Journal: iScience

Article Title: Adenosine-rich extract of Ganoderma lucidum: A safe and effective lipid-lowering substance

doi: 10.1016/j.isci.2022.105214

Figure Lengend Snippet: The levels of proteins and PTMs in the PPAR signaling pathway

Article Snippet: FABP5 Rabbit Polyclonal Antibody , proteintech , 12348-1-AP.

Techniques: Expressing, Significance Assay, Modification

Journal: iScience

Article Title: Adenosine-rich extract of Ganoderma lucidum: A safe and effective lipid-lowering substance

doi: 10.1016/j.isci.2022.105214

Figure Lengend Snippet:

Article Snippet: FABP5 Rabbit Polyclonal Antibody , proteintech , 12348-1-AP.

Techniques: Software

Journal: Cancer Biology & Therapy

Article Title: Fatty acid binding protein 5 promotes the proliferation, migration, and invasion of hepatocellular carcinoma cells by degradation of Krüppel-like factor 9 mediated by miR-889-5p via cAMP-response element binding protein

doi: 10.1080/15384047.2022.2094670

Figure Lengend Snippet: FABP5 expression was upregulated in HCC. a, expression of FABP5 in HCC samples (N = 369) and normal samples (N = 160) from the TCGA data set. b, relative expression of FABP5 mRNA in HCC tissues compared with adjacent normal tissues were detected by RT-qPCR. c, relative expression of FABP5 protein in HCC tissues and adjacent normal tissues were detected by western blotting. Representative images of FABP5 expression in 48 paired HCC tissues(t) and adjacent normal tissues (n). d, the expression of FABP5 was negatively correlated with overall survival time in HCC patients from the TCGA data set. * P < .05 and *** P < .001 as compared with the vehicle control.

Article Snippet: The sections were incubated at 4°C overnight with rabbit anti-human FABP5 primary antibody (Proteintech, cat. no. 12348-1-AP) and then with goat anti-rabbit secondary antibody (Proteintech, cat. no. PR30009) for 50 min at room temperature.

Techniques: Expressing, Quantitative RT-PCR, Western Blot

Journal: Cancer Biology & Therapy

Article Title: Fatty acid binding protein 5 promotes the proliferation, migration, and invasion of hepatocellular carcinoma cells by degradation of Krüppel-like factor 9 mediated by miR-889-5p via cAMP-response element binding protein

doi: 10.1080/15384047.2022.2094670

Figure Lengend Snippet: Correlation between FABP5 expression and clinicopathological characteristics

Article Snippet: The sections were incubated at 4°C overnight with rabbit anti-human FABP5 primary antibody (Proteintech, cat. no. 12348-1-AP) and then with goat anti-rabbit secondary antibody (Proteintech, cat. no. PR30009) for 50 min at room temperature.

Techniques: Expressing

Journal: Cancer Biology & Therapy

Article Title: Fatty acid binding protein 5 promotes the proliferation, migration, and invasion of hepatocellular carcinoma cells by degradation of Krüppel-like factor 9 mediated by miR-889-5p via cAMP-response element binding protein

doi: 10.1080/15384047.2022.2094670

Figure Lengend Snippet: FABP5 knockdown inhibited cell growth, migration and invasion in HCC cells. a, the mRNA and protein expression levels of FABP5 were analyzed in FABP5-knockdown HepG2 and SK-Hep-1 cells by RT-qPCR and western blotting. b, the growth curve of HCC cells was determined by CCK-8 assay. c, PI fluorescence pattern was applied for cell-cycle distribution in HCC cells transfected with shFABP5 or shNC. d, HCC cells transfected with shFABP5 or shNC were stained with Annexin-V FITC and PI to be measured apoptosis. Apoptotic cells are presented in the right-lower (Q3, early apoptosis) and right-upper (Q2, late apoptosis) quadrants of the plots. e, transwell assay revealed the migration abilities of HCC cells transfected with shFABP5 or shNC. f, transwell assay revealed the invasive abilities of HCC cells transfected with shFABP5 or shNC. Results are presented as mean ± S.E.M. (N = 3). Results were averaged from three independent experiments and presented as percentage of control levels. * p < .05, ** p < .01 and *** p < .001 as compared with the vehicle control. FITC, fluorescein isothiocyanate; PI, propidium iodide.

Article Snippet: The sections were incubated at 4°C overnight with rabbit anti-human FABP5 primary antibody (Proteintech, cat. no. 12348-1-AP) and then with goat anti-rabbit secondary antibody (Proteintech, cat. no. PR30009) for 50 min at room temperature.

Techniques: Migration, Expressing, Quantitative RT-PCR, Western Blot, CCK-8 Assay, Fluorescence, Transfection, Staining, Transwell Assay

Journal: Cancer Biology & Therapy

Article Title: Fatty acid binding protein 5 promotes the proliferation, migration, and invasion of hepatocellular carcinoma cells by degradation of Krüppel-like factor 9 mediated by miR-889-5p via cAMP-response element binding protein

doi: 10.1080/15384047.2022.2094670

Figure Lengend Snippet: Decreased expression of FABP5 inhibited the growth of HCC cells in vivo. a, HepG2 cells and HepG2 cells stably transfected with shFABP5 RNA or shNC were implanted into nude mice by subcutaneous injection. Tumor volume was measured each 7 days by a calliper until the end of the experiment. The tumor growth curve was drawn. b, the mice and the tumors of different groups on the 28th day after injection. c, the weight of each xenograft tumor was measured at the end of the experiment. d, the differences in FABP5 mRNA levels between the shFABP5 group and the control groups were detected by RT-qPCR. e, the protein levels of FABP5 in the shFABP5 group and the control groups were measured by western blotting. f, the tumors were analyzed by immunohistochemical staining with anti-FABP5. Original magnification: 400 × . *** p < .001 as compared with the vehicle control.

Article Snippet: The sections were incubated at 4°C overnight with rabbit anti-human FABP5 primary antibody (Proteintech, cat. no. 12348-1-AP) and then with goat anti-rabbit secondary antibody (Proteintech, cat. no. PR30009) for 50 min at room temperature.

Techniques: Expressing, In Vivo, Stable Transfection, Transfection, Injection, Quantitative RT-PCR, Western Blot, Immunohistochemical staining, Staining

Journal: Cancer Biology & Therapy

Article Title: Fatty acid binding protein 5 promotes the proliferation, migration, and invasion of hepatocellular carcinoma cells by degradation of Krüppel-like factor 9 mediated by miR-889-5p via cAMP-response element binding protein

doi: 10.1080/15384047.2022.2094670

Figure Lengend Snippet: The expression of KLF9 was negatively correlated with FABP5. a, gene microarray analysis were applied to examine the differential expression genes in SK-Hep-1 cells before and after FABP5 knockdown (FC > |2|; P < .01). b, gene ontology (GO) enrichment analysis of differential expression genes in accordance with the biological processes, cellular component, and molecular function. c, KLF9 mRNA expression in HepG2 and SK-Hep-1 cells with FABP5 knockdown determined by RT-qPCR. d, the KLF9 expression in HCC tissue and adjacent tissues were detected by RT-qPCR. e, relative expression of KLF9 protein in HCC tissues and adjacent normal tissues were detected by Western blotting. Representative images of KLF9 expression in 48 paired HCC tissues (t) and adjacent normal tissues (n). e, the expression of KLF9 was positively correlated with overall survival time in HCC patients from the TCGA data set. F, A negative correlation was found between the protein expression level of FABP5 and KLF9 in HCC samples. *p < .05, **p < .01, and ***p < .001 as compared with the vehicle control.

Article Snippet: The sections were incubated at 4°C overnight with rabbit anti-human FABP5 primary antibody (Proteintech, cat. no. 12348-1-AP) and then with goat anti-rabbit secondary antibody (Proteintech, cat. no. PR30009) for 50 min at room temperature.

Techniques: Expressing, Microarray, Quantitative RT-PCR, Western Blot

Journal: Cancer Biology & Therapy

Article Title: Fatty acid binding protein 5 promotes the proliferation, migration, and invasion of hepatocellular carcinoma cells by degradation of Krüppel-like factor 9 mediated by miR-889-5p via cAMP-response element binding protein

doi: 10.1080/15384047.2022.2094670

Figure Lengend Snippet: KLF9 knockdown reversed the phenotypes caused by FABP5 knockdown in HCC cells. a-b, HepG2 and SK-Hep-1 cells upon co-transfection of shFABP5 with shKLF9 showed significantly reduced KLF9 expression at the mRNA and protein levels. c, CCK-8 assay showed that shKLF9 significantly reversed the inhibition of proliferation induced by FABP5 knockdown in HCC cells. d, transwell migration assay revealed that shKLF9 significantly rescued the migratory inhibition induced FABP5 knockdown in HCC cells. e, transwell invasion assay revealed that shKLF9 significantly rescued the inhibition of invasion induced by FABP5 knockdown in HCC cells. F, KLF9 knockdown reversed the cell cycle arrest caused by FABP5 knockdown in HCC cells. PI fluorescence pattern was applied for cell-cycle distribution. G, KLF9 knockdown reversed the apoptosis caused by FABP5 knockdown in HCC cells. HepG2 and SK-Hep-1 cells were stained with Annexin-V FITC and PI to be measured apoptosis. Apoptotic cells are presented in the right-lower (Q3, early apoptosis) and right-upper (Q2, late apoptosis) quadrants of the plots. Results are presented as mean ± S.E.M. (N = 3). * p < .05, ** p < .01, and ***p < .001 as compared with the vehicle control. Results were averaged from three independent experiments and presented as percentage of control levels. FITC, fluorescein isothiocyanate; PI, propidium iodide.

Article Snippet: The sections were incubated at 4°C overnight with rabbit anti-human FABP5 primary antibody (Proteintech, cat. no. 12348-1-AP) and then with goat anti-rabbit secondary antibody (Proteintech, cat. no. PR30009) for 50 min at room temperature.

Techniques: Cotransfection, Expressing, CCK-8 Assay, Inhibition, Transwell Migration Assay, Transwell Invasion Assay, Fluorescence, Staining

Journal: Cancer Biology & Therapy

Article Title: Fatty acid binding protein 5 promotes the proliferation, migration, and invasion of hepatocellular carcinoma cells by degradation of Krüppel-like factor 9 mediated by miR-889-5p via cAMP-response element binding protein

doi: 10.1080/15384047.2022.2094670

Figure Lengend Snippet: PI3K/AKT signaling pathway was involved in FABP5-induced KLF9 downregulation. a, KEGG pathway enrichment analysis of top 20 differentially expressed signaling pathways in FABP5 knockdown group compared with control group in SK-Hep-1 cells. b, PI3K/AKT signaling pathway was analyzed by western blotting in the indicated groups.

Article Snippet: The sections were incubated at 4°C overnight with rabbit anti-human FABP5 primary antibody (Proteintech, cat. no. 12348-1-AP) and then with goat anti-rabbit secondary antibody (Proteintech, cat. no. PR30009) for 50 min at room temperature.

Techniques: Western Blot

Journal: Cancer Biology & Therapy

Article Title: Fatty acid binding protein 5 promotes the proliferation, migration, and invasion of hepatocellular carcinoma cells by degradation of Krüppel-like factor 9 mediated by miR-889-5p via cAMP-response element binding protein

doi: 10.1080/15384047.2022.2094670

Figure Lengend Snippet: miR-889-5p suppressed KLF9 expression by directly targeted KLF9. a, the expression of miR-889-5p was significantly decreased by FABP5 knockdown in HCC cells. b, bioinformatics analysis predicted that the 3ʹUTR sequence of KLF9 is complementary to the seed sequence of miR-889-5p.The core binding sequences of KLF9 were mutated (in red text). c, dual-luciferase reporter assay was performed to verify that miR-889-5p directly bound to the 3’-UTR sequences of KLF9 in the cells co-transfected with miR-889 mimics or NC with pGL3-KLF9-WT or pGL3-KLF9-Mut. d, relative expression of miR-889-5p in 48 HCC tissues compared with adjacent normal tissues was detected by RT-qPCR. E, miR-889-5p expression was negatively correlated with KLF9 expression and positively correlated with FABP5 expression in HCC tissues. f-g, increased expression of miR-889-5p attenuated KLF9 mRNA and protein expression of in HCC cells with shFABP5 treatment. *p < .05, **p < .01, and ***p < .001 as compared with the vehicle control.

Article Snippet: The sections were incubated at 4°C overnight with rabbit anti-human FABP5 primary antibody (Proteintech, cat. no. 12348-1-AP) and then with goat anti-rabbit secondary antibody (Proteintech, cat. no. PR30009) for 50 min at room temperature.

Techniques: Expressing, Sequencing, Binding Assay, Luciferase, Reporter Assay, Transfection, Quantitative RT-PCR

Journal: Cancer Biology & Therapy

Article Title: Fatty acid binding protein 5 promotes the proliferation, migration, and invasion of hepatocellular carcinoma cells by degradation of Krüppel-like factor 9 mediated by miR-889-5p via cAMP-response element binding protein

doi: 10.1080/15384047.2022.2094670

Figure Lengend Snippet: FABP5 inhibits the expression of miR-889-5p by regulating CREB protein phosphorylation. a, the siRNA of NF-κB, c-Myc and CREB were transiently transferred into HepG2 and SK-Hep-1 cells. The expression of miR-889-5p was detected by RT-qPCR. b, the designed mutative model of miR-889-5p promoter. c, a luciferase reporter promoter system was employed to confirm CREB bind to miR-889-5p promoter. d, the expression of total and phosphorylated CREB protein were detected by western blotting in the indicated groups. e, CREB inhibitor KG-501 reverse the effect of FABP5 overexpression on miR-889-5p expression in HCC cells. **p < .01 and ***p < .001 as compared with the vehicle control. e, the scheme of the mechanism by which FABP5 affects HCC tumorigenesis. FABP5 improves CREB protein phosphorylation to upregulate the miR-889-5p expression by CREB binding to the miR-889-5p promoter region, whereby leading to downregulation of KLF9 by miR-889-5p binding to the 3ʹ-UTR of the KLF9 mRNA, potentiating the PI3K/AKT signaling pathway and promoting the proliferation, migration, and invasion of HCC cells.

Article Snippet: The sections were incubated at 4°C overnight with rabbit anti-human FABP5 primary antibody (Proteintech, cat. no. 12348-1-AP) and then with goat anti-rabbit secondary antibody (Proteintech, cat. no. PR30009) for 50 min at room temperature.

Techniques: Expressing, Quantitative RT-PCR, Luciferase, Western Blot, Over Expression, Binding Assay, Migration

Journal: Nature Communications

Article Title: Oxidative stress-induced FABP5 S-glutathionylation protects against acute lung injury by suppressing inflammation in macrophages

doi: 10.1038/s41467-021-27428-9

Figure Lengend Snippet: a Schematic of the enrichment and quantitative analysis for SSG-modified Cys-peptides. b Enrichment of protein SSG and LC–MS/MS in BMDMs exposed to H 2 O 2 (200 μM) for 0, 15 min. Gene ontology annotations for biological process of proteins with significant S-glutathionylation alterations found to be increased or decreased in any stage by >1.2-fold compared to control. The slice for metabolic process is slightly displaced from the rest of the pie chart. c Alterations in S-glutathionylation sites under exposure to H 2 O 2 [shown as fold changes of protein S-glutathionylation (H 2 O 2 treated/control). Gray, identified protein sites; blue, metabolism-related differential proteins; orange, FABP5]. d mRNA levels of Ptbp1 , Otub1 , Ctsz , Gapdh , and Fabp5 in BMDMs as evaluated by qPCR, n = 3 biologically independent samples over 3 independent experiments, data are shown as mean ± SEM and analyzed with a 95% confidence interval, P < 0.0001, one-way ANOVA is followed by Tukey’s post-hoc test. Source data are provided as a Source Data file.

Article Snippet: Samples were incubated with Streptavidin-coupled Dynabeads (Invitrogen, 65801D) for 30 min at room temperature on a magnetic rack (Bio-Rad) and were analyzed by immunoblotting using anti-FABP5 antibody (Proteintech, 12348-1-AP; Cell Signaling Technology, 39926).

Techniques: Modification, Liquid Chromatography with Mass Spectroscopy

Journal: Nature Communications

Article Title: Oxidative stress-induced FABP5 S-glutathionylation protects against acute lung injury by suppressing inflammation in macrophages

doi: 10.1038/s41467-021-27428-9

Figure Lengend Snippet: a mRNA levels of FABP family members in RAW264.7 cells as determined by qPCR, n = 3 biologically independent samples over three independent experiments, data are presented as mean ± SEM and analyzed with a 95% confidence interval, P <0.0001, N.D, not detected, one-way ANOVA is followed by Tukey’s post-hoc test. b Protein levels of FABP5 in macrophages (MH-S), lung cancer cells (A549), epithelial cells (MLE-12), endothelial cells (HUVEC), fibroblasts (HFL1), and neutrophils (primary neutrophils) as evaluated by immunoblot. c Levels of FABP5 evaluated by immunoblot in RAW264.7 cells exposed to H 2 O 2 (200 μM) for indicated time (β-actin, loading control). d mRNA levels of Fabp5 in RAW264.7 cells after exposure to H 2 O 2 (200 μM) for indicated time as determined by qPCR, n = 3 biologically independent samples over three independent experiments, data are shown as mean ± SEM and analyzed with a 95% confidence interval, one-way ANOVA is followed by Tukey’s post-hoc test. e Co-IP of S-glutathionylation of FABP5 in COS-7 cells co-transfected with pXJ40-3xFlag-FABP5 and pcDNA3.1-3xFlag-Grx1 (or vector) and exposed for 15 min to H 2 O 2 (200 μM) at 24 h post-transfection (IP, GSH; IB, FABP5). Whole cell lysates confirm the expression of FABP5, Grx1, and β-actin (DTT, negative control). f Co-IP showing S-glutathionylation of FABP5 in BMDMs (from control or Grx1 KO mice) after exposure to H 2 O 2 (200 μM) for 15 min (IP, GSH; IB, FABP5). Whole cell lysates confirm the expression of FABP5, Grx1, and β-actin (DTT, negative control). g Immunoblotting analysis of reducing or non-reducing SDS–PAGE of RAW264.7 cells after exposure to H 2 O 2 (at the indicated concentration) for 15 min. h COS-7 cell lysates subjected to SDS-PAGE under reducing or non-reducing conditions after transfection of pXJ40-3xFlag vector or pXJ40-3xFlag-FABP5 and exposure to H 2 O 2 (200 μM) for 15 min. i Co-IP for S-glutathionylation of FABP5 in WT BMDMs after treatment with LPS (100 ng/mL) for 15 min (IP, GSH; IB, FABP5). Whole cell lysates confirm the expression of FABP5 and β-actin. j S-glutathionylation of FABP5 in lung tissues from Grx1 fl/fl and Grx1 fl/fl LysM cre mouse 24 h following intratracheal administration of PBS or LPS. Source data are provided as a Source Data file.

Article Snippet: Samples were incubated with Streptavidin-coupled Dynabeads (Invitrogen, 65801D) for 30 min at room temperature on a magnetic rack (Bio-Rad) and were analyzed by immunoblotting using anti-FABP5 antibody (Proteintech, 12348-1-AP; Cell Signaling Technology, 39926).

Techniques: Western Blot, Co-Immunoprecipitation Assay, Transfection, Plasmid Preparation, Expressing, Negative Control, SDS Page, Concentration Assay

Journal: Nature Communications

Article Title: Oxidative stress-induced FABP5 S-glutathionylation protects against acute lung injury by suppressing inflammation in macrophages

doi: 10.1038/s41467-021-27428-9

Figure Lengend Snippet: a – c Immunoblot analysis of FABP5 binding to ULCFA ( a ), SLCFA ( b ) or ULCFA with Grx1 treatment ( c ). Purified recombinant FABP5 protein was incubated with GSH plus H 2 O 2 or GSSG for 15 min, then mixed with Biotin-ULCFA or Biotin-SLCFA in the absence or presence of BMS309403 or linoleic acid or Grx1. FABP5 interacted with the Biotin-ULCFA or Biotin-SLCFA was captured using streptavidin magnetic beads. d Immunofluorescence staining of BMDMs after exposure to H 2 O 2 (200 μM) for 30 min (green, FABP5; blue, DAPI; scale bars, 10 μm). e Quantitative analysis of the ratio of nuclear/cytoplasmic fluorescence intensity in BMDMs as in ( d ), n = 10 in each group, P (ctr vs. ctr H2O2) = 0.0172, P (ctr H2O2 vs. Grx1 KO H2O2) <0.0001. f Immunoblot analysis of cytoplasmic and nuclear FABP5 in COS-7 cells. COS-7 cells were transfected with pXJ40-3xFlag vector or pXJ40-3xFlag-FABP5 followed by exposure to H 2 O 2 (200 μM) for 1 h. Lamin B1 (nuclear fraction) and GAPDH (cytoplasmic fraction) are loading controls. g Immunoblots with antibodies against the indicated proteins in nuclear and cytoplasmic fractions from COS-7 cells co-transfected with pXJ40-3xFlag-FABP5 (or vector) and pcDNA3.1-3xFlag-Grx1 (or vector) for 24 h, then exposed to H 2 O 2 (200 μM) for 1 h. h Immunoblots against the indicated proteins of cytoplasmic and nuclear extracts from BMDMs exposed to H 2 O 2 (200 μM) for 1 h. i mRNA expression of Il1b , Il6 , and Tnfα in BMDMs from mice stimulated with LPS (100 ng/mL) for 4 h, as evaluated by qPCR, n = 3 in each group, P ( Il1b , Ctr vs. Ctr+LPS) = 0.0013, P ( Il1b , Ctr+LPS vs. Grx1 KO+LPS) = 0.0091, *** P < 0.0001. j mRNA levels of Il1b , Il6 , and Tnfα in BMDMs pre-treated with an FABP5 inhibitor (SBFI26, 100 μM) for 2 h and stimulated with LPS (100 ng/mL) for 4 h, as determined by qPCR, n = 3 in each group, *** P < 0.0001. k mRNA levels of Il1b , Il6 , and Tnfα in BMDMs after treated with SBFI26 (100 μM) for 2 h and stimulation with LPS (100 ng/mL) for 4 h, as evaluated by qPCR, n = 3 in each group, P ( Il1b , Ctr+LPS vs. Ctr+LPS+inhibitor) < 0.0001, P ( Il16 , Ctr+LPS vs. Ctr+LPS+inhibitor) < 0.0001, P ( Tnfα , Ctr+LPS vs. Ctr+LPS+inhibitor) = 0.0046. All samples were biologically independent and three or more independent experiments were performed. All quantitative data are shown as mean ± SEM and analyzed with a 95% confidence interval. One-way ANOVA followed by Tukey’s post-hoc test for (e, i-k). * P < 0.05, ** P < 0.01, *** P < 0.001, ns = no significance. Source data are provided as a Source Data file.

Article Snippet: Samples were incubated with Streptavidin-coupled Dynabeads (Invitrogen, 65801D) for 30 min at room temperature on a magnetic rack (Bio-Rad) and were analyzed by immunoblotting using anti-FABP5 antibody (Proteintech, 12348-1-AP; Cell Signaling Technology, 39926).

Techniques: Western Blot, Binding Assay, Purification, Recombinant, Incubation, Magnetic Beads, Immunofluorescence, Staining, Fluorescence, Transfection, Plasmid Preparation, Expressing

Journal: Nature Communications

Article Title: Oxidative stress-induced FABP5 S-glutathionylation protects against acute lung injury by suppressing inflammation in macrophages

doi: 10.1038/s41467-021-27428-9

Figure Lengend Snippet: a Model structure of FABP5 and its 6 cysteines, generated by Pymol and based on the crystal structure of Homo sapiens FABP5 (PDB ID,4LKT). b Mass spectra of a peptide from FABP5 including glutathionylated cysteine 120 and cysteine127. c Immunofluorescence staining (with FABP5) and confocal microscopy imaging of COS-7 cells with overexpression of FABP5 WT, C67S, C87S, C120S, and C127S after exposure to H 2 O 2 (200 μM) for 1 h (green, FABP5; blue, DAPI; scale bars, 10 μm). d Immunoblot analysis of cytoplasmic and nuclear FABP5 in COS-7 cells transfected with FABP5 WT or C127S after exposure to H 2 O 2 (200 μM) for 1 h. Lamin B1 (nuclear fraction) and GAPDH (cytoplasmic fraction) are loading controls. e Model structure of FABP5 interaction with an FA (Fatty Acid, Linoleic Acid), generated by Pymol and based on the crystal structure of FABP5 (PDB ID, 4LKT). f Docking structure of FABP5 in complex with S-glutathionylated Cys127 interacting with an FA (Fatty Acid, Linoleic Acid). g Docking structure of dimer FABP5 (PDB ID, 4AZM, Homo sapiens ) in complex with reduced glutathione (GSH) on Cys127. h Co-IP for S-glutathionylation of FABP5 in COS-7 cells overexpressing pXJ40-3xFlag-FABP5 WT or C127S and exposed to H 2 O 2 (200 μM) for 15 min (IP, GSH; IB, FABP5). Whole-cell lysates confirm the expression of FABP5 and β-actin (DTT, negative control). i Immunoblot analysis of FABP5 fatty acid binding. Purified recombinant FABP5 WT or C127S protein (1 μg) was incubated with GSSG (1 mM) for 15 min, then mixed with Biotin-linoleic acid in the absence or presence of BMS309403 (5 mM) for 30 min. FABP5 that associated with the Biotin-linoleic acid was captured using streptavidin magnetic beads. Source data are provided as a Source Data file.

Article Snippet: Samples were incubated with Streptavidin-coupled Dynabeads (Invitrogen, 65801D) for 30 min at room temperature on a magnetic rack (Bio-Rad) and were analyzed by immunoblotting using anti-FABP5 antibody (Proteintech, 12348-1-AP; Cell Signaling Technology, 39926).

Techniques: Generated, Immunofluorescence, Staining, Confocal Microscopy, Imaging, Over Expression, Western Blot, Transfection, Co-Immunoprecipitation Assay, Expressing, Negative Control, Binding Assay, Purification, Recombinant, Incubation, Magnetic Beads

Journal: Nature Communications

Article Title: Oxidative stress-induced FABP5 S-glutathionylation protects against acute lung injury by suppressing inflammation in macrophages

doi: 10.1038/s41467-021-27428-9

Figure Lengend Snippet: a Volcano plot of RNA-seq transcriptome data displaying the pattern of gene expression values for FABP5 KO BMDMs with overexpression of FABP5 C127S relative to FABP5 WT after exposure to LPS (500 ng/mL) for 24 h [red and blue dots, significantly up-and down-regulated genes ( P < 0.01, |log 2 FC|>1); black dashed lines, boundary for identification of up- or down-regulated genes]. b Heatmap of regulation of the inflammatory response and cytokine transcript production in LPS-stimulated (24 h) BMDMs nucleofected with either pXJ40-3xFlag-FABP5 WT or C127S. Data shown are relative to the calculated Z scores across the samples. Red, relatively high levels of expression; blue, relatively low levels of expression. Each column represents one individual (for a total of 3 per group) and each row represents the expression of a single gene. c Diagram of GO analysis classifying DEGs into biological process groups using Panther ( http://www.pantherdb.org ). Some GO categories with FDR <0.05 are included. GO, gene ontology; DEGs, differentially-expressed genes. d mRNA expression of Il1b , Il6 , and Tnfα in FABP5 KO BMDMs after nucleofection with pXJ40-3xFlag-FABP5 WT or C127S and stimulation with LPS (1 μg/mL) for 24 h, as identified by qPCR, n = 3 in each group, P ( Il6 , FABP5 WT+LPS vs. FABP5 C127S+LPS) = 0.0045, *** P < 0.0001. e mRNA expression of Il1b , Il6 , and Tnfα in Grx1 KO BMDMs after nucleofection of pXJ40-3xFlag-FABP5 WT or C127S and treatment with LPS (500 ng/mL) for 24 h, as determined by qPCR, n = 3 in each group, P ( Il6 , Ctr+FABP5 C127S vs. Grx1 KO+FABP5 C127S) = 0.0004, P ( TNFα , Ctr+FABP5 WT vs. Grx1 KO+FABP5 WT) = 0.0123, *** P < 0.0001(except group Il6 , Ctr+FABP5 C127S vs. Grx1 KO+FABP5 C127S). f H&E staining showing lung histopathological changes in FABP5 fl/fl and FABP5 fl/fl LysM cre mice 24 h after intratracheal administration of PBS or LPS (5 mg/kg). Scale bars, 50 μm. g Total numbers of leukocytes in BALF from FABP5 fl/fl and FABP5 fl/fl LysM cre mice 24 h after intratracheal administration of PBS or LPS (5 mg/kg), n = 4, 4, 9, 8, respectively, *** P < 0.0001. All samples were biologically independent and three or more independent experiments were performed. All quantitative data are shown as mean ± SEM and analyzed with a 95% confidence interval. One-way ANOVA followed by Tukey’s post-hoc test for ( d – e , g ). * P < 0.05, ** P < 0.01, *** P < 0.001. Source data are provided as a Source Data file.

Article Snippet: Samples were incubated with Streptavidin-coupled Dynabeads (Invitrogen, 65801D) for 30 min at room temperature on a magnetic rack (Bio-Rad) and were analyzed by immunoblotting using anti-FABP5 antibody (Proteintech, 12348-1-AP; Cell Signaling Technology, 39926).

Techniques: RNA Sequencing Assay, Expressing, Over Expression, Staining

Journal: Nature Communications

Article Title: Oxidative stress-induced FABP5 S-glutathionylation protects against acute lung injury by suppressing inflammation in macrophages

doi: 10.1038/s41467-021-27428-9

Figure Lengend Snippet: a In vitro pull-down assays. The mouse recombinant FABP5 (WT or C127S) protein with 3xFlag tag purified from E. coli treated with GSSG (1 mM) for 15 min was incubated with COS-7 lysates and precipitated with Flag-magnetic beads, respectively. Precipitates were subjected to SDS­PAGE and examined by immunoblot with anti­ PPARβ/δ antibodies. b Transactivation assays in Raw264.7 cells transfected with a PPRE-driven luciferase reporter. After nucleofection with PPRE X3-TK-luc and pRL-TK, Raw264.7 cells were serum starved for 8 h, then exposed to H 2 O 2 (200 μM), GW0742 (1 μM), or both for 24 h, and luciferase activity was determined, n = 3 in each group, P (ctr vs. H2O2) = 0.0003, P (ctr vs.GW0742) < 0.0001, P (GW0742 vs.H2O2+GW0742) = 0.0047. c Transactivation assays in COS-7 cells transfected with a PPRE-driven luciferase reporter. After transfection with PPRE X3-TK-luc, FABP5, and pRL-TK, cells were serum starved for 8 h, then treated with H 2 O 2 (200 μM), GW0742 (1 μM), or both for 24 h, and luciferase activity was measured, n = 3 in each group, *** P < 0.0001. d ChIP analysis of FABP5 recruitment to the PPREs of PPARβ/δ target gene promoters in Raw 264.7 cells after exposure to H 2 O 2 (200 μM) for 1 h. ChIP signals were quantified by qPCR, n = 3 in each group, P (ADRP, ctr vs. H2O2) = 0.001, P (FIAF, ctr vs. H2O2) < 0.0001. e mRNA levels of Adrp , Fiaf , and Cpt1a in RAW264.7 cells after treatment with BMS309403 (50 μM) for 2 h, stimulation with GW0742 (1 μM) for 30 min, and exposure to H 2 O 2 (200 μM) for 4 h, as determined by qPCR, n = 3 in each group, P ( Adrp , ctr vs. GW0742) < 0.0001, P ( Adrp , GW0742 vs. GW0742+BMS309403) = 0.0009, P ( Fiaf , ctr vs. GW0742) = 0.001, P ( Fiaf , GW0742 vs. GW0742+BMS309403) = 0.001, P ( Cpt1α , ctr vs. GW0742) < 0.0001, P ( Cpt1α , GW0742 vs. GW0742+BMS309403) < 0.0001. f Co-IP for S-glutathionylation of FABP5 in COS-7 cells overexpressing pXJ40-3xFlag-FABP5 WT or G114R and exposed to H 2 O 2 (200 μM) for 15 min (IP, GSH; IB, FABP5). Whole-cell lysates confirm the expression of FABP5 and β-actin (DTT, negative control). All samples were biologically independent and three or more independent experiments were performed. All quantitative data are shown as mean ± SEM and analyzed with a 95% confidence interval. One-way ANOVA followed by Tukey’s post-hoc test for ( b – e ). * P < 0.05, ** P < 0.01, *** P < 0.001. Source data are provided as a Source Data file.

Article Snippet: Samples were incubated with Streptavidin-coupled Dynabeads (Invitrogen, 65801D) for 30 min at room temperature on a magnetic rack (Bio-Rad) and were analyzed by immunoblotting using anti-FABP5 antibody (Proteintech, 12348-1-AP; Cell Signaling Technology, 39926).

Techniques: In Vitro, Recombinant, Purification, Incubation, Magnetic Beads, Western Blot, Transfection, Luciferase, Activity Assay, Co-Immunoprecipitation Assay, Expressing, Negative Control

Journal: Nature Communications

Article Title: Oxidative stress-induced FABP5 S-glutathionylation protects against acute lung injury by suppressing inflammation in macrophages

doi: 10.1038/s41467-021-27428-9

Figure Lengend Snippet: a Schematic of the enrichment and quantitative analysis for SSG-modified Cys-peptides. b Enrichment of protein SSG and LC–MS/MS in BMDMs exposed to H 2 O 2 (200 μM) for 0, 15 min. Gene ontology annotations for biological process of proteins with significant S-glutathionylation alterations found to be increased or decreased in any stage by >1.2-fold compared to control. The slice for metabolic process is slightly displaced from the rest of the pie chart. c Alterations in S-glutathionylation sites under exposure to H 2 O 2 [shown as fold changes of protein S-glutathionylation (H 2 O 2 treated/control). Gray, identified protein sites; blue, metabolism-related differential proteins; orange, FABP5]. d mRNA levels of Ptbp1 , Otub1 , Ctsz , Gapdh , and Fabp5 in BMDMs as evaluated by qPCR, n = 3 biologically independent samples over 3 independent experiments, data are shown as mean ± SEM and analyzed with a 95% confidence interval, P < 0.0001, one-way ANOVA is followed by Tukey’s post-hoc test. Source data are provided as a Source Data file.

Article Snippet: Total cell lysates were used for Western blot using antibodies against FABP5 (Cell Signaling Technology, 39926, 1:1000), FABP5 (Proteintech, 12348-1-AP, 1:500), Grx1 (Abcam, ab45953, 1:250), β-actin (Huabio, M1210-2, 1:2000), PPARβ/δ (Santa Cruz, sc-74517, 1:500), Lamin A/C (Cell Signaling Technology, 4777, 1:2000) and Lamin B1 (Proteintech, 66059-1-Ig, 1:1000).

Techniques: Modification, Liquid Chromatography with Mass Spectroscopy

Journal: Nature Communications

Article Title: Oxidative stress-induced FABP5 S-glutathionylation protects against acute lung injury by suppressing inflammation in macrophages

doi: 10.1038/s41467-021-27428-9

Figure Lengend Snippet: a mRNA levels of FABP family members in RAW264.7 cells as determined by qPCR, n = 3 biologically independent samples over three independent experiments, data are presented as mean ± SEM and analyzed with a 95% confidence interval, P <0.0001, N.D, not detected, one-way ANOVA is followed by Tukey’s post-hoc test. b Protein levels of FABP5 in macrophages (MH-S), lung cancer cells (A549), epithelial cells (MLE-12), endothelial cells (HUVEC), fibroblasts (HFL1), and neutrophils (primary neutrophils) as evaluated by immunoblot. c Levels of FABP5 evaluated by immunoblot in RAW264.7 cells exposed to H 2 O 2 (200 μM) for indicated time (β-actin, loading control). d mRNA levels of Fabp5 in RAW264.7 cells after exposure to H 2 O 2 (200 μM) for indicated time as determined by qPCR, n = 3 biologically independent samples over three independent experiments, data are shown as mean ± SEM and analyzed with a 95% confidence interval, one-way ANOVA is followed by Tukey’s post-hoc test. e Co-IP of S-glutathionylation of FABP5 in COS-7 cells co-transfected with pXJ40-3xFlag-FABP5 and pcDNA3.1-3xFlag-Grx1 (or vector) and exposed for 15 min to H 2 O 2 (200 μM) at 24 h post-transfection (IP, GSH; IB, FABP5). Whole cell lysates confirm the expression of FABP5, Grx1, and β-actin (DTT, negative control). f Co-IP showing S-glutathionylation of FABP5 in BMDMs (from control or Grx1 KO mice) after exposure to H 2 O 2 (200 μM) for 15 min (IP, GSH; IB, FABP5). Whole cell lysates confirm the expression of FABP5, Grx1, and β-actin (DTT, negative control). g Immunoblotting analysis of reducing or non-reducing SDS–PAGE of RAW264.7 cells after exposure to H 2 O 2 (at the indicated concentration) for 15 min. h COS-7 cell lysates subjected to SDS-PAGE under reducing or non-reducing conditions after transfection of pXJ40-3xFlag vector or pXJ40-3xFlag-FABP5 and exposure to H 2 O 2 (200 μM) for 15 min. i Co-IP for S-glutathionylation of FABP5 in WT BMDMs after treatment with LPS (100 ng/mL) for 15 min (IP, GSH; IB, FABP5). Whole cell lysates confirm the expression of FABP5 and β-actin. j S-glutathionylation of FABP5 in lung tissues from Grx1 fl/fl and Grx1 fl/fl LysM cre mouse 24 h following intratracheal administration of PBS or LPS. Source data are provided as a Source Data file.

Article Snippet: Total cell lysates were used for Western blot using antibodies against FABP5 (Cell Signaling Technology, 39926, 1:1000), FABP5 (Proteintech, 12348-1-AP, 1:500), Grx1 (Abcam, ab45953, 1:250), β-actin (Huabio, M1210-2, 1:2000), PPARβ/δ (Santa Cruz, sc-74517, 1:500), Lamin A/C (Cell Signaling Technology, 4777, 1:2000) and Lamin B1 (Proteintech, 66059-1-Ig, 1:1000).

Techniques: Western Blot, Co-Immunoprecipitation Assay, Transfection, Plasmid Preparation, Expressing, Negative Control, SDS Page, Concentration Assay

Journal: Nature Communications

Article Title: Oxidative stress-induced FABP5 S-glutathionylation protects against acute lung injury by suppressing inflammation in macrophages

doi: 10.1038/s41467-021-27428-9

Figure Lengend Snippet: a – c Immunoblot analysis of FABP5 binding to ULCFA ( a ), SLCFA ( b ) or ULCFA with Grx1 treatment ( c ). Purified recombinant FABP5 protein was incubated with GSH plus H 2 O 2 or GSSG for 15 min, then mixed with Biotin-ULCFA or Biotin-SLCFA in the absence or presence of BMS309403 or linoleic acid or Grx1. FABP5 interacted with the Biotin-ULCFA or Biotin-SLCFA was captured using streptavidin magnetic beads. d Immunofluorescence staining of BMDMs after exposure to H 2 O 2 (200 μM) for 30 min (green, FABP5; blue, DAPI; scale bars, 10 μm). e Quantitative analysis of the ratio of nuclear/cytoplasmic fluorescence intensity in BMDMs as in ( d ), n = 10 in each group, P (ctr vs. ctr H2O2) = 0.0172, P (ctr H2O2 vs. Grx1 KO H2O2) <0.0001. f Immunoblot analysis of cytoplasmic and nuclear FABP5 in COS-7 cells. COS-7 cells were transfected with pXJ40-3xFlag vector or pXJ40-3xFlag-FABP5 followed by exposure to H 2 O 2 (200 μM) for 1 h. Lamin B1 (nuclear fraction) and GAPDH (cytoplasmic fraction) are loading controls. g Immunoblots with antibodies against the indicated proteins in nuclear and cytoplasmic fractions from COS-7 cells co-transfected with pXJ40-3xFlag-FABP5 (or vector) and pcDNA3.1-3xFlag-Grx1 (or vector) for 24 h, then exposed to H 2 O 2 (200 μM) for 1 h. h Immunoblots against the indicated proteins of cytoplasmic and nuclear extracts from BMDMs exposed to H 2 O 2 (200 μM) for 1 h. i mRNA expression of Il1b , Il6 , and Tnfα in BMDMs from mice stimulated with LPS (100 ng/mL) for 4 h, as evaluated by qPCR, n = 3 in each group, P ( Il1b , Ctr vs. Ctr+LPS) = 0.0013, P ( Il1b , Ctr+LPS vs. Grx1 KO+LPS) = 0.0091, *** P < 0.0001. j mRNA levels of Il1b , Il6 , and Tnfα in BMDMs pre-treated with an FABP5 inhibitor (SBFI26, 100 μM) for 2 h and stimulated with LPS (100 ng/mL) for 4 h, as determined by qPCR, n = 3 in each group, *** P < 0.0001. k mRNA levels of Il1b , Il6 , and Tnfα in BMDMs after treated with SBFI26 (100 μM) for 2 h and stimulation with LPS (100 ng/mL) for 4 h, as evaluated by qPCR, n = 3 in each group, P ( Il1b , Ctr+LPS vs. Ctr+LPS+inhibitor) < 0.0001, P ( Il16 , Ctr+LPS vs. Ctr+LPS+inhibitor) < 0.0001, P ( Tnfα , Ctr+LPS vs. Ctr+LPS+inhibitor) = 0.0046. All samples were biologically independent and three or more independent experiments were performed. All quantitative data are shown as mean ± SEM and analyzed with a 95% confidence interval. One-way ANOVA followed by Tukey’s post-hoc test for (e, i-k). * P < 0.05, ** P < 0.01, *** P < 0.001, ns = no significance. Source data are provided as a Source Data file.

Article Snippet: Total cell lysates were used for Western blot using antibodies against FABP5 (Cell Signaling Technology, 39926, 1:1000), FABP5 (Proteintech, 12348-1-AP, 1:500), Grx1 (Abcam, ab45953, 1:250), β-actin (Huabio, M1210-2, 1:2000), PPARβ/δ (Santa Cruz, sc-74517, 1:500), Lamin A/C (Cell Signaling Technology, 4777, 1:2000) and Lamin B1 (Proteintech, 66059-1-Ig, 1:1000).

Techniques: Western Blot, Binding Assay, Purification, Recombinant, Incubation, Magnetic Beads, Immunofluorescence, Staining, Fluorescence, Transfection, Plasmid Preparation, Expressing

Journal: Nature Communications

Article Title: Oxidative stress-induced FABP5 S-glutathionylation protects against acute lung injury by suppressing inflammation in macrophages

doi: 10.1038/s41467-021-27428-9

Figure Lengend Snippet: a Model structure of FABP5 and its 6 cysteines, generated by Pymol and based on the crystal structure of Homo sapiens FABP5 (PDB ID,4LKT). b Mass spectra of a peptide from FABP5 including glutathionylated cysteine 120 and cysteine127. c Immunofluorescence staining (with FABP5) and confocal microscopy imaging of COS-7 cells with overexpression of FABP5 WT, C67S, C87S, C120S, and C127S after exposure to H 2 O 2 (200 μM) for 1 h (green, FABP5; blue, DAPI; scale bars, 10 μm). d Immunoblot analysis of cytoplasmic and nuclear FABP5 in COS-7 cells transfected with FABP5 WT or C127S after exposure to H 2 O 2 (200 μM) for 1 h. Lamin B1 (nuclear fraction) and GAPDH (cytoplasmic fraction) are loading controls. e Model structure of FABP5 interaction with an FA (Fatty Acid, Linoleic Acid), generated by Pymol and based on the crystal structure of FABP5 (PDB ID, 4LKT). f Docking structure of FABP5 in complex with S-glutathionylated Cys127 interacting with an FA (Fatty Acid, Linoleic Acid). g Docking structure of dimer FABP5 (PDB ID, 4AZM, Homo sapiens ) in complex with reduced glutathione (GSH) on Cys127. h Co-IP for S-glutathionylation of FABP5 in COS-7 cells overexpressing pXJ40-3xFlag-FABP5 WT or C127S and exposed to H 2 O 2 (200 μM) for 15 min (IP, GSH; IB, FABP5). Whole-cell lysates confirm the expression of FABP5 and β-actin (DTT, negative control). i Immunoblot analysis of FABP5 fatty acid binding. Purified recombinant FABP5 WT or C127S protein (1 μg) was incubated with GSSG (1 mM) for 15 min, then mixed with Biotin-linoleic acid in the absence or presence of BMS309403 (5 mM) for 30 min. FABP5 that associated with the Biotin-linoleic acid was captured using streptavidin magnetic beads. Source data are provided as a Source Data file.

Article Snippet: Total cell lysates were used for Western blot using antibodies against FABP5 (Cell Signaling Technology, 39926, 1:1000), FABP5 (Proteintech, 12348-1-AP, 1:500), Grx1 (Abcam, ab45953, 1:250), β-actin (Huabio, M1210-2, 1:2000), PPARβ/δ (Santa Cruz, sc-74517, 1:500), Lamin A/C (Cell Signaling Technology, 4777, 1:2000) and Lamin B1 (Proteintech, 66059-1-Ig, 1:1000).

Techniques: Generated, Immunofluorescence, Staining, Confocal Microscopy, Imaging, Over Expression, Western Blot, Transfection, Co-Immunoprecipitation Assay, Expressing, Negative Control, Binding Assay, Purification, Recombinant, Incubation, Magnetic Beads

Journal: Nature Communications

Article Title: Oxidative stress-induced FABP5 S-glutathionylation protects against acute lung injury by suppressing inflammation in macrophages

doi: 10.1038/s41467-021-27428-9

Figure Lengend Snippet: a Volcano plot of RNA-seq transcriptome data displaying the pattern of gene expression values for FABP5 KO BMDMs with overexpression of FABP5 C127S relative to FABP5 WT after exposure to LPS (500 ng/mL) for 24 h [red and blue dots, significantly up-and down-regulated genes ( P < 0.01, |log 2 FC|>1); black dashed lines, boundary for identification of up- or down-regulated genes]. b Heatmap of regulation of the inflammatory response and cytokine transcript production in LPS-stimulated (24 h) BMDMs nucleofected with either pXJ40-3xFlag-FABP5 WT or C127S. Data shown are relative to the calculated Z scores across the samples. Red, relatively high levels of expression; blue, relatively low levels of expression. Each column represents one individual (for a total of 3 per group) and each row represents the expression of a single gene. c Diagram of GO analysis classifying DEGs into biological process groups using Panther ( http://www.pantherdb.org ). Some GO categories with FDR <0.05 are included. GO, gene ontology; DEGs, differentially-expressed genes. d mRNA expression of Il1b , Il6 , and Tnfα in FABP5 KO BMDMs after nucleofection with pXJ40-3xFlag-FABP5 WT or C127S and stimulation with LPS (1 μg/mL) for 24 h, as identified by qPCR, n = 3 in each group, P ( Il6 , FABP5 WT+LPS vs. FABP5 C127S+LPS) = 0.0045, *** P < 0.0001. e mRNA expression of Il1b , Il6 , and Tnfα in Grx1 KO BMDMs after nucleofection of pXJ40-3xFlag-FABP5 WT or C127S and treatment with LPS (500 ng/mL) for 24 h, as determined by qPCR, n = 3 in each group, P ( Il6 , Ctr+FABP5 C127S vs. Grx1 KO+FABP5 C127S) = 0.0004, P ( TNFα , Ctr+FABP5 WT vs. Grx1 KO+FABP5 WT) = 0.0123, *** P < 0.0001(except group Il6 , Ctr+FABP5 C127S vs. Grx1 KO+FABP5 C127S). f H&E staining showing lung histopathological changes in FABP5 fl/fl and FABP5 fl/fl LysM cre mice 24 h after intratracheal administration of PBS or LPS (5 mg/kg). Scale bars, 50 μm. g Total numbers of leukocytes in BALF from FABP5 fl/fl and FABP5 fl/fl LysM cre mice 24 h after intratracheal administration of PBS or LPS (5 mg/kg), n = 4, 4, 9, 8, respectively, *** P < 0.0001. All samples were biologically independent and three or more independent experiments were performed. All quantitative data are shown as mean ± SEM and analyzed with a 95% confidence interval. One-way ANOVA followed by Tukey’s post-hoc test for ( d – e , g ). * P < 0.05, ** P < 0.01, *** P < 0.001. Source data are provided as a Source Data file.

Article Snippet: Total cell lysates were used for Western blot using antibodies against FABP5 (Cell Signaling Technology, 39926, 1:1000), FABP5 (Proteintech, 12348-1-AP, 1:500), Grx1 (Abcam, ab45953, 1:250), β-actin (Huabio, M1210-2, 1:2000), PPARβ/δ (Santa Cruz, sc-74517, 1:500), Lamin A/C (Cell Signaling Technology, 4777, 1:2000) and Lamin B1 (Proteintech, 66059-1-Ig, 1:1000).

Techniques: RNA Sequencing Assay, Expressing, Over Expression, Staining

Journal: Nature Communications

Article Title: Oxidative stress-induced FABP5 S-glutathionylation protects against acute lung injury by suppressing inflammation in macrophages

doi: 10.1038/s41467-021-27428-9

Figure Lengend Snippet: a In vitro pull-down assays. The mouse recombinant FABP5 (WT or C127S) protein with 3xFlag tag purified from E. coli treated with GSSG (1 mM) for 15 min was incubated with COS-7 lysates and precipitated with Flag-magnetic beads, respectively. Precipitates were subjected to SDS­PAGE and examined by immunoblot with anti­ PPARβ/δ antibodies. b Transactivation assays in Raw264.7 cells transfected with a PPRE-driven luciferase reporter. After nucleofection with PPRE X3-TK-luc and pRL-TK, Raw264.7 cells were serum starved for 8 h, then exposed to H 2 O 2 (200 μM), GW0742 (1 μM), or both for 24 h, and luciferase activity was determined, n = 3 in each group, P (ctr vs. H2O2) = 0.0003, P (ctr vs.GW0742) < 0.0001, P (GW0742 vs.H2O2+GW0742) = 0.0047. c Transactivation assays in COS-7 cells transfected with a PPRE-driven luciferase reporter. After transfection with PPRE X3-TK-luc, FABP5, and pRL-TK, cells were serum starved for 8 h, then treated with H 2 O 2 (200 μM), GW0742 (1 μM), or both for 24 h, and luciferase activity was measured, n = 3 in each group, *** P < 0.0001. d ChIP analysis of FABP5 recruitment to the PPREs of PPARβ/δ target gene promoters in Raw 264.7 cells after exposure to H 2 O 2 (200 μM) for 1 h. ChIP signals were quantified by qPCR, n = 3 in each group, P (ADRP, ctr vs. H2O2) = 0.001, P (FIAF, ctr vs. H2O2) < 0.0001. e mRNA levels of Adrp , Fiaf , and Cpt1a in RAW264.7 cells after treatment with BMS309403 (50 μM) for 2 h, stimulation with GW0742 (1 μM) for 30 min, and exposure to H 2 O 2 (200 μM) for 4 h, as determined by qPCR, n = 3 in each group, P ( Adrp , ctr vs. GW0742) < 0.0001, P ( Adrp , GW0742 vs. GW0742+BMS309403) = 0.0009, P ( Fiaf , ctr vs. GW0742) = 0.001, P ( Fiaf , GW0742 vs. GW0742+BMS309403) = 0.001, P ( Cpt1α , ctr vs. GW0742) < 0.0001, P ( Cpt1α , GW0742 vs. GW0742+BMS309403) < 0.0001. f Co-IP for S-glutathionylation of FABP5 in COS-7 cells overexpressing pXJ40-3xFlag-FABP5 WT or G114R and exposed to H 2 O 2 (200 μM) for 15 min (IP, GSH; IB, FABP5). Whole-cell lysates confirm the expression of FABP5 and β-actin (DTT, negative control). All samples were biologically independent and three or more independent experiments were performed. All quantitative data are shown as mean ± SEM and analyzed with a 95% confidence interval. One-way ANOVA followed by Tukey’s post-hoc test for ( b – e ). * P < 0.05, ** P < 0.01, *** P < 0.001. Source data are provided as a Source Data file.

Article Snippet: Total cell lysates were used for Western blot using antibodies against FABP5 (Cell Signaling Technology, 39926, 1:1000), FABP5 (Proteintech, 12348-1-AP, 1:500), Grx1 (Abcam, ab45953, 1:250), β-actin (Huabio, M1210-2, 1:2000), PPARβ/δ (Santa Cruz, sc-74517, 1:500), Lamin A/C (Cell Signaling Technology, 4777, 1:2000) and Lamin B1 (Proteintech, 66059-1-Ig, 1:1000).

Techniques: In Vitro, Recombinant, Purification, Incubation, Magnetic Beads, Western Blot, Transfection, Luciferase, Activity Assay, Co-Immunoprecipitation Assay, Expressing, Negative Control

Journal: Nature Communications

Article Title: Oxidative stress-induced FABP5 S-glutathionylation protects against acute lung injury by suppressing inflammation in macrophages

doi: 10.1038/s41467-021-27428-9

Figure Lengend Snippet: a Schematic of the enrichment and quantitative analysis for SSG-modified Cys-peptides. b Enrichment of protein SSG and LC–MS/MS in BMDMs exposed to H 2 O 2 (200 μM) for 0, 15 min. Gene ontology annotations for biological process of proteins with significant S-glutathionylation alterations found to be increased or decreased in any stage by >1.2-fold compared to control. The slice for metabolic process is slightly displaced from the rest of the pie chart. c Alterations in S-glutathionylation sites under exposure to H 2 O 2 [shown as fold changes of protein S-glutathionylation (H 2 O 2 treated/control). Gray, identified protein sites; blue, metabolism-related differential proteins; orange, FABP5]. d mRNA levels of Ptbp1 , Otub1 , Ctsz , Gapdh , and Fabp5 in BMDMs as evaluated by qPCR, n = 3 biologically independent samples over 3 independent experiments, data are shown as mean ± SEM and analyzed with a 95% confidence interval, P < 0.0001, one-way ANOVA is followed by Tukey’s post-hoc test. Source data are provided as a Source Data file.

Article Snippet: And DNA-bound protein was immunoprecipitated using anti-FABP5 (Proteintech, 12348-1-AP) and anti-IgG (Thermo Fisher Scientific, 26157) antibodies.

Techniques: Modification, Liquid Chromatography with Mass Spectroscopy

Journal: Nature Communications

Article Title: Oxidative stress-induced FABP5 S-glutathionylation protects against acute lung injury by suppressing inflammation in macrophages

doi: 10.1038/s41467-021-27428-9

Figure Lengend Snippet: a mRNA levels of FABP family members in RAW264.7 cells as determined by qPCR, n = 3 biologically independent samples over three independent experiments, data are presented as mean ± SEM and analyzed with a 95% confidence interval, P <0.0001, N.D, not detected, one-way ANOVA is followed by Tukey’s post-hoc test. b Protein levels of FABP5 in macrophages (MH-S), lung cancer cells (A549), epithelial cells (MLE-12), endothelial cells (HUVEC), fibroblasts (HFL1), and neutrophils (primary neutrophils) as evaluated by immunoblot. c Levels of FABP5 evaluated by immunoblot in RAW264.7 cells exposed to H 2 O 2 (200 μM) for indicated time (β-actin, loading control). d mRNA levels of Fabp5 in RAW264.7 cells after exposure to H 2 O 2 (200 μM) for indicated time as determined by qPCR, n = 3 biologically independent samples over three independent experiments, data are shown as mean ± SEM and analyzed with a 95% confidence interval, one-way ANOVA is followed by Tukey’s post-hoc test. e Co-IP of S-glutathionylation of FABP5 in COS-7 cells co-transfected with pXJ40-3xFlag-FABP5 and pcDNA3.1-3xFlag-Grx1 (or vector) and exposed for 15 min to H 2 O 2 (200 μM) at 24 h post-transfection (IP, GSH; IB, FABP5). Whole cell lysates confirm the expression of FABP5, Grx1, and β-actin (DTT, negative control). f Co-IP showing S-glutathionylation of FABP5 in BMDMs (from control or Grx1 KO mice) after exposure to H 2 O 2 (200 μM) for 15 min (IP, GSH; IB, FABP5). Whole cell lysates confirm the expression of FABP5, Grx1, and β-actin (DTT, negative control). g Immunoblotting analysis of reducing or non-reducing SDS–PAGE of RAW264.7 cells after exposure to H 2 O 2 (at the indicated concentration) for 15 min. h COS-7 cell lysates subjected to SDS-PAGE under reducing or non-reducing conditions after transfection of pXJ40-3xFlag vector or pXJ40-3xFlag-FABP5 and exposure to H 2 O 2 (200 μM) for 15 min. i Co-IP for S-glutathionylation of FABP5 in WT BMDMs after treatment with LPS (100 ng/mL) for 15 min (IP, GSH; IB, FABP5). Whole cell lysates confirm the expression of FABP5 and β-actin. j S-glutathionylation of FABP5 in lung tissues from Grx1 fl/fl and Grx1 fl/fl LysM cre mouse 24 h following intratracheal administration of PBS or LPS. Source data are provided as a Source Data file.

Article Snippet: And DNA-bound protein was immunoprecipitated using anti-FABP5 (Proteintech, 12348-1-AP) and anti-IgG (Thermo Fisher Scientific, 26157) antibodies.

Techniques: Western Blot, Co-Immunoprecipitation Assay, Transfection, Plasmid Preparation, Expressing, Negative Control, SDS Page, Concentration Assay

Journal: Nature Communications

Article Title: Oxidative stress-induced FABP5 S-glutathionylation protects against acute lung injury by suppressing inflammation in macrophages

doi: 10.1038/s41467-021-27428-9

Figure Lengend Snippet: a – c Immunoblot analysis of FABP5 binding to ULCFA ( a ), SLCFA ( b ) or ULCFA with Grx1 treatment ( c ). Purified recombinant FABP5 protein was incubated with GSH plus H 2 O 2 or GSSG for 15 min, then mixed with Biotin-ULCFA or Biotin-SLCFA in the absence or presence of BMS309403 or linoleic acid or Grx1. FABP5 interacted with the Biotin-ULCFA or Biotin-SLCFA was captured using streptavidin magnetic beads. d Immunofluorescence staining of BMDMs after exposure to H 2 O 2 (200 μM) for 30 min (green, FABP5; blue, DAPI; scale bars, 10 μm). e Quantitative analysis of the ratio of nuclear/cytoplasmic fluorescence intensity in BMDMs as in ( d ), n = 10 in each group, P (ctr vs. ctr H2O2) = 0.0172, P (ctr H2O2 vs. Grx1 KO H2O2) <0.0001. f Immunoblot analysis of cytoplasmic and nuclear FABP5 in COS-7 cells. COS-7 cells were transfected with pXJ40-3xFlag vector or pXJ40-3xFlag-FABP5 followed by exposure to H 2 O 2 (200 μM) for 1 h. Lamin B1 (nuclear fraction) and GAPDH (cytoplasmic fraction) are loading controls. g Immunoblots with antibodies against the indicated proteins in nuclear and cytoplasmic fractions from COS-7 cells co-transfected with pXJ40-3xFlag-FABP5 (or vector) and pcDNA3.1-3xFlag-Grx1 (or vector) for 24 h, then exposed to H 2 O 2 (200 μM) for 1 h. h Immunoblots against the indicated proteins of cytoplasmic and nuclear extracts from BMDMs exposed to H 2 O 2 (200 μM) for 1 h. i mRNA expression of Il1b , Il6 , and Tnfα in BMDMs from mice stimulated with LPS (100 ng/mL) for 4 h, as evaluated by qPCR, n = 3 in each group, P ( Il1b , Ctr vs. Ctr+LPS) = 0.0013, P ( Il1b , Ctr+LPS vs. Grx1 KO+LPS) = 0.0091, *** P < 0.0001. j mRNA levels of Il1b , Il6 , and Tnfα in BMDMs pre-treated with an FABP5 inhibitor (SBFI26, 100 μM) for 2 h and stimulated with LPS (100 ng/mL) for 4 h, as determined by qPCR, n = 3 in each group, *** P < 0.0001. k mRNA levels of Il1b , Il6 , and Tnfα in BMDMs after treated with SBFI26 (100 μM) for 2 h and stimulation with LPS (100 ng/mL) for 4 h, as evaluated by qPCR, n = 3 in each group, P ( Il1b , Ctr+LPS vs. Ctr+LPS+inhibitor) < 0.0001, P ( Il16 , Ctr+LPS vs. Ctr+LPS+inhibitor) < 0.0001, P ( Tnfα , Ctr+LPS vs. Ctr+LPS+inhibitor) = 0.0046. All samples were biologically independent and three or more independent experiments were performed. All quantitative data are shown as mean ± SEM and analyzed with a 95% confidence interval. One-way ANOVA followed by Tukey’s post-hoc test for (e, i-k). * P < 0.05, ** P < 0.01, *** P < 0.001, ns = no significance. Source data are provided as a Source Data file.

Article Snippet: And DNA-bound protein was immunoprecipitated using anti-FABP5 (Proteintech, 12348-1-AP) and anti-IgG (Thermo Fisher Scientific, 26157) antibodies.

Techniques: Western Blot, Binding Assay, Purification, Recombinant, Incubation, Magnetic Beads, Immunofluorescence, Staining, Fluorescence, Transfection, Plasmid Preparation, Expressing

Journal: Nature Communications

Article Title: Oxidative stress-induced FABP5 S-glutathionylation protects against acute lung injury by suppressing inflammation in macrophages

doi: 10.1038/s41467-021-27428-9

Figure Lengend Snippet: a Model structure of FABP5 and its 6 cysteines, generated by Pymol and based on the crystal structure of Homo sapiens FABP5 (PDB ID,4LKT). b Mass spectra of a peptide from FABP5 including glutathionylated cysteine 120 and cysteine127. c Immunofluorescence staining (with FABP5) and confocal microscopy imaging of COS-7 cells with overexpression of FABP5 WT, C67S, C87S, C120S, and C127S after exposure to H 2 O 2 (200 μM) for 1 h (green, FABP5; blue, DAPI; scale bars, 10 μm). d Immunoblot analysis of cytoplasmic and nuclear FABP5 in COS-7 cells transfected with FABP5 WT or C127S after exposure to H 2 O 2 (200 μM) for 1 h. Lamin B1 (nuclear fraction) and GAPDH (cytoplasmic fraction) are loading controls. e Model structure of FABP5 interaction with an FA (Fatty Acid, Linoleic Acid), generated by Pymol and based on the crystal structure of FABP5 (PDB ID, 4LKT). f Docking structure of FABP5 in complex with S-glutathionylated Cys127 interacting with an FA (Fatty Acid, Linoleic Acid). g Docking structure of dimer FABP5 (PDB ID, 4AZM, Homo sapiens ) in complex with reduced glutathione (GSH) on Cys127. h Co-IP for S-glutathionylation of FABP5 in COS-7 cells overexpressing pXJ40-3xFlag-FABP5 WT or C127S and exposed to H 2 O 2 (200 μM) for 15 min (IP, GSH; IB, FABP5). Whole-cell lysates confirm the expression of FABP5 and β-actin (DTT, negative control). i Immunoblot analysis of FABP5 fatty acid binding. Purified recombinant FABP5 WT or C127S protein (1 μg) was incubated with GSSG (1 mM) for 15 min, then mixed with Biotin-linoleic acid in the absence or presence of BMS309403 (5 mM) for 30 min. FABP5 that associated with the Biotin-linoleic acid was captured using streptavidin magnetic beads. Source data are provided as a Source Data file.

Article Snippet: And DNA-bound protein was immunoprecipitated using anti-FABP5 (Proteintech, 12348-1-AP) and anti-IgG (Thermo Fisher Scientific, 26157) antibodies.

Techniques: Generated, Immunofluorescence, Staining, Confocal Microscopy, Imaging, Over Expression, Western Blot, Transfection, Co-Immunoprecipitation Assay, Expressing, Negative Control, Binding Assay, Purification, Recombinant, Incubation, Magnetic Beads

Journal: Nature Communications

Article Title: Oxidative stress-induced FABP5 S-glutathionylation protects against acute lung injury by suppressing inflammation in macrophages

doi: 10.1038/s41467-021-27428-9

Figure Lengend Snippet: a Volcano plot of RNA-seq transcriptome data displaying the pattern of gene expression values for FABP5 KO BMDMs with overexpression of FABP5 C127S relative to FABP5 WT after exposure to LPS (500 ng/mL) for 24 h [red and blue dots, significantly up-and down-regulated genes ( P < 0.01, |log 2 FC|>1); black dashed lines, boundary for identification of up- or down-regulated genes]. b Heatmap of regulation of the inflammatory response and cytokine transcript production in LPS-stimulated (24 h) BMDMs nucleofected with either pXJ40-3xFlag-FABP5 WT or C127S. Data shown are relative to the calculated Z scores across the samples. Red, relatively high levels of expression; blue, relatively low levels of expression. Each column represents one individual (for a total of 3 per group) and each row represents the expression of a single gene. c Diagram of GO analysis classifying DEGs into biological process groups using Panther ( http://www.pantherdb.org ). Some GO categories with FDR <0.05 are included. GO, gene ontology; DEGs, differentially-expressed genes. d mRNA expression of Il1b , Il6 , and Tnfα in FABP5 KO BMDMs after nucleofection with pXJ40-3xFlag-FABP5 WT or C127S and stimulation with LPS (1 μg/mL) for 24 h, as identified by qPCR, n = 3 in each group, P ( Il6 , FABP5 WT+LPS vs. FABP5 C127S+LPS) = 0.0045, *** P < 0.0001. e mRNA expression of Il1b , Il6 , and Tnfα in Grx1 KO BMDMs after nucleofection of pXJ40-3xFlag-FABP5 WT or C127S and treatment with LPS (500 ng/mL) for 24 h, as determined by qPCR, n = 3 in each group, P ( Il6 , Ctr+FABP5 C127S vs. Grx1 KO+FABP5 C127S) = 0.0004, P ( TNFα , Ctr+FABP5 WT vs. Grx1 KO+FABP5 WT) = 0.0123, *** P < 0.0001(except group Il6 , Ctr+FABP5 C127S vs. Grx1 KO+FABP5 C127S). f H&E staining showing lung histopathological changes in FABP5 fl/fl and FABP5 fl/fl LysM cre mice 24 h after intratracheal administration of PBS or LPS (5 mg/kg). Scale bars, 50 μm. g Total numbers of leukocytes in BALF from FABP5 fl/fl and FABP5 fl/fl LysM cre mice 24 h after intratracheal administration of PBS or LPS (5 mg/kg), n = 4, 4, 9, 8, respectively, *** P < 0.0001. All samples were biologically independent and three or more independent experiments were performed. All quantitative data are shown as mean ± SEM and analyzed with a 95% confidence interval. One-way ANOVA followed by Tukey’s post-hoc test for ( d – e , g ). * P < 0.05, ** P < 0.01, *** P < 0.001. Source data are provided as a Source Data file.

Article Snippet: And DNA-bound protein was immunoprecipitated using anti-FABP5 (Proteintech, 12348-1-AP) and anti-IgG (Thermo Fisher Scientific, 26157) antibodies.

Techniques: RNA Sequencing Assay, Expressing, Over Expression, Staining

Journal: Nature Communications

Article Title: Oxidative stress-induced FABP5 S-glutathionylation protects against acute lung injury by suppressing inflammation in macrophages

doi: 10.1038/s41467-021-27428-9

Figure Lengend Snippet: a In vitro pull-down assays. The mouse recombinant FABP5 (WT or C127S) protein with 3xFlag tag purified from E. coli treated with GSSG (1 mM) for 15 min was incubated with COS-7 lysates and precipitated with Flag-magnetic beads, respectively. Precipitates were subjected to SDS­PAGE and examined by immunoblot with anti­ PPARβ/δ antibodies. b Transactivation assays in Raw264.7 cells transfected with a PPRE-driven luciferase reporter. After nucleofection with PPRE X3-TK-luc and pRL-TK, Raw264.7 cells were serum starved for 8 h, then exposed to H 2 O 2 (200 μM), GW0742 (1 μM), or both for 24 h, and luciferase activity was determined, n = 3 in each group, P (ctr vs. H2O2) = 0.0003, P (ctr vs.GW0742) < 0.0001, P (GW0742 vs.H2O2+GW0742) = 0.0047. c Transactivation assays in COS-7 cells transfected with a PPRE-driven luciferase reporter. After transfection with PPRE X3-TK-luc, FABP5, and pRL-TK, cells were serum starved for 8 h, then treated with H 2 O 2 (200 μM), GW0742 (1 μM), or both for 24 h, and luciferase activity was measured, n = 3 in each group, *** P < 0.0001. d ChIP analysis of FABP5 recruitment to the PPREs of PPARβ/δ target gene promoters in Raw 264.7 cells after exposure to H 2 O 2 (200 μM) for 1 h. ChIP signals were quantified by qPCR, n = 3 in each group, P (ADRP, ctr vs. H2O2) = 0.001, P (FIAF, ctr vs. H2O2) < 0.0001. e mRNA levels of Adrp , Fiaf , and Cpt1a in RAW264.7 cells after treatment with BMS309403 (50 μM) for 2 h, stimulation with GW0742 (1 μM) for 30 min, and exposure to H 2 O 2 (200 μM) for 4 h, as determined by qPCR, n = 3 in each group, P ( Adrp , ctr vs. GW0742) < 0.0001, P ( Adrp , GW0742 vs. GW0742+BMS309403) = 0.0009, P ( Fiaf , ctr vs. GW0742) = 0.001, P ( Fiaf , GW0742 vs. GW0742+BMS309403) = 0.001, P ( Cpt1α , ctr vs. GW0742) < 0.0001, P ( Cpt1α , GW0742 vs. GW0742+BMS309403) < 0.0001. f Co-IP for S-glutathionylation of FABP5 in COS-7 cells overexpressing pXJ40-3xFlag-FABP5 WT or G114R and exposed to H 2 O 2 (200 μM) for 15 min (IP, GSH; IB, FABP5). Whole-cell lysates confirm the expression of FABP5 and β-actin (DTT, negative control). All samples were biologically independent and three or more independent experiments were performed. All quantitative data are shown as mean ± SEM and analyzed with a 95% confidence interval. One-way ANOVA followed by Tukey’s post-hoc test for ( b – e ). * P < 0.05, ** P < 0.01, *** P < 0.001. Source data are provided as a Source Data file.

Article Snippet: And DNA-bound protein was immunoprecipitated using anti-FABP5 (Proteintech, 12348-1-AP) and anti-IgG (Thermo Fisher Scientific, 26157) antibodies.

Techniques: In Vitro, Recombinant, Purification, Incubation, Magnetic Beads, Western Blot, Transfection, Luciferase, Activity Assay, Co-Immunoprecipitation Assay, Expressing, Negative Control