Journal: PLOS One

Article Title: Human resistin is critical to activation of the NLRP3 inflammasome in macrophages

doi: 10.1371/journal.pone.0337682

Figure Lengend Snippet: ( A ) Schematic presentation of proposed pathway. ( B ) Human macrophages were cultured in 6-well plates under the conditions shown for 15 minutes before being washed and lysed for western blot analysis with antibodies to phospho-tyrosine (p-Y) and NLRP3 (image is representative of 6 repeats). ( C ) Human macrophages were cultured under the conditions shown for 12 hours. PBS was used as a control. Cells were washed, lysed, and assayed for their ability to cleave a fluorescent caspase-1 substrate, YVAD-AFC. Values were normalized to PBS controls. All conditions were run in duplicate wells, and two independent experiments were performed. Error bars represent the mean ± SD (n = 4). **** p < 0.001 versus PBS. ( D ) Hypoxia upregulates BTK and NLRP3 colocalization in C57BL/6 WT mice but not in RELMα KO mice. Immunofluorescence images of NLRP3 and BTK in lung tissues of mice kept under normoxic or hypoxic conditions for 4 days. Lung sections were stained with anti-NLRP3 (red) and BTK (green). The arrowheads point to cells positively stained for BTK and NLRP3 (yellow). The upper images are shown at higher magnification (400×); the lower panels display separate channels. Representative photograph of n = 6 mice per group. ( E ) Human resistin colocalized with BTK and NLRP3 in patients with PH. Immunofluorescence images of lung tissue slices from PH patients. Sections were stained with anti-hResistin (red) and co-stained with anti-BTK (green) and anti-NLRP3 (cyan) antibodies. The arrowheads point to cells positively stained for hResistin, BTK, and NLRP3. Separate channels are displayed in the lower panels. Original magnification: 100 × , 200 × , and 400 × . 4DHx, 4-day hypoxic; ab, antibody; BTK, Bruton’s tyrosine kinase; Con, control; Hres, human resistin; Ib, ibrutinib; KO, knockout; NLRP3, nucleotide-binding domain–like receptor protein 3; Nx, normoxic; PBS, phosphate-buffered saline; PH, pulmonary hypertension; RELMα, resistin-like molecule alpha; WT, wild-type. Created in BioRender. Lam, W. (2026) https://BioRender.com/1zhhut0 .

Article Snippet: After deparaffinization of tissue, rehydration, and antigen retrieval, sections were treated with anti-NLRP3 (Abcam, ab214185) and anti-Mac2 (Cedarlane, CL8942LE), anti-MPO (R&D Systems, AF3667), anti-CD79b (Abcam, ab134147), anti-BTK (Sigma-Aldrich, SAB4502936), anti-hResistin (R&D Systems, AF1359), or anti-RELMα (R&D Systems, MAB1523) antibodies overnight at 4°C and then with Alexa Fluor 488-donkey anti-mouse IgG (Jackson ImmunoResearch, 715-545-150) and Cy3-donkey anti-rabbit IgG (Jackson ImmunoResearch, 711-166-152) for double fluorescence staining.

Techniques: Cell Culture, Western Blot, Control, Immunofluorescence, Staining, Knock-Out, Binding Assay, Saline

Journal: Nature Communications

Article Title: The REDD1–NF-κB–miRNAs–eNOS/SIRT1 axis mediates obesity-induced endothelial cell senescence and hypertension

doi: 10.1038/s41467-026-70601-1

Figure Lengend Snippet: a – c Aortic vessels were isolated from male mice fed either a chow diet (lean) or an HFD (DIO) for 16 weeks. Vessels were either intact (E+) or de-endothelialized (E−). REDD1 expression was assessed by Western blotting ( a , b ) and confocal microscopy ( c ). Representative images from four mice per group with similar results. Scale bar = 50 µm. d , e REDD1 mRNA ( d , n = 4 independent experiments) and REDD1 protein levels ( e , n = 3 independent experiments) were measured after treatment with BSA-conjugated palmitic acid (PA, 300 µM), cholesterol (Chol, 100 µM, dissolved in DMSO), leptin (10 µg/ml), resistin (Retn, 100 ng/ml), oxLDL (50 µg/ml), or high glucose (HG, 25 mM) using qRT-PCR and Western blotting. f – h HAECs were transfected with 80 nM control siRNA (siC) or REDD1 siRNA (siREDD1) and then treated with PA ( f ), oxLDL ( g ), or high glucose (HG, h ) for 48 h. SA-β-gal + cells were detected using a SA-β-gal staining kit. Scale bar = 100 µm. The percentage of SA-β-gal + cells was calculated as the ratio of blue-stained to total cells ( n = 4 independent experiments). Western blotting for REDD1, p53, and p21 was performed in three independent experiments with similar results. Data are presented as mean ± s.e.m. Statistical significance was determined using one-way ANOVA with Holm–Sidak’s multiple comparisons test ( d , f – h ).

Article Snippet: Cells were then treated with or without oxLDL (50 μg/ml), prepared by incubating native LDL (1.019 mg protein) with 10 μM CuSO4 for 4 h , cholesterol (100 μM, #C8667; Sigma-Aldrich, St. Louis, MO, USA), glucose (25 mM, 25 mM L -glucose used for osmotic control), recombinant human resistin (100 ng/ml, #1359-RN; R&D systems, Minneapolis, MN, USA), recombinant human leptin (10 μg/ml, #398-LP; R&D Systems), recombinant human TNF-α (10 ng/ml, #10291-TA; R&D Systems), recombinant human IL-6 (40 ng/ml, #206-IL; R&D Systems), palmitate-BSA (300 μM), or H 2 O 2 (50–150 μM) for 48 h. Palmitic acid-BSA solution (5 mM) was prepared by mixing 10 μL of palmitate solution (500 mM in ethanol, #P9767; Sigma-Aldrich) with 1 mL of BSA solution (10%, w/v in serum-free M199, #A8806; Sigma-Aldrich).

Techniques: Isolation, Expressing, Western Blot, Confocal Microscopy, Quantitative RT-PCR, Transfection, Control, Staining

Journal: Nature Communications

Article Title: The REDD1–NF-κB–miRNAs–eNOS/SIRT1 axis mediates obesity-induced endothelial cell senescence and hypertension

doi: 10.1038/s41467-026-70601-1

Figure Lengend Snippet: a – c Representative H&E-stained images of renal tissues from lean and DIO mice: WT and Redd1 −/− ( a , n = 8 mice/group), Redd1 fl/fl and Redd1 ΔEC ( b , n = 7 mice/group), or WT and miR-214-3p −/− ( c , n = 6 mice/group). Glomerular size was quantified in four randomly selected fields per section using ImageJ. Scale bar = 50 µm. d , e Fibrotic area in renal tissues (Masson′s trichrome staining; ( d )) and serum creatinine levels ( e ) were quantified using ImageJ and ELISA, respectively ( n = 8, 7, and 6 mice for Redd1 −/− , Redd1 ΔEC , miR-214-3p −/− groups, respectively; same mice as in ( a – c )). f Representative Western blots using target-specific antibodies from four mice per group with similar results. Target proteins were analyzed independently three times, with tubulin probed on the same membrane as a loading control. g Graphical representation illustrating the role of REDD1 in obesity-induced vascular senescence and hypertension based on our current and previous findings . FFA free fatty acid, IR insulin resistance, TG triglyceride, VLDL very-low-density lipoprotein, LPL lipoprotein lipase, CE cholesteryl ester, ROS reactive oxygen species, Glc glucose, Retn resistin, GLG glycogen. Data are presented as mean ± s.e.m. Statistical significance was determined using two-way ANOVA with Holm–Sidak’s multiple comparisons test ( a – e ).

Article Snippet: Cells were then treated with or without oxLDL (50 μg/ml), prepared by incubating native LDL (1.019 mg protein) with 10 μM CuSO4 for 4 h , cholesterol (100 μM, #C8667; Sigma-Aldrich, St. Louis, MO, USA), glucose (25 mM, 25 mM L -glucose used for osmotic control), recombinant human resistin (100 ng/ml, #1359-RN; R&D systems, Minneapolis, MN, USA), recombinant human leptin (10 μg/ml, #398-LP; R&D Systems), recombinant human TNF-α (10 ng/ml, #10291-TA; R&D Systems), recombinant human IL-6 (40 ng/ml, #206-IL; R&D Systems), palmitate-BSA (300 μM), or H 2 O 2 (50–150 μM) for 48 h. Palmitic acid-BSA solution (5 mM) was prepared by mixing 10 μL of palmitate solution (500 mM in ethanol, #P9767; Sigma-Aldrich) with 1 mL of BSA solution (10%, w/v in serum-free M199, #A8806; Sigma-Aldrich).

Techniques: Staining, Enzyme-linked Immunosorbent Assay, Western Blot, Membrane, Control