Vcam 1

Lung tissue was excised 24 h after LPS-induced lung injury, fixed in 4% paraformaldehyde for 10 min, embedded in paraffin, and cut into 4-µm-thick sections. Staining for Ly6G (LS-C36561, 1:100; LifeSpan Biosciences, Seattle, WA), MPO (SC-52707, 1:100, Santa Cruz Biotechnology, Dallas, TX), VLA-4 (#8440S, 1:1000; Cell Signaling, Danvers, MA), VCAM-1 (#14694, 1:1000; Cell Signaling), and TGF-β (ab66043, 1:100, Abcam, Cambridge, UK) was performed using Envision® + Dual Link System-HRP (DAB+) kits (K4065; DAKO, Carpinteria, CA). The sections were deparaffinized in xylene, dehydrated in ethanol, and then heated in 0.01 M citrate buffer (pH 6.0). Endogenous peroxidase activity was inactivated in 3% H₂O₂ for 10 min at room temperature (RT), and the sections were blocked with blocking buffer. Secondary anti-rabbit antibody-coated polymer peroxidase complexes were applied for 30 min at RT, followed by substrate/chromogen incubation for 5–15 s at RT. The sections were counterstained with hematoxylin (109249; Merck) for 10 s and then washed in running water for 10 min. They were observed and photographed with an Olympus AX80 fluorescence microscope (Olympus America, Melville, NY). The percentage of IHC signal per photographed field was determined with Image-Pro Plus software (Media Cybernetics, Inc., Silver Spring, MD).

TDD was synthesized in our laboratory as previously reported (Supplementary Material online, Figure S1, 99% purity).14 LPS was purchased from Sigma‐Aldrich (St. Louis, MO, USA). Antibodies for phosphor (p)‐ERK1/2, ERK1/2, p‐p38, p38, p‐JNK, JNK, ICAM‐1, VCAM‐1, E‐selectin and NF‐κB/p65 were purchased from Cell Signaling Technology (Beverly, MA, USA). TRIzol reagent, a PrimeScript™ RT reagent kit and an SYBR Premix Ex Taq™ II kit were obtained from TaKaRa Bio, Inc. (Shiga, Japan). Lamin B Rabbit Monoclonal Antibody, a nuclear protein extraction kit, and an NF‐κB Activation‐Nuclear Translocation Assay Kit were obtained from Biotechnology Co. (Shanghai, China). The antibody for HMBOX1 was obtained from Proteintech Group Inc. (Wuhan, China). ELISA kits for IL‐6 and TNF‐α, β‐actin antibody, horseradish peroxidase‐conjugated secondary antibodies and enhanced chemiluminescence (ECL) detection kit were obtained from Boster Biotech Co., Ltd. (Wuhan, China).

Formalin-fixed tissues were embedded in paraffin and cut into 5 μm sections. Sections were evaluated by H&E and immunohistochemical analysis following our previously reported protocol (48 (link)) using antibodies specific for CD31 (Cell Signaling Technology, 77699), NG2 (MilliporeSigma, ab5320), VCAM-1 (Cell Signaling Technology, 32653S), ICAM-1 (Abcam, ab179707), FasL (Santa Cruz Biotechnology, NOK-1), CC3 (Cell Signaling Technology, 9664), Ki67 (Abcam, ab15580), CD3 (Thermo Fisher Scientific, PA1-29547), CD8 (Cell Signaling Technology, 98941S), FoxP3 (R&D Systems, MAB8214), F4/80 (Cell Signaling Technology, 70076S), iNOS (Thermo Fisher Scientific, PA1-21054), Arg-1 (Cell Signaling Technology, 93668S), human CD3 (Thermo Fisher Scientific, PA1-29547), human CD8 (Abcam, ab93278), human CD206 (Cell Signaling Technology, 91992S), and human PD-L1 (Cell Signaling Technology, 13684S). Negative controls included omission of primary antibody. Color images were obtained with Hamamatsu Nanozoomer 2.0HT using NDPview2 software. Pictures were analyzed using NIS Elements (Nikon) and Fiji software. Quantification is shown as percentage of area fraction.

Immunoblotting was generally carried out as described in the previous studies.^[62 (link)
^] The primary antibody of TXNIP (1:2000) was purchased from Abcam, the other primary antibodies were obtained from Cell Signaling Technology and were listed below: ICAM‐1 (1:1000), YAP1 (1:1000), P‐YAP1 (1:1000), Cyr61 (1:1000), and VCAM‐1 (1:1000). Secondary HRP‐conjugated antibodies (1:1000 – 1:3000) used for IB analysis were purchased from Cell Signaling Technology (CST, Beverly, MA, USA).

HRMECs were harvested and lysed for 10 minutes in cold lysis buffer containing protease inhibitors (Cat, P8340, Sigma, St. Louis, MO). 50 μg total proteins were immunoblotted with primary antibodies against Serine⁴⁷³ phosphorylated Akt (RRID, AB_329825), total Akt (RRID, AB_915783), Threonine¹⁷² phosphorylated AMPK (RRID, AB_330330), total AMPK (RRID, AB_330331), HRP conjuncted anti-rabbit IgG (RRID, AB_2099233), HRP conjuncted anti-mouse IgG (RRID, AB_330924) and VCAM-1 (Cat, 13662) (all from Cell Signaling, Danvers, MA). The membrane was exposed with Super-Signal Reagent (Cat, 34080, Pierce, Illinois, USA), and imaged by a Bio-Rad ChemiDoc Touch station.