Journal: Nutrients

Article Title: Tryptophan Attenuates Chronic Restraint Stress-Induced Intestinal Injury Through Modulation of Intestinal Barrier Integrity and Gut Microbiota Homeostasis

doi: 10.3390/nu17060975

Figure Lengend Snippet: Figure 3. Tryptophan alleviates CRS-induced intestine leakage and epithelial apoptosis. (A) Serum intestinal fatty acid-binding protein content by ELISA; (B) FITC-dextran content in serum; (C) TUNEL staining plots; (D) TUNEL-positive cell counts; different letters denote significant differences between groups, p < 0.05. Ctrl refers to control group.

Article Snippet: We obtained the Mouse Corticosterone (CORT) ELISA Kit (Elabscience, Shanghai, China), FITC—dextran (4 kD) (Sigma, Saint Louis, MI, USA); the mouse intestinal fatty acid-binding protein (FABP) ELISA Kit (CUSABIO, Wuhan, China); and all of the 11 SCFA standards (ZZ Standards Co., Ltd., Shanghai, China).

Techniques: Binding Assay, Enzyme-linked Immunosorbent Assay, TUNEL Assay, Staining, Control

Journal: The Journal of Veterinary Medical Science

Article Title: Cystine transporter expression is a marker to identify a subpopulation of canine adipose-derived stem cells

doi: 10.1292/jvms.19-0373

Figure Lengend Snippet: Primers used in this study

Article Snippet: The cells were then incubated for 1 hr in DPBS containing 10 μg /m l of goat anti-mouse fatty acid-binding protein 4 (FABP4) polyclonal antibody (accessories of mouse mesenchymal stem cell functional identification kit, lot no. IOG0516031; R&D Systems) to label adipocytes.

Techniques:

Journal: The Journal of Veterinary Medical Science

Article Title: Cystine transporter expression is a marker to identify a subpopulation of canine adipose-derived stem cells

doi: 10.1292/jvms.19-0373

Figure Lengend Snippet: Immunofluorescence analysis of adipogenic and osteogenic differentiation. (A) After 15 days of culture in adipogenic differentiation medium, adipose-derived stem cells (ADSCs) appeared as large, round cells with lipid-rich cytoplasmic vacuoles, with both xCT Hi and xCT Lo subpopulations fatty acid-binding protein 4 (FABP4) + , indicating adipogenic differentiation. (B) After 15 days of culture in osteogenic differentiation medium, ADSCs appeared spindle-shaped with cytoplasmic granules, with both xCT Hi and xCT Lo subpopulations osteopontin + , indicating osteogenic differentiation.

Article Snippet: The cells were then incubated for 1 hr in DPBS containing 10 μg /m l of goat anti-mouse fatty acid-binding protein 4 (FABP4) polyclonal antibody (accessories of mouse mesenchymal stem cell functional identification kit, lot no. IOG0516031; R&D Systems) to label adipocytes.

Techniques: Immunofluorescence, Derivative Assay, Binding Assay

Journal: The Journal of Veterinary Medical Science

Article Title: Cystine transporter expression is a marker to identify a subpopulation of canine adipose-derived stem cells

doi: 10.1292/jvms.19-0373

Figure Lengend Snippet: Comparison of the expression of adipogenic and osteogenic differentiation markers. (A) Levels of adipogenic differentiation-specific mRNA ( FABP4 and PPARγ ) for xCT Hi and xCT Lo subpopulations determined by Quantitative reverse transcription polymerase chain reaction (qRT-PCR). xCT Hi showed significantly higher marker levels than xCT Lo . (B) Levels of osteogenic differentiation-specific mRNA ( BMP1 and SPP ) in xCT Hi and xCT Lo subpopulations detected by qRT-PCR. xCT Lo showed significantly higher marker levels than xCT Hi . Values are expressed as the mean ± standard error ( n =6). Scale bar: 100 µ m. * P <0.05.

Article Snippet: The cells were then incubated for 1 hr in DPBS containing 10 μg /m l of goat anti-mouse fatty acid-binding protein 4 (FABP4) polyclonal antibody (accessories of mouse mesenchymal stem cell functional identification kit, lot no. IOG0516031; R&D Systems) to label adipocytes.

Techniques: Comparison, Expressing, Reverse Transcription, Polymerase Chain Reaction, Quantitative RT-PCR, Marker

Journal: Bone Research

Article Title: FABP4 secreted by M1-polarized macrophages promotes synovitis and angiogenesis to exacerbate rheumatoid arthritis

doi: 10.1038/s41413-022-00211-2

Figure Lengend Snippet: The expression of FABP4 in the RA mouse model, BMDMs and RA patients. a , b FABP4 concentrations in the synovial fluid ( a ) and serum ( b ) of controls and RA patients ( n = 8 per group) were assessed by ELISA. C Representative images and quantification of HE staining and FABP4 immunohistochemical staining in the synovium of control and C57BL/6 J mice at 4 and 8 weeks after AIA modeling ( n = 10 per group). Scale bars: 50 μm, 100 μm and 200 μm. d Representative images and quantitative analysis of coimmunostaining of FABP4 with F4/80 or NOS2 in the synovium of control and C57BL/6 J mice at 4 and 8 weeks after AIA modeling ( n = 10 per group). Scale bars: 12.5 μm. e Representative images and quantification of safranin O and fast green staining and FABP4 immunohistochemical staining in knee cartilage from controls and C57BL/6 J mice at 4 and 8 weeks after AIA modeling ( n = 10 per group). Scale bars: 50 μm and 100 μm. f Western blot showing FABP4 and NOS2 in BMDMs stimulated with 50, 200, or 500 ng·mL −1 LPS for 12 h or 24 h. g Quantitative PCR analysis of NOS2, Arg-1, and FABP4 mRNA expression in BMDMs ( n = 3 per group). h FABP4 concentrations in the supernatant of BMDMs stimulated with 50, 200, or 500 ng·mL −1 LPS for 12 h or 24 h ( n = 3 per group) were assessed by ELISA. Student’s t -test or one-way analysis of variance (ANOVA) and Tukey’s multiple comparison test. * P < 0.05, ** P < 0.01; ns, no significance. The data are shown as the mean ± SEM

Article Snippet: We used human and mouse FABP4 ELISA kits (Elabscience Biotechnology, Bethesda, MD, USA: #E-EL-H0285c and #E-EL-M2404c) to analyze the level of FABP4 in the supernatant of macrophages stimulated with lipopolysaccharide (LPS), the serum of C57BL/6 J and TSC1KO mice, and human serum and synovial fluid.

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Staining, Immunohistochemical staining, Control, Western Blot, Real-time Polymerase Chain Reaction, Comparison

Journal: Bone Research

Article Title: FABP4 secreted by M1-polarized macrophages promotes synovitis and angiogenesis to exacerbate rheumatoid arthritis

doi: 10.1038/s41413-022-00211-2

Figure Lengend Snippet: The effect of FABP4 and M1-polarized macrophage supernatant on HUVECs, FLSs, and chondrocytes in vitro. a , b Tube formation assay and quantification of HUVECs cultured with vehicle (C), rhFABP4 (P), rhFABP4 + BMS309403 (PB), M1-polarized macrophage supernatant (S), or M1-polarized macrophage supernatant+BMS309403 (SB) ( n = 3 per group). Scale bar: 100 µm. c Immunoblot analysis of FABP4 and VEGFα in HUVECs cultured with C, P, PB, S, or SB for 24 h. d , g Representative images and quantification of BrdU (green) immunofluorescence ( d ) and Transwell assays ( g ) in HUVECs and FLSs treated with C, P, PB, S, or SB for 24 h ( n = 3 per group). Scale bar: 25 µm, 100 µm. e , f Immunoblot analysis of ERK1/2, p-ERK1/2, P65, and p-P65 in HUVECs ( e ) and FLSs ( f ) treated with C, P, PB, S, or SB for 1 h. h Toluidine blue staining of ATDC5 cells treated with C, P, PB, S, or SB for 24 h ( n = 3 per group). i Immunoblot analysis of FABP4, MMP13, Sox9, and Col2a1 in primary chondrocytes treated with C, P, PB, S, or SB for 24 h. j Immunoblot analysis of P65 and p-P65 in primary chondrocytes treated with C, P, PB, S, or SB for 1 h. One-way ANOVA and Tukey’s multiple comparison test. * P < 0.05, ** P < 0.01; # P < 0.05, ## P < 0.01 compared to the control. The data are shown as the mean ± SEM

Article Snippet: We used human and mouse FABP4 ELISA kits (Elabscience Biotechnology, Bethesda, MD, USA: #E-EL-H0285c and #E-EL-M2404c) to analyze the level of FABP4 in the supernatant of macrophages stimulated with lipopolysaccharide (LPS), the serum of C57BL/6 J and TSC1KO mice, and human serum and synovial fluid.

Techniques: In Vitro, Tube Formation Assay, Cell Culture, Western Blot, Immunofluorescence, Staining, Comparison, Control

Journal: Bone Research

Article Title: FABP4 secreted by M1-polarized macrophages promotes synovitis and angiogenesis to exacerbate rheumatoid arthritis

doi: 10.1038/s41413-022-00211-2

Figure Lengend Snippet: Recombinant FABP4 exacerbates the development of RA in C57BL/6 J mice. a Representative images and quantitative analysis of FABP4 were assessed by immunohistochemical staining and coimmunostaining of FABP4 with F4/80 or NOS2 in the synovium of control and RA mice treated with vehicle or rmFABP4 for 4 and 8 weeks ( n = 10 per group). Scale bars: 12.5 µm and 100 µm. b – i Representative images and quantification of Vimentin and MMP3 coimmunostaining ( b , f ), CD31 and EMCN coimmunostaining ( c , g ), Col2a1 immunofluorescence staining ( d , h ), and MMP13 immunohistochemical staining ( e , i ) in the knee joints of control and RA mice treated with vehicle or rmFABP4 for 4 and 8 weeks ( n = 10 per group). Scale bars: 12.5 µm, 25 µm, and 100 µm. One-way ANOVA and Tukey’s multiple comparison test. ** P < 0.01. The data are shown as the mean ± SEM

Article Snippet: We used human and mouse FABP4 ELISA kits (Elabscience Biotechnology, Bethesda, MD, USA: #E-EL-H0285c and #E-EL-M2404c) to analyze the level of FABP4 in the supernatant of macrophages stimulated with lipopolysaccharide (LPS), the serum of C57BL/6 J and TSC1KO mice, and human serum and synovial fluid.

Techniques: Recombinant, Immunohistochemical staining, Staining, Control, Immunofluorescence, Comparison

Journal: Bone Research

Article Title: FABP4 secreted by M1-polarized macrophages promotes synovitis and angiogenesis to exacerbate rheumatoid arthritis

doi: 10.1038/s41413-022-00211-2

Figure Lengend Snippet: Activation of the mTORC1 pathway enhances the secretion of FABP4 by M1-polarized macrophages to exacerbate RA progression. a , b Representative images and quantification of F4/80 and pS6 coimmunostaining in the mouse ( n = 10 per group) ( a ) and human synovium ( n = 16 per group) ( b ) of the control and RA groups. Scale bar: 12.5 µm. c Western blot showing S6 and pS6 in human synovial tissue. d Western blot showing FABP4, NOS2, S6 and pS6 in BMDMs treated with LPS or rapamycin. e Representative immunofluorescence staining images and quantitative analysis of FABP4 in BMDMs stimulated with LPS or rapamycin ( n = 3 per group). Scale bar: 100 µm. f Western blot showing FABP4, NOS2, S6 and pS6 in BMDMs treated with MHY1485 or rapamycin. g Representative immunofluorescence staining images and quantitative analysis of FABP4 in BMDMs treated with MHY1485 or rapamycin ( n = 3 per group). Scale bar: 100 µm. h Western blot showing FABP4, NOS2, S6 and pS6 in BMDMs from control and TSC1KO mice. i Representative images and quantification of F4/80 and pS6 coimmunostaining in the synovium of wild-type and TSC1KO mice at 4 and 8 weeks after AIA modeling ( n = 10 per group). Scale bar: 12.5 µm. j Representative images and quantification of FABP4 immunohistochemical staining and coimmunostaining of FABP4 with F4/80 or NOS2 in the synovium of control and TSC1KO mice at 4 and 8 weeks after AIA modeling ( n = 10 per group). Scale bars: 12.5 µm and 100 µm. f–j Representative images ( f ) and quantification of Vimentin and MMP3 coimmunostaining ( g ), CD31 and EMCN coimmunostaining ( h ), Col2a1 immunofluorescence staining ( i ), and MMP13 immunohistochemical staining ( j ) in the knee joints of controls and TSC1KO mice at 4 and 8 weeks after AIA modeling ( n = 10 per group). Scale bar: 12.5 µm, 25 µm, and 100 µm. Student’s t -test or one-way ANOVA and Tukey’s multiple comparison test. ** P < 0.01. The data are shown as the mean ± SEM

Article Snippet: We used human and mouse FABP4 ELISA kits (Elabscience Biotechnology, Bethesda, MD, USA: #E-EL-H0285c and #E-EL-M2404c) to analyze the level of FABP4 in the supernatant of macrophages stimulated with lipopolysaccharide (LPS), the serum of C57BL/6 J and TSC1KO mice, and human serum and synovial fluid.

Techniques: Activation Assay, Control, Western Blot, Immunofluorescence, Staining, Immunohistochemical staining, Comparison

Journal: Bone Research

Article Title: FABP4 secreted by M1-polarized macrophages promotes synovitis and angiogenesis to exacerbate rheumatoid arthritis

doi: 10.1038/s41413-022-00211-2

Figure Lengend Snippet: Inhibition of the mTORC1 pathway reduces FABP4 secretion by M1-polarized macrophages to attenuate RA progression. a Representative images and quantification of F4/80 and pS6 coimmunostaining in the synovium of controls and Rheb1KO mice at 4 and 8 weeks after AIA modeling ( n = 10 per group). Scale bar: 12.5 µm. b – e Representative images ( b ) and quantitative analysis of FABP4 immunohistochemical staining ( c ) and coimmunostaining of FABP4 with F4/80 ( d ) or NOS2 ( e ) in the synovium of controls and Rheb1KO mice at 4 and 8 weeks after AIA modeling ( n = 10 per group). Scale bars: 12.5 µm and 100 µm. f – j Representative images ( f ) and quantitative analysis of Vimentin and MMP3 coimmunostaining ( g ), CD31 and EMCN coimmunostaining ( h ), Col2a1 immunofluorescence staining ( i ), and MMP13 immunohistochemical staining ( j ) in the knee joints of controls and Rheb1KO mice at 4 and 8 weeks after AIA modeling ( n = 10 per group). Scale bar: 12.5 µm, 25 µm, and 100 µm. Student’s t -test. ** P < 0.01. The data are shown as the mean ± SEM

Article Snippet: We used human and mouse FABP4 ELISA kits (Elabscience Biotechnology, Bethesda, MD, USA: #E-EL-H0285c and #E-EL-M2404c) to analyze the level of FABP4 in the supernatant of macrophages stimulated with lipopolysaccharide (LPS), the serum of C57BL/6 J and TSC1KO mice, and human serum and synovial fluid.

Techniques: Inhibition, Immunohistochemical staining, Staining, Immunofluorescence

Journal: Bone Research

Article Title: FABP4 secreted by M1-polarized macrophages promotes synovitis and angiogenesis to exacerbate rheumatoid arthritis

doi: 10.1038/s41413-022-00211-2

Figure Lengend Snippet: BMS309403 and anagliptin reduce FABP4 expression in murine synovial macrophages. a – c Representative images and quantification of FABP4 immunohistochemical staining and coimmunostaining of FABP4 with F4/80 or NOS2 in the synovium of TSC1KO mice treated with vehicle, BMS309403 or anagliptin for 4 and 8 weeks after AIA modeling ( n = 10 per group). Scale bar: 12.5 µm and 100 µm. d – f Representative images and quantification of FABP4 immunohistochemical staining and coimmunostaining of FABP4 and F4/80 or NOS2 in the synovium of RA mice treated with vehicle or BMS309403 for 4 and 8 weeks ( n = 10 per group). Scale bar: 12.5 µm and 100 µm. Student’s t -test or one-way ANOVA and Tukey’s multiple comparison test. * P < 0.05, ** P < 0.01. The data are shown as the mean ± SEM

Article Snippet: We used human and mouse FABP4 ELISA kits (Elabscience Biotechnology, Bethesda, MD, USA: #E-EL-H0285c and #E-EL-M2404c) to analyze the level of FABP4 in the supernatant of macrophages stimulated with lipopolysaccharide (LPS), the serum of C57BL/6 J and TSC1KO mice, and human serum and synovial fluid.

Techniques: Expressing, Immunohistochemical staining, Staining, Comparison

Journal: Bone Research

Article Title: FABP4 secreted by M1-polarized macrophages promotes synovitis and angiogenesis to exacerbate rheumatoid arthritis

doi: 10.1038/s41413-022-00211-2

Figure Lengend Snippet: Inhibiting FABP4 prevents RA development. a – d Representative images of Vimentin and MMP3 coimmunostaining ( a ), CD31 and EMCN coimmunostaining ( b ), Col2a1 immunofluorescence staining ( c ), and immunohistochemical staining of MMP13 ( D ) in the knee joints of TSC1KO mice treated with vehicle, BMS309403 or anagliptin for 4 and 8 weeks after AIA modeling. Scale bar: 12.5 µm, 25 µm, and 100 µm. e – h Representative images of Vimentin and MMP3 coimmunostaining ( e ), CD31 and EMCN coimmunostaining ( f ), Col2a1 immunofluorescence staining ( g ), and immunohistochemical staining of MMP13 ( h ) in the knee joints of RA mice treated with vehicle or BMS309403 for 4 and 8 weeks. Scale bar: 12.5 µm, 25 µm, and 100 µm. i – l Quantification of Vimentin-MMP3 ( i ), CD31-EMCN ( j ), Col2a1 ( k ), and MMP13 ( l ) in TSC1KO mice treated with vehicle, BMS309403, or anagliptin for 4 and 8 weeks after AIA surgery ( n = 10 per group). m – p Quantification of Vimentin-MMP3 ( m ), CD31-EMCN ( n ), Col2a1 ( o ), and MMP13 ( p ) in RA mice treated with vehicle or BMS309403 for 4 and 8 weeks ( n = 10 per group). Student’s t -test or one-way ANOVA and Tukey’s multiple comparison test were used. ** P < 0.01. The data are shown as the mean ± SEM

Article Snippet: We used human and mouse FABP4 ELISA kits (Elabscience Biotechnology, Bethesda, MD, USA: #E-EL-H0285c and #E-EL-M2404c) to analyze the level of FABP4 in the supernatant of macrophages stimulated with lipopolysaccharide (LPS), the serum of C57BL/6 J and TSC1KO mice, and human serum and synovial fluid.

Techniques: Immunofluorescence, Staining, Immunohistochemical staining, Comparison

Journal: Oncotarget

Article Title: Fatty acid binding protein 4 enhances prostate cancer progression by upregulating matrix metalloproteinases and stromal cell cytokine production

doi: 10.18632/oncotarget.22908

Figure Lengend Snippet: (A and B) Expression and secretion of FABP4 in PCa cells. Overall, 1 × 10 5 PC-3 cells were cultured in a 35-mm dish, and treated with or without 50 nM FABP4 siRNA-1 for 24 hours. Equal amounts of proteins (10 μg) from the cultured cells were subjected to anti-FABP4 and anti-beta-actin staining (A). The mean FABP4 level in the conditioned medium (CM) was significantly lower in PC-3 cells treated with FABP4 siRNA-1 (235.4 ± 11.6 pg per 10 5 cells) compared with control PC-3 cells (938.8 ± 29.0 pg per 10 5 cells) ( P = 0.007, B). ** P < 0.01. (C) Cytokine secretion of PrSC cells stimulated with FABP4. Overall, 2 × 10 4 PrSC cells were treated with or without 100 ng ml -1 rFABP4 or PC-3 conditioned medium (PC-3 CM) obtained from (A), in the presence or absence of 30 μM BMS309403 (BMS) for 24 hours. IL-8 and IL-6 levels were significantly higher in PrSC treated with rFABP4 or PC-3 CM (6662.0 ± 457.1 and 2678.1 ± 342.4 pg (10 4 cells) -1 , P = 0.0003 and P = 0.0002; and 2506.1 ± 218.7 and 654.8 ± 51.0 pg (10 4 cells) -1 , P = 0.0044 and P = 0.021; respectively), and the effect was markedly inhibited in the presence of BMS309403. In addition, IL-8 and IL-6 levels were significantly lower in the conditioned medium of PrSC cultured in PC-3 CM treated with FABP4 siRNA-1 compared with untreated PC-3 CM. Mean ± S.D., ** P < 0.01, *** P < 0.001. (D) Western blotting using anti-αSMA and anti-beta-actin antibodies of PrSC proteins treated with various agents described in (C). (E and F) PrSC augmented PC-3 cell invasiveness by secreting IL-8 and IL-6 in response to FABP4 secreted by PC-3 cells. (E) The relative value (%) of in vitro Matrigel invasion assay under each conditioned media is shown based on PC-3 cells with PrSC CM. PrSC-rFABP4 CM: conditioned media of PrSC treated with 100 ng ml-1 rFABP4. IL-8 blocking Ab: conditioned media of PrSC treated with rFABP4 in the presence of neutralizing IL-8 antibodies. IL-6 blocking Ab: conditioned media of PrSC treated with rFABP4 in the presence of neutralizing IL-6 antibodies. Ctrl mouse IgG: conditioned media of PrSC treated with rFABP4 in the presence of isotype control mouse IgG. Ctrl goat IgG: conditioned media of PrSC treated with rFABP4 in the presence of isotype control goat IgG. (F) The relative value (%) of in vitro Matrigel invasion assay under each condition is shown based on the condition with PrSC cells cultured in the lower chamber. PC-3: PC-3 cells were placed in the upper chamber. PC-3 siFABP4: FABP4 siRNA1 treated PC-3 cells were placed in the upper chamber. PrSC: PrSC cells were seeded in the lower chamber. IL-8 blocking Ab, IL-6 blocking Ab, Ctrl mouse IgG, and Ctrl goat IgG as shown in (E). Bar plots represent the percentage of invading cells relative to the invasion of PC-3 with PrSC cells cultured in the lower chamber. Mean ± S.D., * P < 0.05, ** P < 0.01.

Article Snippet: Mouse serum FABP4 levels were measured using a mouse FABP4 ELISA kit (CycLex Co., Ltd., Nagoya, Japan), according to the manufacturer’s instructions.

Techniques: Expressing, Cell Culture, Staining, Control, Western Blot, In Vitro, Invasion Assay, Blocking Assay

Journal: Oncotarget

Article Title: Fatty acid binding protein 4 enhances prostate cancer progression by upregulating matrix metalloproteinases and stromal cell cytokine production

doi: 10.18632/oncotarget.22908

Figure Lengend Snippet: (A) Histogram showing the relative value (%) of invading cells of each condition based on untreated PC-3 cells. PC-3 cells were treated with 50 nM FABP4 siRNAs or rFABP4 at the indicated concentration. All invading cells were counted and compared with untreated PC-3 cells (Ctrl); * P < 0.05 and ** P < 0.01. (B, C, D and E) Altered matrix metalloproteinase (MMPs) levels by FABP4. (B and C) Overall, 1 × 10 5 PC-3 cells were cultured in a 35-mm dish, and treated with 50 nM FABP4 siRNA-1. Each conditioned medium (CM) was then collected and analyzed for MMP levels using the RayBio Human MMP Antibody Array kit. The signal intensity in the array images was quantified and scored by Densitograph software (ATTO). Fold changes among samples were analyzed and compared using RayBio Antibody Array Analysis Tool provided by RayBiotech (B). MMP2 and MMP9 signals in the array images are indicated by rectangular boxes (C). Fold changes of signals in control PC-3 cells (upper) and FABP4 siRNA-1 treated PC-3 cells (lower) were analyzed using the RayBio Antibody Array Analysis Tool (C). (D) Zymography analysis of MMP2 and MMP9 activity. MMP2 and MMP9 enzymatic activity in condition medium from PC-3 cells treated with 50 nM FABP4 siRNAs or 100 ng ml -1 rFABP4. (E) Relative MMP2 and MMP9 mRNA levels. PC-3 cells were treated with 50 nM FABP4 siRNAs and 100 ng ml -1 rFABP4 for 24 hours. MMP2, MMP9 and beta-actin mRNA levels were assessed by quantitative RT–PCR, and compared with untreated cells. Mean ± S.D., * P < 0.05 and ** P < 0.01.

Article Snippet: Mouse serum FABP4 levels were measured using a mouse FABP4 ELISA kit (CycLex Co., Ltd., Nagoya, Japan), according to the manufacturer’s instructions.

Techniques: Concentration Assay, Cell Culture, Ab Array, Software, Control, Zymography, Activity Assay, Quantitative RT-PCR

Journal: Oncotarget

Article Title: Fatty acid binding protein 4 enhances prostate cancer progression by upregulating matrix metalloproteinases and stromal cell cytokine production

doi: 10.18632/oncotarget.22908

Figure Lengend Snippet: (A and B) Requirement of ERK activation for enhanced IL-8 and IL-6 in PrSC by FABP4. PrSC cells were treated with rFABP4 at 30 and 100 ng ml -1 for 3 hours. Cells cultured previously in the presence or absence of 1 μM U0126 for 1 hour before treatment with rFABP4 (A), IL-8 and IL-6 mRNA levels were measured by quantitative RT-PCR, and compared with untreated cells (B). ** P < 0.01. (C) AKT and ERK activation of PC-3 by FABP4. PC-3 cells were treated with 0–100 ng ml -1 rFABP4 for 3 hours. Then, an equal amount of protein (10 μg) from the cells was subjected to anti-AKT, anti-pAKT, anti-ERK, anti-pERK, and anti-beta-actin antibodies. (D) Requirement of AKT and ERK activation for enhanced invasion of PC-3 cells by FABP4. The relative value (%) of in vitro Matrigel invaded PC-3 cells is shown based on untreated PC-3 cells. rFABP4: PC-3 cells treated with 100 ng ml -1 rFABP4 for 24 hours. The cells were treated with 10 μM LY294002 (rFABP4+LY) or 1 μM U0126 (rFABP4+U) for 1 hour before treatment with rFABP4. rFABP4+BMS: PC-3 cells treated with 100 ng ml -1 rFABP4 for 24 hours with 30 μM BMS309403. Then, all invading cells were counted and compared with untreated cells; * P < 0.05 and ** P < 0.01. (E and F) Requirement of AKT and ERK activation for MMP activation of PC-3 cells by FABP4. PC-3 cells were treated with 100 ng ml -1 rFABP4 alone (rFABP4) and 10 μM LY294002 (rFABP4+LY), 1 μM U0126 (rFABP4+U), or 30 μM BMS309403 (rFABP4+BMS). (E) MMP2 and MMP9 enzymatic activity in culture medium was determined by Zymography analysis. (F) Relative values of MMP2 and MMP9 mRNA levels were measured by quantitative RT-PCR, and the levels were compared with untreated cells. ** P < 0.01 and *** P < 0.001.

Article Snippet: Mouse serum FABP4 levels were measured using a mouse FABP4 ELISA kit (CycLex Co., Ltd., Nagoya, Japan), according to the manufacturer’s instructions.

Techniques: Activation Assay, Cell Culture, Quantitative RT-PCR, In Vitro, Activity Assay, Zymography

Journal: Oncotarget

Article Title: Fatty acid binding protein 4 enhances prostate cancer progression by upregulating matrix metalloproteinases and stromal cell cytokine production

doi: 10.18632/oncotarget.22908

Figure Lengend Snippet: For in vivo metastatic tumor studies, PC-3M-luc-C6 cells were intraperitoneallyinjected into mice randomly assigned to four groups (5 mice per group): control (Ctrl), PrSC, HFD, and HFD+BMS. For the PrSC group, PrSC cells were injected with the tumor cells. Mice in the HFD and HFD+BMS groups were fed HFD, and mice in the Ctrl and PrSC groups were fed a control diet. Mice in the HFD+BMS group were administrated BMS309403 by dissolution in drinking water. (A and B) Increased luciferase activity by PrSC or HFD. At 14 and 28 days inoculation, bioluminescence was used to detect intraperitoneal tumor growth and metastases by the intraperitoneal injection of luciferin. (C) Increased tumor metastasis in the PrSC and HFD groups. At 29 days after the injection of cells, mice were sacrificed, and the proportion of metastatic tumor burden in peritoneal organs, peritoneum, intestine, stomach, liver, and diaphragm were evaluated. (D, E and F) Increased invasive capacity in the PrSC and HFD groups by the increased adipocyte infiltration, stromal fibroblast activation, and upregulation of FABP4 and MMPs. (D) Slides of mouse tumor tissues were subjected to hematoxylin and eosin staining (H & E), and immunohistochemistry to detect the expression of FABP4 and αSMA. (E and F) mRNA expressions of FABP4 and MMP2 and 9 in tumors from each group were analyzed by quantitative RT-PCR. The mRNA expression levels of FABP4 and MMP2 and 9 was normalized to the levels of beta-actin , and the relative values were compared with the level of the Ctrl group. * P < 0.05, ** P < 0.01, and *** P < 0.001. (G) HFD stimulated FABP4 expression. The serum FABP4 concentration was measured by a mouse FABP4 ELISA kit. The serum FABP4 level was significantly higher in the HFD and HFD+BMS groups compared with the Ctrl group. * P < 0.05. (H) Increased serum IL-8 levels in the PrSC and HFD groups. The serum cytokine concentrations were measured by a Cytometric bead array kit. The serum level of IL-8 was significantly higher in the PrSC and HFD groups compared with the Ctrl group, and in the HFD group compared with the HFD+BMS group. * P < 0.05, ** P < 0.01. (I and J) Stimulation of PC-3 cell invasiveness by mouse serum containing higher concentrations of FABP4 and IL-8. In vitro cell invasion is shown (I), and in the presence of 30 μM BMS309403 or 10 μg ml -1 IL-8 blocking antibody (J). All invading cells were counted and compared with control cells. Mean ± S.D., * P < 0.05, ** P < 0.01.

Article Snippet: Mouse serum FABP4 levels were measured using a mouse FABP4 ELISA kit (CycLex Co., Ltd., Nagoya, Japan), according to the manufacturer’s instructions.

Techniques: In Vivo, Control, Injection, Dissolution, Luciferase, Activity Assay, Activation Assay, Staining, Immunohistochemistry, Expressing, Quantitative RT-PCR, Concentration Assay, Enzyme-linked Immunosorbent Assay, In Vitro, Blocking Assay

Journal: Oncotarget

Article Title: Fatty acid binding protein 4 enhances prostate cancer progression by upregulating matrix metalloproteinases and stromal cell cytokine production

doi: 10.18632/oncotarget.22908

Figure Lengend Snippet: (A) Immunohistology staining of tissue samples from PCa radical prostatectomy using an anti-human FABP4-specific antibody. (B, C, and D) Serum FABP4 levels from 40 controls and 104 patients with PCa were measured using a human FABP4 ELISA Kit. (B) The serum FABP4 level was significantly higher in PCa patients than in the controls (16.6 ± 6.6 and 13.6 ± 4.0 ng ml -1 , respectively, P = 0.001). (C) The serum FABP4 level was significantly higher in patients with a GS of ≥ 7 ( n =79) compared with a GS of ≤ 6 ( n =25) (17.1 ± 7.2 and 14.6 ± 2.6 ng ml -1 , respectively, P = 0.018). (D) The serum FABP4 level in surgical specimens was significantly higher in patients with pathological T (pT) stages (≥ pT3, n =34) of PCa than in those with more localized disease (≤ pT2, n =70) (18.2 ± 10.1 and 15.8 ± 3.7 ng ml -1 , respectively, P = 0.022). (E) The percentage of αSMA-positive staining stromal cells in the surrounding of cancer cells was calculated, and the staining level was determined by the staining percentage score characterized as low-staining (≤10%) and high-staining (>10%). (F) The relationship between αSMA expression level and pathological T stage (pT) was evaluated. Patients with low αSMA staining were comprised 77.4% of pT1, 19.4% of pT2, and 3.2% of pT3 or 4. Patients with high αSMA staining were comprised 45.9% of pT1, 36.5% of pT2, and 17.6% of pT3 or 4 ( P = 0.031). (G) The serum FABP4 level was higher in patients with high αSMA staining compared with low αSMA staining (FABP4, 14.8 ± 0.3 and 16.2 ± 0.5 ng/ml, P = 0.068; MIC-1, 891.4 ± 218.4 and 1075.9 ± 354.8 pg/ml, P = 0.056, respectively).

Article Snippet: Mouse serum FABP4 levels were measured using a mouse FABP4 ELISA kit (CycLex Co., Ltd., Nagoya, Japan), according to the manufacturer’s instructions.

Techniques: Staining, Enzyme-linked Immunosorbent Assay, Expressing

Journal: Oncotarget

Article Title: Fatty acid binding protein 4 enhances prostate cancer progression by upregulating matrix metalloproteinases and stromal cell cytokine production

doi: 10.18632/oncotarget.22908

Figure Lengend Snippet: FABP4 secreted from PCa cells directly stimulates PCa cell invasiveness via the upregulation of MMPs. FABP4 activates PrSCs resulting in the augmentation of PCa metastasis and invasiveness via the secretion of IL-8 and IL-6. HFD influences PCa metastasis and invasiveness by upregulating FABP4 and IL-8, suggesting circulating FABP4 might have an important role in PCa progression.

Article Snippet: Mouse serum FABP4 levels were measured using a mouse FABP4 ELISA kit (CycLex Co., Ltd., Nagoya, Japan), according to the manufacturer’s instructions.

Techniques:

Journal: eLife

Article Title: Identification of a weight loss-associated causal eQTL in MTIF3 and the effects of MTIF3 deficiency on human adipocyte function

doi: 10.7554/eLife.84168

Figure Lengend Snippet:

Article Snippet: Antibody , Anti-FABP4 (rabbit polyclonal antibody) , Cell Signalling Technology , Cat: 12589 , WB (1:1000).

Techniques: Recombinant, Plasmid Preparation, Sequencing, TaqMan Assay, DNA Extraction, Isolation, Sample Prep, Picogreen Assay

Journal: The Journal of Veterinary Medical Science

Article Title: Cystine transporter expression is a marker to identify a subpopulation of canine adipose-derived stem cells

doi: 10.1292/jvms.19-0373

Figure Lengend Snippet: Primers used in this study

Article Snippet: The cells were then incubated for 1 hr in DPBS containing 10 µg /m l of goat anti-mouse fatty acid-binding protein 4 (FABP4) polyclonal antibody (accessories of mouse mesenchymal stem cell functional identification kit, lot no. IOG0516031; R&D Systems) to label adipocytes.

Techniques:

Journal: The Journal of Veterinary Medical Science

Article Title: Cystine transporter expression is a marker to identify a subpopulation of canine adipose-derived stem cells

doi: 10.1292/jvms.19-0373

Figure Lengend Snippet: Immunofluorescence analysis of adipogenic and osteogenic differentiation. (A) After 15 days of culture in adipogenic differentiation medium, adipose-derived stem cells (ADSCs) appeared as large, round cells with lipid-rich cytoplasmic vacuoles, with both xCT Hi and xCT Lo subpopulations fatty acid-binding protein 4 (FABP4) + , indicating adipogenic differentiation. (B) After 15 days of culture in osteogenic differentiation medium, ADSCs appeared spindle-shaped with cytoplasmic granules, with both xCT Hi and xCT Lo subpopulations osteopontin + , indicating osteogenic differentiation.

Article Snippet: The cells were then incubated for 1 hr in DPBS containing 10 µg /m l of goat anti-mouse fatty acid-binding protein 4 (FABP4) polyclonal antibody (accessories of mouse mesenchymal stem cell functional identification kit, lot no. IOG0516031; R&D Systems) to label adipocytes.

Techniques: Immunofluorescence, Derivative Assay, Binding Assay

Journal: The Journal of Veterinary Medical Science

Article Title: Cystine transporter expression is a marker to identify a subpopulation of canine adipose-derived stem cells

doi: 10.1292/jvms.19-0373

Figure Lengend Snippet: Comparison of the expression of adipogenic and osteogenic differentiation markers. (A) Levels of adipogenic differentiation-specific mRNA ( FABP4 and PPARγ ) for xCT Hi and xCT Lo subpopulations determined by Quantitative reverse transcription polymerase chain reaction (qRT-PCR). xCT Hi showed significantly higher marker levels than xCT Lo . (B) Levels of osteogenic differentiation-specific mRNA ( BMP1 and SPP ) in xCT Hi and xCT Lo subpopulations detected by qRT-PCR. xCT Lo showed significantly higher marker levels than xCT Hi . Values are expressed as the mean ± standard error ( n =6). Scale bar: 100 µ m. * P <0.05.

Article Snippet: The cells were then incubated for 1 hr in DPBS containing 10 µg /m l of goat anti-mouse fatty acid-binding protein 4 (FABP4) polyclonal antibody (accessories of mouse mesenchymal stem cell functional identification kit, lot no. IOG0516031; R&D Systems) to label adipocytes.

Techniques: Comparison, Expressing, Reverse Transcription, Polymerase Chain Reaction, Quantitative RT-PCR, Marker

Journal: Cell Death & Disease

Article Title: RETRACTED ARTICLE: FABP4 contributes to renal interstitial fibrosis via mediating inflammation and lipid metabolism

doi: 10.1038/s41419-019-1610-5

Figure Lengend Snippet: a , b The protein levels of FABP4 in NRK-52E and NRK-49F cells based on immunofluorescence staining and western blotting assay ( n = 3). c , d The protein levels of FABP4 in mice and rats based on immunofluorescence staining and western blotting assay ( n = 3). e Serum protein levels of FABP4 in mice and rats based on Elisa assay ( n = 7). Data are presented as the mean ± SD. ** p < 0.01 compared with sham groups or control groups

Article Snippet: Mouse and Rat FABP4 ELISA Kits were purchased from Boster Biological Technology Co., Ltd. (California, USA).

Techniques: Immunofluorescence, Staining, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: Cell Death & Disease

Article Title: RETRACTED ARTICLE: FABP4 contributes to renal interstitial fibrosis via mediating inflammation and lipid metabolism

doi: 10.1038/s41419-019-1610-5

Figure Lengend Snippet: a The protein levels of FABP4 and PPARγ based on immunefluorescence staining (400× magnification) and western blotting assay in FABP4 siRNA transfected NRK-52E and NRK-49F cells. b The mRNA levels of IL-1β, IL-6, and TNF-α, and the protein levels of p65 and ICAM-1 in NRK-52E and NRK-49F cells after FABP4 siRNA transfection. c The protein levels of EHHADH, ACOX1, SCP-2, CPT1, ACADM, and ACADL in NRK-52E and NRK-49F cells after FABP4 siRNA transfection. d The mRNA levels of α-SMA, COL1A1 and COL3A1 in NRK-52E and NRK-49F cells after FABP4 siRNA transfection and the protein levels of α-SMA and COL1A based on immunofluorescence assay (400× magnification) in NRK-52E and NRK-49F cells after FABP4 siRNA transfection. e The levels of TC, TG, and FFAs in NRK-52E and NRK-49F cells after FABP4 siRNA transfection and the lipid droplets based on Oil O Red staining (400× magnification) in NRK-52E and NRK-49F cells after FABP4 siRNA transfection. Data are presented as the mean ± SD ( n = 3). ** p < 0.01 control groups compared with LPS-groups; ## p < 0.01 and # p < 0.05 LPS groups compared with FABP4 siRNA groups

Article Snippet: Mouse and Rat FABP4 ELISA Kits were purchased from Boster Biological Technology Co., Ltd. (California, USA).

Techniques: Staining, Western Blot, Transfection, Immunofluorescence

Journal: Cell Death & Disease

Article Title: RETRACTED ARTICLE: FABP4 contributes to renal interstitial fibrosis via mediating inflammation and lipid metabolism

doi: 10.1038/s41419-019-1610-5

Figure Lengend Snippet: a The protein levels of FABP4, PPARγ, p65, and ICAM based on immunefluorescence staining (400× magnification) and western blotting assay after treatment by FABP4 inhibitor in NRK-52E and NRK-49F cells. b The protein levels of EHHADH, ACOX1, SCP-2, CPT1, ACADM, and ACADL in NRK-52E and NRK-49F cells treated by FABP4 inhibitor. c The protein levels of α-SMA and COL1A based on immunofluorescence assay (400× magnification), and the mRNA levels of α-SMA, COL1A1, and COL3A1 in NRK-52E and NRK-49F cells after treatment by FABP4 inhibitor in NRK-52E and NRK-49F cells. d The lipid droplets based on Oil O Red staining (400× magnification) and the levels of TC, TG, and FFAs in NRK-52E and NRK-49F cells after FABP4 inhibitor treatment. Data are presented as the mean ± SD ( n = 3). ** p < 0.01 control groups compared with LPS groups, ## p < 0.01 and # p < 0.05 LPS groups compared with FABP4 inhibitor groups

Article Snippet: Mouse and Rat FABP4 ELISA Kits were purchased from Boster Biological Technology Co., Ltd. (California, USA).

Techniques: Staining, Western Blot, Immunofluorescence

Journal: Cell Death & Disease

Article Title: RETRACTED ARTICLE: FABP4 contributes to renal interstitial fibrosis via mediating inflammation and lipid metabolism

doi: 10.1038/s41419-019-1610-5

Figure Lengend Snippet: a The protein levels of FABP4 based on immunofluorescence staining (400× magnification) and western blotting assay in FABP4 KO mice. b The protein levels of PPARγ based on immunofluorescence staining (400× magnification) and western blotting assay in FABP4 KO mice. c The mRNA levels of IL-1β, IL-6, and TNF-α in FABP4 KO mice. d The protein levels of p65, ICAM-1, EHHADH, ACOX1, SCP-2, CPT1, ACADM, and ACADL in FABP4 KO mice. Data are presented as the mean ± SD ( n = 3). ** p < 0.01 Sham group compared with UUO mice; ## p < 0.01 and # p < 0.05 UUO WT mice compared with UUO FABP4 −/− mice

Article Snippet: Mouse and Rat FABP4 ELISA Kits were purchased from Boster Biological Technology Co., Ltd. (California, USA).

Techniques: Immunofluorescence, Staining, Western Blot

Journal: Cell Death & Disease

Article Title: RETRACTED ARTICLE: FABP4 contributes to renal interstitial fibrosis via mediating inflammation and lipid metabolism

doi: 10.1038/s41419-019-1610-5

Figure Lengend Snippet: a The serum levels of Cr, BUN, and H&E staining (400× original magnification) of the kidney tissue in FABP4 KO mice ( n = 5). b Degree of fibrosis based on Masson and Sirius Red staining (400× original magnification), and Sirius Red polarized light observation (400× original magnification) in FABP4 KO mice. c The mRNA levels of α-SMA, COL1A1, COL3A1, and the protein levels of α-SMA and COL1A based on immunofluorescence staining (400× magnification) in FABP4 KO mice ( n = 3). d The lipid droplets based on Oil O Red staining (400× original magnification), and the levels of TG, TC, and FFA in FABP4 KO mice. Data are presented as the mean ± SD. ** p < 0.01 and * p < 0.05 Sham group compared with UUO FABP4 KO mice; ## p < 0.01 and # p < 0.05 UUO WT mice group compared with UUO FABP4 −/− mice

Article Snippet: Mouse and Rat FABP4 ELISA Kits were purchased from Boster Biological Technology Co., Ltd. (California, USA).

Techniques: Staining, Immunofluorescence

Journal: Cell Death & Disease

Article Title: RETRACTED ARTICLE: FABP4 contributes to renal interstitial fibrosis via mediating inflammation and lipid metabolism

doi: 10.1038/s41419-019-1610-5

Figure Lengend Snippet: FABP4 downregulated PPAR-γ expression, up-regulated the protein levels of p65 and ICAM-1, and decreased the protein levels of ACADM, ACADL, SCP-2, CPT1, EHHADH, and ACOX1 to promote inflammation and lipid metabolism

Article Snippet: Mouse and Rat FABP4 ELISA Kits were purchased from Boster Biological Technology Co., Ltd. (California, USA).

Techniques: Expressing