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) Western blot and qPCR analysis of Syn1 expression in PC3 cells transfected to express Syn1f (PC3-Syn1). (B) Fluorescence microscopy images of PC3 cells labeled with green cell tracker and PC3 cells expressing Syn1f and labeled with red cell tracker labelling. Arrows mark the colabeled cells. Bar, 25 μm. Content mixing between PC3 cells and between PC3-Syn1 cells was quantified by flow cytometry. (C). Representative images (scale bar 90 μm) and quantification of tumorspheres developed by PC3 cells and by PC3-Syn1 cells. (D) Quantification of CD133 gene expression detected as GFP expression driven by the human CD133 promoter for PC3 cells and for PC3-Syn1 cells. (E) Flow cytometry analysis of cell surface CD133 expression for the co-cultured PC3 cells labeled with green cell tracker and the PC3-Syn1 cells labeled with red cell tracker. Results averaged over 3 independent experiments are presented as percentages of CD133+ cells among green PC3 cells, red PC3-Syn-1 cells and yellow cells generated by PC3/PC3-Syn1 fusion cells (F) Western blot analysis of expression of pAKT, AKT, MMP-9, Vimentin, E-Cadherin, and tubulin, as a loading control, in PC3 cells and PC3-Syn1 cells. (G) PC3 cells stably expressing lentiviral GFP (PC3-GFP cells), and PC3-GFP cells co-cultured with hMYO in the presence of either Syn1 peptide or scrambled Syn1 peptide. After co-culturing the cells were treated or not treated with doxorubicin. The dependence of the co-culturing-induced increase in doxorubicin resistance of PC3 cells on cell fusion. Numbers of viable PC3 cells after a 24 h application of the drug were normalized to the numbers in corresponding controls not treated with the drug. PC3 cells (1); PC3 cells grown in the presence of either Syn1 peptide (3) or scrambled Syn1 peptide (2); PC3 cells co-cultured in a direct contact with hMYO (4), PC3 cells co-cultured with hMYO in the presence of either Syn1 peptide (6) or scrambled Syn1 peptide (5). All results are shown as means ± SEM (n ≥ 3). (H) Fluorescent images of PC3 cells stably expressing lentiviral GFP (PC3-GFP cells), and PC3-GFP cells co-cultured in a direct contact with hMYO in the presence of either Syn1 peptide or scrambled Syn1 peptide. Cell were either treated with doxorumicin or not treated with doxorubicin. (I) Representative fluorescent images of prostate tissue microarray samples including BPH, PIN, Gleason grade 5–6, 7 and 8–10 stained with Syncytin 1 and Annexin A5. (J) Quantification of Syncytin 1 and Annexin A5 in BPH, PIN, Gleason grade 5–6, 7 and 8–10 prostate tissue samples. Means ± SEM (n ≥ 10). (K) Suggested pathway by which muscle cells in the microenvironment of prostate cancer cells promote its progression.

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: Western Blot, Expressing, Transfection, Fluorescence, Microscopy, Labeling, Flow Cytometry, Cell Culture, Generated, Stable Transfection, Microarray, Staining

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

Journal: PLoS ONE

Article Title: MicroRNAs Overexpressed in Growth-Restricted Rat Skeletal Muscles Regulate the Glucose Transport in Cell Culture Targeting Central TGF-β Factor SMAD4

doi: 10.1371/journal.pone.0034596

Figure Lengend Snippet: PremiR transfections (25 nM) of rat L6 myoblast-myocytes were done and the cell-extracts were subjected to Western Blot analysis for SMAD4, nucleolin and DnaJ-B1 level after 48 hours (I). The triplicate experiments were run in SDS-PAGE/Western Blot to have statistical significant data (T-test) and expressed as relative intensities (%) (I).

Article Snippet: Mammalian skeletal muscle cell-lines (bought from ATCC) of rat origin L6 myoblasts and H9c2 cardiomyocytes (embryonic heart tissue, myoblast morphology) were used in this study.

Techniques: Transfection, Western Blot, SDS Page

Journal: PLoS ONE

Article Title: MicroRNAs Overexpressed in Growth-Restricted Rat Skeletal Muscles Regulate the Glucose Transport in Cell Culture Targeting Central TGF-β Factor SMAD4

doi: 10.1371/journal.pone.0034596

Figure Lengend Snippet: Small inhibitory RNA (25 nM) was used to deplete SMAD4 in L6 cell culture system prior to glucose transport assay using 14 C-2-deoxyglucose as described in . Both skeletal muscle cells (I & II) and cardiomyocytes cells (III) were used for this experiment. The transport assay condition is maintained in the same way as was done for . (IV) The Western Blot analysis in L6 cell-lines to show the specificity of siRNA against SMAD4. Dharmacon-designed SMARTpool siRNAs (4 sets) specific to SMAD4 and non-targeting negative control siRNA were used for this experiment according to the manufacturer's instruction.

Article Snippet: Mammalian skeletal muscle cell-lines (bought from ATCC) of rat origin L6 myoblasts and H9c2 cardiomyocytes (embryonic heart tissue, myoblast morphology) were used in this study.

Techniques: Cell Culture, Transport Assay, Western Blot, Negative Control