Calponin

Whole-cell lysates extracted using RIPA (Radio Immunoprecipitation Assay) Lysis Buffer (Huaxingbio Biotechnology, Beijing, China), and exosome fractions in PBS were solubilized in SDS-PAGE loading buffer and heated (95°C, 5 min) for western blot. Antibodies against Hsp70 and CD63 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA), anti-ARF6, NCX1, α-SMA and Calponin antibodies were obtained from Abcam (Cambridge, UK), anti-SRF antibodies were obtained from CST Inc. (Danvers, MA, USA). SDS-PAGE was performed using 12% Tris-Glycine Gels (Huaxingbio, Beijing, China), and the protein was transferred to Polyvinylidene Difluoride (PVDF) membranes (Millipore Corporation, Bedford, MA, USA). The membranes were blocked in 5% nonfat dried milk powder (Huaxingbio Biotechnology, Beijing, China) in TBST for 1 h at room temperature and incubated with primary antibodies overnight at 4°C. Secondary antibodies conjugated with horseradish peroxidase (HRP, Sigma-Aldrich, St. Louis, MO, USA) were incubated with the membranes for 2 h. The blots were visualized by ECL chemiluminescence reagents from Pierce Biotechnology (Rockford, IL, USA).

Calponin, pancytokeratins AE1/AE3, collagen IV (Abcam), collagen 1, collagen 2 (Cedarlane), PPARγ, Sox9, Runx2 (Santa Cruz Biotechnology), osteocalcin (AbD Serotec), FGFR3, laminin (Novus Biologicals), CD31, CD44, CD45, CD73, CD105, Mac1 (BD Biosciences), α-SMA, Ki67 (Dako).

SMC marker proteins in control and patient cell lines were quantified using western blot. Cells were seeded in 6 well plates at a seeding density of 150.000 cells/well and incubated overnight. The next day, they were washed with Earl’s Balanced Salt Solution (EBSS; Thermo Fisher Scientific) and each well was lysed in 100ul of Nu Page Sample buffer and reducing agent (9:1) (Thermo Fisher Scientific. After boiling, 8ul of each sample was loaded onto either NuPAGE 4–12% Bis-Tris or NuPAGE 3–8% TA gels depending on the size of the protein in question. Western blotting was performed as previously published^{38 (link)}. Primary antibodies were incubated overnight at 4 °C. Primary antibody against tubulin (1:8000) was used as loading control. Primary antibodies against Calponin (1:4000; Abcam), Smooth muscle actin (1:4000; Dako), SM22 (1:4000; Abcam) were incubated overnight at 4 °C. Secondary antibodies IRDye® 800CW Goat anti-Rabbit IgG and IRDye® 680CW Goat anti-Mouse IgG (1:5000; LI-COR Biosciences). The fluorescent signal was visualized using the Odyssey Infrared Imaging System (Odyssey version 4 software; LI-COR Biosciences). Intensity of bands was quantified using Fiji^{39 (link)}.

Western blot test was used to identify expression of SMC proteins. Cells were lysed in RIPA buffer with protease and phenylmethylsulfonyl fluoride (PMSF; Roche, Indianapolis, IN, USA). The protein concentration was assayed using the BCA method (Bio-Rad). Approximately 20 to 50 μg of total protein samples was loaded on a 10% SDS-PAGE after denaturation by boiling for 10 min. The separated proteins transferred to polyvinylidene difluoride membranes. Membranes were blocked by incubation in Tris-buffered saline containing 0.05% Tween 20 and 5% skimmed milk with constant shaking for 1 h. The membrane was probed with antibodies against a-SMA (1 : 1000, Abcam, Cambridge, UK), calponin (1 : 1000, Abcam, Cambridge, UK), SM22a (1 : 1000, Abcam, Cambridge, UK), SM-MHC (1 : 1000, Abcam, Cambridge, UK), smoothelin-like 2 (1 : 2000, Santa Cruz, Dallas, USA), ACLP (1 : 1000, Abcam, Cambridge, UK), CaMKIIα (1 : 5000, Abcam, Cambridge, UK), CaMKIIβ (1 : 1000, Abcam, Cambridge, UK), CaMKIIγ (1 : 500, Abcam, Cambridge, UK), and CaMKIIδ (1 : 1000, Santa Cruz, Dallas, USA) overnight at 4°C. GAPDH was used as an internal loading control. The membranes were washed three times with TBST and incubated with the appropriate secondary antibodies at room temperature for 1 h and detected using enhanced chemiluminescence.

Cells were fixed with 4% PFA, permeablilised with 0.1% Triton X-100, blocked in 1% BSA/PBS and incubated with primary antibodies (Calponin, Abcam, ab46794; αSMA,Sigma; phosphorylated myosin light chain 2, Cell Signalling, 3675S and anti-total Ucma/GRP antibody (Cterm-GRP, GenoGla Diagnostics, Faro, Portugal). The following secondary antibodies were used: anti-rabbit-FITC (Dako, F0205) and anti-mouse-FITC (Dako, F0232). Nuclei were stained with DAPI (Sigma–Aldrich). Cells were mounted in a glycerol DABCO mounting medium. Pictures were taken with a Leica DM2000 widefield microscope (Leica Microsystems).

Cultures of ESC were fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100 in PBS and blocked with 10% normal swine serum (Dako). Incubation of cell or tissue samples with primary antibodies was performed at 4°C overnight, followed by incubation with secondary antibodies for 30 mins at 37°C after three thorough washes with PBS. Subsequently, the cells were counterstained with DAPI (1:1000 in PBS) for 3 mins at room temperature and mounted with fluorescent mounting media (Dako) before image acquisition using an Axio Imager. M2 microscope and AxioVision Digital Imaging System (Carl Zeiss Ltd.). Primary antibodies used were Calponin, SM-22α (both from Abcam), SM-αA (Sigma Aldrich), HSP47 (Abcam) and DAPI (Sigma Aldrich). The appropriate fluorescent-conjugated (Alexa 488 and Alexa 546) IgG antibodies were used as secondary antibodies (Invitrogen).