primary bronchial smooth muscle cells bsmcs  (PromoCell)

Journal: Nature Communications

Article Title: SARS-CoV-2 infection of human pluripotent stem cell-derived vascular cells reveals smooth muscle cells as key mediators of vascular pathology during infection

doi: 10.1038/s41467-024-54917-4

Figure Lengend Snippet: A Schematic of directed differentiation of hPSCs to vascular endothelial cells (ECs), smooth muscle cells (SMCs), and pericytes (PCs) (Created using BioRender). B Bulk RNA sequencing was performed on hPSC-derived ECs, PCs, and SMCs. The normalized expression values of known EC, PC, or SMC marker genes were quantitated over three biological replicates. The values plotted represent scaled normalized expression values for each gene across samples ( C ) (left) Principal component analysis (PCA) on bulk-RNA sequencing data from hPSCs, hPSC-ECs, hPSC-PCs, and hPSC-SMCs and primary endothelial cells (HUVECs), primary pericytes, and primary bronchial smooth muscle cells (BSMCs). (right) Mural cell-only PCA analysis (Primary pericytes, BSMCs, hPSC-PCs, and hPSC-SMCs) ( D ) Expression of PECAM1 and VE-Cadherin was quantitated by flow cytometry on hPSCs or on two independent differentiations of ECs. Data points on the bar graph represent values from two independent differentiations. Expression of VE-Cadherin, VWF, and PECAM1 in hPSC-derived ECs was observed by immunofluorescence. E Expression of NG2 was quantitated by flow cytometry on hPSCs or on two independent differentiations of PCs. Data points on bar graph represent values from two independent differentiations. Expression of PDGFR-B, and PDGFR-A was quantitated by flow cytometry on HEK293 cells, human dermal fibroblast (hDF) and two independent PC differentiation. Expression of SMA, PDGFR-B, and NG2 in hPSC derived PCs was observed by immunofluorescence. F Expression of PDGFR-B or SMA was quantitated by flow cytometry on hPSCs or on two independent differentiations of SMCs. Data points on the bar graph represent values from two independent differentiations. Expression of SMA, PDGFR-B, and NG2 in hPSC-derived SMCs was observed by immunofluorescence. Scale bar = 50 µm for all immunofluorescence images.

Article Snippet: Primary bronchial smooth muscle cells (BSMCs) were obtained from PromoCell (Cat# C12561).

Techniques: RNA Sequencing Assay, Derivative Assay, Expressing, Marker, Flow Cytometry, Immunofluorescence

Journal: Molecular cancer research : MCR

Article Title: INTERACTIONS WITH MUSCLE CELLS BOOST FUSION, STEMNESS AND DRUG RESISTANCE OF PROSTATE CANCER CELLS

doi: 10.1158/1541-7786.MCR-18-0500

Figure Lengend Snippet: (A) Representative images of single-cell clones of PC3 cells (tumorspheres, marked by yellow arrows) developed in an anchorage-independent manner, in a serum-free medium enriched in growth factors by PC3 cells and PC3 cells that were co-cultured with either human skeletal muscle cells (hMYO), prostate smooth muscle cells (pSMC), or mouse myoblasts (mMYO). Scale bar, 90 μm (B) Numbers of tumorspheres formed by PC3 cells and PC3 cells that had been co-cultured with muscle cell of the indicated types. (C) Percentages of CD133 gene expressing cells among PC3 cells grown on their own (1), with hMYO (2), pSMC (3) or mMYO (4). To detect transcriptional activity of the CD133 gene in fluorescence microscopy as GFP expression, PC3 cells were transfected with pLenti lentiviral construct with GFP reporter driven by the human CD133 promoter. The transfection efficiency was consistent from experiment to experiment at 80–85 % of the cells, as estimated using the GFP empty vector. The cells used for all conditions within the same series of experiments were transfected in parallel 24h before the beginning of co-culturing experiments. Thus, both the percentage of the CD133 promoter–driven GFP reporter transfected prostate cancer cells and the distribution of the numbers of the reporter construct copies per cell within cell population are expected to be the same for all conditions. (D) Flow cytometry analysis of CD133 expression at the surface of PC3 cells or of PC3 cells co-cultured with either hMYO, pSMC, or mMYO. (E) qPCR analysis of RNA level expression of CD133 in PC3 cells. (F,G) PC3 cells and PC3 cells co-cultured with either hMYO cells or pSMC for 72 hours were treated with different concentrations of either doxorubicin (F) or cisplatin (G) for 24 h. The cell viability was determined from MTT assay. (H,I) Immunoblot of PC3 cells and PC3 cells co-cultured with hMYO (H) or pSMC (I) for different EMT markers: phosphorylated AKT (pAKT), AKT, MMP-9, Vimentin, E-Cadherin, Slug (I), and for Tubulin, as a loading control. (A-I) In all co-culturing experiments, cancer cells were grown on the inserts. All results are shown as means ± SEM (n ≥ 3).

Article Snippet: PC3 cells (ATCC, Manassas, VA), primary human myoblasts (hMYO) and primary prostate smooth muscle cells (pSMC), both obtained from Lonza, were maintained according to the manufacturer’s instructions.

Techniques: Clone Assay, Cell Culture, Expressing, Activity Assay, Fluorescence, Microscopy, Transfection, Construct, Plasmid Preparation, Flow Cytometry, MTT Assay, Western Blot

Journal: Molecular cancer research : MCR

Article Title: INTERACTIONS WITH MUSCLE CELLS BOOST FUSION, STEMNESS AND DRUG RESISTANCE OF PROSTATE CANCER CELLS

doi: 10.1158/1541-7786.MCR-18-0500

Figure Lengend Snippet: (A) An increase in doxorubicin resistance observed for PC3 cells cultured in the conditioned medium collected from a 72-h co-culturing of PC3 and hMYO or mMYO or pSMC. We assessed changes in cell viability from counts of viable cells at the end of the 24 h treatment. IL-4 (B) and IL-13 (C) levels in the conditioned media collected from pPCa, pSMC, hMYO, PC3 cultures as well as from pPCa/hMYO, pPCa/pSMC, PC3/hMYO and PC3/pSMC co-cultures were evaluated using human IL-4 (B) or IL-13 (C) ELISA kits. (D) qPCR analysis of RNA level expression of IL-4 and IL-13 in PC3 cells and PC3 cells co-cultured with hMYO. (E) qPCR analysis of RNA level expression of IL-4 and IL-13 in hMYO and hMYO co-cultured with PC3 cells. (F) Numbers of tumorspheres formed by PC3 cells and by PC3 cells co-cultured with hMYO in the presence or in the absence of neutralizing antibodies to either IL-4 or IL-13. (G) Numbers of tumorspheres formed by PC3 cells, PC3 cells co-cultured with pSMC in the presence or in the absence of neutralizing antibodies to either IL-4 or IL-13. (H) Numbers of tumorspheres formed by PC3 cells and by PC3 cells co-cultured with mMYO in the presence or in the absence of neutralizing antibodies to either IL-4 or IL-13. (I) Flow cytometry analysis of CD133 expression at the surfaces of PC3 cells and of PC3 cells co-cultured with mMYO in the presence or in the absence of neutralizing antibodies to either IL-4 or IL-13. (B-I) In all co-culturing experiments, cancer cells were grown on the inserts. All results are shown as means ± SEM (n ≥ 3).

Article Snippet: PC3 cells (ATCC, Manassas, VA), primary human myoblasts (hMYO) and primary prostate smooth muscle cells (pSMC), both obtained from Lonza, were maintained according to the manufacturer’s instructions.

Techniques: Cell Culture, Enzyme-linked Immunosorbent Assay, Expressing, Flow Cytometry

Journal: Molecular cancer research : MCR

Article Title: INTERACTIONS WITH MUSCLE CELLS BOOST FUSION, STEMNESS AND DRUG RESISTANCE OF PROSTATE CANCER CELLS

doi: 10.1158/1541-7786.MCR-18-0500

Figure Lengend Snippet: (A). Fluorescence microscopy images of PC3 cells labeled with either green cell tracker or red cell tracker and co-cultured with either pSMC or hMYO for 72 h. Yellow and white arrows mark, respectively the co-labeled and multinucleated cells (white arrows). Bar, 25 μm. (B, C) PC3 cells labeled with either green cell tracker or red cell tracker and cultured by themselves, or co-cultured with hMYO (B) or pSMC (C) for 72h. Content mixing (bars 1 and 2) and multinucleation (bars 3 and 4) are quantified as the ratio of the number of nuclei either in double-labeled cells (fusion) or in multinucleated cells (multinucleation) to the total number of nuclei in the field of view. (D, E) Multinucleation (D) and content mixing (E) were quantified for pPCa cells cultured by themselves or co-cultured with hMYO or co-cultured with hMYO in the presence of FdUrd for 72 h. (F). Fluorescence microscopy images of co-cultured pPCa labeled with green cell-tracker and either pSMC or hMYO labeled with red cell-tracker. Arrows mark cells generated by pPCa-muscle cell fusion and labeled with both cell trackers. Bar, 25 μm. (G) Quantification of fusion between pPCa and pSMC and between pPCa and hMYO. (H) Quantification of fusion between PC3 cells and either pSMC (1), hMYO (2) or mMYO (3). (A-H). Cancer and muscle cells were co-cultured in a direct contact. All results are shown as means ± SEM (n ≥ 3).

Article Snippet: PC3 cells (ATCC, Manassas, VA), primary human myoblasts (hMYO) and primary prostate smooth muscle cells (pSMC), both obtained from Lonza, were maintained according to the manufacturer’s instructions.

Techniques: Fluorescence, Microscopy, Labeling, Cell Culture, Generated

Journal: Molecular cancer research : MCR

Article Title: INTERACTIONS WITH MUSCLE CELLS BOOST FUSION, STEMNESS AND DRUG RESISTANCE OF PROSTATE CANCER CELLS

doi: 10.1158/1541-7786.MCR-18-0500

Figure Lengend Snippet: (A) qPCR analysis of Syn1 and AnxA5 expression in PC3 cells grown on the inserts on their own or co-cultured with hMYO (A) grown on the bottom of the same wells. (B,C) Western blot analysis of expression of Syn1 and AnxA5 in PC3 lysates with tubulin as a loading control for PC3 and for PC3 co-cultured with either hMYO (B) or pSMC (C). (D) Western blot analysis of AnxA5 expression in PC3 or PC3/hMYO cocultures transfected either with AnxA5 siRNA or non-targeting (NT) siRNA, and tubulin as a loading control. (E, F) The effects of siRNA suppression of AnxA5 expression in PC3 cells and in PC3 cells cocultured for 72 h in direct contact with hMYO (PC3/hMYO) on content mixing (E) and on multinucleation of PC3 cells (F). 1 – PC3 cells transfected with NT siRNA; 2 – PC3/hMYO coculture transfected with NT siRNA. 3 – PC3 transfected with AnxA5 siRNA and cocultured with hMYO. 4 - PC3/hMYO coculture transfected with AnxA5 siRNA. (G) Western blots for PC3 cells cultured on their own or with mMYO. (H) Multinucleation for PC3 cells (1), PC3 cells co-cultured for 72 h with mMYO (2), and PC3 cells co-cultured with AnxA5−/− mMYO (3). (I) Multinucleation for PC3 cells (1), PC3 cells co-cultured for 72h with mMYO (2), and PC3 co-cultured for 72 h with mMYO in the presence of 50 μM Syn1 peptide (4) or 50 μM scrambled Syn1 peptide (3). (J) At the top, Western blots of PC3 cells transduced with either non-targeting control shRNA (PC3-NT) or with Syn1 shRNAs (PC3-Syn1 shRNA) to evaluate levels of expression of Syn1 with tubulin as a loading control. Below, multinucleation of PC3-NT cells (1), for PC3-NT cells co-cultured for 72 h with hMYO (2), and for PC3-Syn1 shRNA co-cultured with hMYO (3). (K) Quantification of heterotypic fusion between hMYO and either PC3 cells or PC3-Syn1 shRNA after 72h of co-culturing. (A-D) Cancer cells were grown on the inserts. (E-I) In all fusion experiments, cancer and muscle cells were co-cultured in a direct contact. All results are shown as means ± SEM (n ≥ 3).

Article Snippet: PC3 cells (ATCC, Manassas, VA), primary human myoblasts (hMYO) and primary prostate smooth muscle cells (pSMC), both obtained from Lonza, were maintained according to the manufacturer’s instructions.

Techniques: Expressing, Cell Culture, Western Blot, Transfection, Transduction, shRNA