Lamin Type B

Samples were fixed, paraffin embedded, sectioned, and stained with hematoxylin/eosin for histological evaluation as described [57 (link)]. Tissue sections were subject to immunological staining with avidin:biotinylated enzyme complex as described [18 (link),58 (link)]. Proteins were extracted from TS cells using M-PER reagent (PIERCE) with the addition of protease inhibitor cocktail (Sigma-Aldrich), 1 mM sodium molybdate, 1 mM sodium vanadate, and 10 mM N-ethylmaleimide, or SDS lysis buffer (2% SDS, 10% glycerol, and 50 mM Tris, pH 6.8). Protein extracts were subject to immunoblotting as described [54 (link)]. Bound primary antibodies were detected with horseradish peroxidase-conjugated secondary antibodies (Vector Lab), followed by ECL-mediated visualization (GE HealthCare) and autoradiography. Mouse monoclonal antibodies anti-actin (Thermo Fisher; 1:1,000), anti-BrdU (Thermo Fisher; 1:300), anti-Cdx2 (BioGenex; 1:1), anti-MDM2 (Santa Cruz; 1:100), and anti-SUMO-1 (Zymed; 1:2,000); rabbit polyclonal antibodies anti-calnexin (Stressgene; 1:2,000), anti-cyclin D1 (Neomarker; 1:100), anti-Ki67 (Neomarker; 1:400), anti-laminin (Sigma-Aldrich; 1:25), anti-Myc tag (CalBioChem; 1:400), anti-Oct4 (Santa Cruz; 1:200), anti-p53 (Santa Cruz; 1:50), and anti-p450scc (Chemicon; 1:200); and goat polyclonal antibody anti-lamin B (Santa Cruz; 1:100) were used as primary antibodies. BrdU incorporation analysis was performed by intraperitoneal injection of BrdU (250 μg/g of body weight) into pregnant females for 1 h. Placentas were recovered, fixed, embedded, sectioned, and subject to immunostaining as described [18 (link),57 (link)].

Cytosolic and nuclear extracts were prepared as previously described on colon tissues [38 (link)]. The following primary antibodies were used: anti-IκB-α (SCB, 1:500 #sc1643, D.B.A, Milan, Italy), anti-NF-κB p65 (SCB; 1:500 #sc8008), anti-iNOS (BD Transduction Laboratories, 1:500), anti-MnSOD (Millipore, 1:500, Cat 06-984, D.B.A, Milan, Italy) in 1× phosphate-buffer saline (Biogenerica srl, Catania, Italy), 5% w/v non-fat dried milk, 0.1% Tween-20 at 4 °C overnight. Membranes were incubated with peroxidase-conjugated bovine anti-mouse IgG secondary antibody or peroxidase-conjugated goat anti-rabbit IgG (Jackson ImmunoResearch, West Grove, PA, USA; 1:2,000) for 1 h at room temperature. Anti-β-actin or anti-lamin A/C (D.B.A, Milan, Italy) antibodies were used as controls. Protein expression was analyzed as previously reported [38 (link)].

The reagents used in this study are as follows: Drugs Honokiol (MCE,CAT#HY-N0003, purity is 99.90%), ATP (MCE,CAT#HY-B0345A/CS-2387), ASC oligomerization inhibitor MCC950 (MCE,CAT#HY-12815A), Caspase-1 inhibitor VX-765 (MCE,CAT#HY-13205), LPS (Sigma,CAT#L2880), collagenase IV (BioFroxx,CAT#2091MG100), DNase I (SAITONG,CAT#PS0825), single-cell suspensions kit (Solarbio,CAT#P5380), RPMI-1640 media (Gibco, REF:C11875500BT), FBS (Multicell, CAT NO:086-150), p3*flag-NLRP3 (PPL, CAT#PPL00151-2b), P3 Primary Cell 4D-Nucleofector X Kit L (Lonza,CAT#V4XP-3024), Bradford Assay kit(Beyotime,CAT#P0006), Blood Urea Nitrogen (BUN) Detection Kit (Nanjing Jiancheng Bioengineering Institute,CAT#C013-2-1), Creatinine Assay Kit (Nanjing Jiancheng Bioengineering Institute,CAT#C011-2-1), IL-33 (Affinity,CAT#DF8319),NLRP3 (Affinity, CAT#DF74), PrimeScript RT Reagent Kit (Takara, CAT#RR037A), HieffTM qPCR SYBR Green Master Mix (YEASEN, CAT#11201ES08), RIPA lysis buffer (Beyotime,CAT#P0013B), Nuclear protein Extraction Kit (Solarbio,CAT#R0050), BCA Protein Assay kit (Beyotime, CAT#P0012), PVDF membranes (Millipore,CAT#ISEQ00010), NLRP3 (CST,CAT#20836T), Cleaved caspase-1 (Affinity,CAT#AF4005), Cleaced IL-1β(CST,CAT#20839T), Caspase-1 (CST,CAT#20842T), ASC (CST,CAT#20838T), IL-33 (Affinity,CAT#DF8319), ST2 (proteintech,CAT#60112-1-Ig), NF-κB (Affinity,CAT#BF8005), Tublinβ (Affinity,CAT#AF7011), Lamin B (Affinity,CAT#BF8009), GAPDH (Abbkine,CAT#KTD101-CN), secondary rabbit-antibody (Immunoway, CAT#RS23920), secondary mouse-antibody (Abbkine,CAT#A23710), Mouse IL-33 ELISA Kit (MultiSciences,CAT#70-EK233/2-48), Mouse IL-1β ELISA Kit (FANKEWEI,CAT#F2923-B), Rat IL-1β ELISA Kit (FANKEWEI,CAT#F2923-B), Rat IL-18 ELISA Kit (FANKEWEI,CAT#F3070-B). Recombinanl NLR Family, Pyrin Domain Containing Protein 3(NLRP3) (Cloud-Clone, CAT#RPK115Hu01).

Kidney and cell samples were lysed using the RIPA lysis buffer, and the nuclear proteins were extracted using a nuclear protein extraction kit. The protein concentrations of the lysates were determined using a BCA Protein Assay Kit. Next, 20 µg total protein per sample was resolved using sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred onto 0.2 µm PVDF membranes by using a semidry Trans-Blot apparatus (BIO-RAD, Hercules, CA, USA). Subsequently, the membranes were blocked at room temperature (20–30 °C) for 1 h with 5% skim milk in Tris-buffered saline containing 0.1% Tween-20 (TBST). Afterward, the membranes were incubated overnight at 4 °C with the following primary antibodies: antibodies against NLRP3, cleaved caspase-1, cleaved IL-1, caspase-1, ASC, IL-33, ST2, NF-κB, tubulin β, lamin B, and GAPDH. Next, the membranes were washed three times with TBST and then incubated at room temperature for 2 h with a secondary anti-rabbit or anti-mouse antibody, as required. The blots were analyzed using an Odyssey fluorescence scanner (LI-COR, Biosciences, Lincoln, NE). The signals were captured by using the supplied Odyssey software v3.0, and the results were expressed as fold changes normalized to the expression levels of tubulin, GAPDH, or lamin B.

Protein was extracted from frozen liver and epididymal white adipose tissues (eWAT). Nuclear protein was prepared from mouse livers as described previously (Alam et al., 2017 (link)). Protein concentration was determined using a Pierce BCA Protein Assay kit (Thermo Fisher Scientific, MA). Western blotting was performed as described previously (Liu et al., 2020 (link)). Primary antibodies (1:500–1:1,000) against Phospho-HSL, ATGL, PPAR-γ, CPT-1a, Nrf2, BAX, BCL-2, Adiponectin, Cleaved-Caspase3, pro-Caspase3 and CYP7A1 were from Cell Signaling Technology (MA, United States). β-Actin and Lamin B antibody were the reference of total protein and nuclear protein, respectively, and purchased from Abcam (Cambridge, MA, United States). Densitometric analysis was performed using UN-SAN-IT Gel (Silk Scientific, Orem, UT) software.