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: iScience

Article Title: The role of epithelial progesterone receptor isoforms in embryo implantation

doi: 10.1016/j.isci.2021.103487

Figure Lengend Snippet: Suppressed LIF transcription by blocking ESR1 binding at Lif promoter (A–E) The 3D representative images of uterine glands in the adult Wnt7acre, ePRAoe, and ePRBoe mice at diestrus stage. Blue is non-cystic gland, and red is cystic gland (A). Real-time PCR showed mRNA levels of Lif and its receptors Lifr and IL6st in the whole uterus of the Wnt7acre, ePRAoe, and ePRBoe mice at D3.5 (B). Immunohistochemistry pictures of STAT3, pSTAT3 (Tyr705), ESR1, and PGR in Wnt7acre, ePRAoe, and ePRBoe mouse uterus at D3.5 (C). UCSC Genome Browser display of signals of ESR1 ChIP-seq of wild-type adult mouse uterus after 1-h E2 or vehicle treatment; PGR ChIP-seq of Pgrcre/+, Pgrcre/+PgrALsL/+, and Pgrcre/+PgrBLsL/+ uterus after 1-h P4 treatment; and H3K27AC ChIP-seq of D3.5 wild-type mouse uterus at Lif promoter (D). ChIP-qPCR indicates enrichment fold of ESR1 binding at the LIF promoter in the ePRAoe and ePRBoe mice at D3.5 (E). Scale bars: 100 μm in (A) and 50 μm in (C). Each dot indicates one mouse; the red line is median level. Student's t test. ∗p < 0.05. N = 6 mice for real-time PCR. N = 3 mice for ChIP-qPCR. See also Figure S6.

Article Snippet: Rabbit monoclonal pSTAT3 (Tyr705, IHC 1:400) , Cell Signaling , Cat# 9145; RRID: AB_2491009.

Techniques: Blocking Assay, Binding Assay, Real-time Polymerase Chain Reaction, Immunohistochemistry, ChIP-sequencing, ChIP-qPCR

Journal: iScience

Article Title: The role of epithelial progesterone receptor isoforms in embryo implantation

doi: 10.1016/j.isci.2021.103487

Figure Lengend Snippet: Key resources table

Article Snippet: Rabbit monoclonal pSTAT3 (Tyr705, IHC 1:400) , Cell Signaling , Cat# 9145; RRID: AB_2491009.

Techniques: Immunohistochemistry-IF, Western Blot, RNAscope, Plasmid Preparation, Recombinant, Modification, Electrophoresis, Multiplex Assay, Bicinchoninic Acid Protein Assay, Staining, Reverse Transcription, SYBR Green Assay, RNA Amplification, Purification, Generated, Software, Magnetic Cell Separation