Ab96723

EVs were lysed in an RIPA buffer (Thermo Scientific, Milan, Italy Pierce RIPA buffer) containing protease inhibitor (Roche, Milan, Italy Protease inhibitor cocktail tablets) and phosphatases inhibitor (Sigma Aldrich Milan, Italy, Phosphatase inhibitor cocktail 3) for 2 h in ice and the protein concentration was determined using the Bradford assay (Bio-Rad, Milan, Italy Protein Assay Dye Reagent Concentrate). Then, 16 μg of protein was separated using 10% SDS-PAGE and blotted onto a nitrocellulose membrane (GE Healthcare, Milan Italy Amersham Protran 0.45 µm NC ). The membranes were stained with a Ponceau S solution (Sigma, P7170-1L) and then washed with Tris-buffered saline containing 0.1% Tween-20 (T-TBS). After blocking with 5% no-fat dry milk in T-TBS, the membranes were incubated at 4 °C overnight with the following primary antibodies: anti-COL1A2 (Abcam, Cambridge, UK ab96723 1:1000), anti-Cytokeratin 2e (KRT2 Abcam, ab170106 1:800), anti-Alix (Santacruz Biotechnology, Texas, USA, 1A12 1:250), anti-Calnexin (Abcam ab10286 1:1000), and anti-TSG101 (Abcam ab83 1:500). Detection was performed using peroxidase-conjugated secondary antibodies using the ultra-enhanced chemiluminescence system (Thermo Scientific, Super Signal West Femto Maximum Sensitivity Substrate #34095).

For immunostaining, cells on glass-bottom dishes were fixed in 4% paraformaldehyde and permeabilized with 0.2% Triton X-100. The samples were blocked in 5% skim milk, followed by incubation with primary antibodies overnight at 4 °C. The corresponding secondary antibodies were labeled with AlexaFluor 546 (Thermo Fisher Scientific). Nuclear staining was performed using 4,6-diamidino-2-phenylindole (DAPI). Images were collected using a confocal microscope (FLUOVIEW FV10i; Olympus, Tokyo, Japan). Antibodies and reagents employed in western blotting and immunostaining included: anti-α SMA (A2547, Sigma-Aldrich; Merck KGaA, Darmstadt, Germany), anti-collagen I (ab34710, Abcam, MA, USA), anti-COL1A2 (ab96723, Abcam), anti-COL1A1 (E8F4L) (Cell signaling, Danvers, MA, USA), anti-phospho-smad2 (Ser465/467)/smad3 (Ser423/425) (D27F4, Cell Signaling), anti-smad2/3 (D7G7) (Cell Signaling), anti-TGF-beta RI (MAB5871, R&D Systems, Minneapolis, MN, USA), anti-TGF-beta RII (R&D Systems), and anti-GAPDH (MAB374, Sigma-Aldrich).

Total protein of colonic tissues was extracted according to the manufacturer’s protocol (Vazyme, USA). Briefly, protein concentrations were determined through BCA Protein Assay Kit (Vazyme, USA). Samples with equal amounts of protein (25 µg) were fractionated on 10% SDS polyacrylamide gels, transferred to polyvinylidene difluoride (PVDF) membranes, and blocked in 5% skim milk in TBST for 1.5 h at 25°C. The membranes were then incubated at 4°C overnight with 1:1,000 dilutions (v/v) of the primary antibodies. After washing the membranes with TBST, the secondary antibodies with 1:1,000 dilutions were added and incubated for another 2 h at 25°C. Protein expressions were examined using an Enhanced Chemiluminescence Detection System. GAPDH was used as a loading control. Antibodies in western blotting were purchased from Abcam (Cambridge, MA, USA), including Col1a2 (ab96723), Col3a2 (ab196613), MMP-1 (ab52631), MMP-3 (ab52915), TIMP-1 (ab86482), E-cadherin (ab76055), N-cadherin (ab202030), Vimentin (ab193555), a-SMA (ab32575), MMP-9 (ab38898) and GAPDH (ab181602).

To obtain total protein, cells were lysed in RIPA buffer with protease inhibitor added. Equal amounts of protein (20 or 30 μg) were separated on a 10% SDS‐PAGE gel and then electroblotted onto PVDF membranes. After blocking with 5% non‐fat‐dried milk in Tris‐buffered saline with 0.1% Tween 20 (TBST) buffer for 1 hours at room temperature, the membranes were incubated overnight at 4°C with primary antibodies. The membranes were washed and incubated with corresponding IgG‐HRP secondary antibody (Jackson ImmunoResearch, USA). The bands were visualized with the AlphaImager HP system. The antibodies used in this study were as follows: anti‐Col1 (Abcam, ab96723, 1:2000), anti‐Col3 (Abcam, ab184993, 1:1000), anti‐α‐SMA (Proteintech, 14395‐1‐AP, 1:1000), anti‐Akt (CST, #4691, 1:1000), anti‐pAkt (CST, #4060, 1:1000), anti‐FoxO1 (CST, #14952, 1:1000), anti‐pFoxO1(Thr24)/FoxO3a (Thr32) (CST, #9464, 1:1000), anti‐p27^Kip1 (CST, #3686, 1:1000) and anti‐GAPDH (Bioss, bs‐2188R, 1:4000).

Total protein were extracted from cells with a protein extraction reagent (Thermo Fisher Scientific, USA). Membranes were exposed to a rabbit anti-PPARγ antibody (Santa Cruz, sc-7196, diluted 1:100), rabbit anti-COL1A2 (Abcam, ab96723, diluted 1:1000) or rabbit anti-α-SMA antibody (Abcam, ab5694, diluted 1:500) or a rabbit β-actin or GAPDH antibody (Cell Signaling Technology, 4970S, 2118S, diluted 1:2000). After incubation with the HRP-conjugated secondary antibody, blots were visualized with the Pierce ECL Western Blotting Substrate (Thermo Fisher Scientific, USA). The relative protein expression levels were normalized to the β-actin or GAPDH levels.

After fixation with 4% PFA for 48 h, the tibias from the WT and Neat1-KO mice were maintained in 18% EDTA solutions for 4 weeks for decalcification and then embedded in paraffin. According to the IHC protocol, paraffin-embedded slices were dewaxed in xylene and rehydrated in graded alcohol solutions. Then, antibodies against osteocalcin (1:500, Proteintech, 23418-1-AP) or Col1α (1:200, Abcam, ab96723) were used for staining. Quantitative histomorphometric analysis was performed on the tibias of male mice, and osteoblast number per bone surface was measured by conducting toluidine blue staining using OsteoMeasure XP Software (OsteoMetric) in a blinded fashion.