1 cells were cultured with or without addition of adipogenesis-inducing factors for 8 days. Undifferentiated or differentiated cells were stimulated by 10 ng/ml of LPS or 10 ng/ml of TNF-α for 3 h. Total RNAs were isolated and reverse transcribed. The expression of cxcl3 and cxcl13 genes 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).

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

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

Journal: Journal of Lipid Research

Article Title: CXCL3 positively regulates adipogenic differentiation

doi: 10.1194/jlr.M067207

Figure Lengend Snippet: Cxcl3 promoter activity is regulated by the binding of PPARγ2. A: Schematic diagram of putative PPARγ2 binding sites in the 5′ upstream region of the mouse cxcl3 gene. Six reporter plasmids containing variable lengths of cxcl3 gene upstream region are shown. A: pGL-1000. B: pGL4-885. C: pGL4-491. D: pGL4-448. E: pGL-420. F: pGL4-336. B: 3T3-L1 cells were stably transfected with one of the six pGL4.17-cxcl3 reporter plasmids described in A. The cells were induced to differentiate into adipocytes for 4 days. Cytoplasmic lysates were analyzed for luciferase activities in triplicate. Error bars represent SD. Three independent cell clones were isolated and analyzed for each reporter plasmid producing similar results. The results from a typical cell clone are shown for each construct. C: The upstream nucleotide sequence of the transcriptional starting point in the mouse cxcl3 gene. Two putative PPRE sites were mutated by site-directed mutagenesis (mD: −436 to −421; mE: −352 to −337) in the pGL4-1000 wild-type cxcl3 promoter construct to analyze their functional significance. The mutated bases are shown in italic lower cases. D: The mutated (mD, mE, and mD + mE) and the wild-type control constructs were stably transfected into 3T3-L1 cells. Luciferase reporter assay was performed as in B. E: 3T3-L1 cells were induced to differentiate for 4 days. Chromatins were extracted and immunoprecipitated with an antibody against PPARγ2. PCR analyses of the immunoprecipitated DNA were carried out. The positions of the PCR primer pairs are shown in C.

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

Techniques: Activity Assay, Binding Assay, Stable Transfection, Transfection, Luciferase, Clone Assay, Isolation, Plasmid Preparation, Construct, Sequencing, Mutagenesis, Functional Assay, Control, Reporter Assay, Immunoprecipitation

Journal: Journal of Lipid Research

Article Title: CXCL3 positively regulates adipogenic differentiation

doi: 10.1194/jlr.M067207

Figure Lengend Snippet: Schematic illustration of CXCL3-induced signaling pathway in adipocytes.

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

Techniques: