For marking and quantifying EdU-positive cells, (Thermo-Fisher Scientific) was used. 200 m. NIHMS973600-supplement-2.tif (45M) GUID:?AA277BB7-FC76-4079-9993-9541E0919B69 Abstract Ca2+ is an important second messenger, and it is involved in many cellular processes such as cell death and proliferation. The rise in intracellular Ca2+ levels can be due to the generation of inositol 1,4,5-trisphosphate (InsP3), which is a product of phosphatidylinositol 4,5-bisphosphate (PIP2) hydrolysis by CAL-130 phospholipases C (PLCs), that leads to Ca2+ release from endoplasmic reticulum by InsP3 receptors (InsP3R). Ca2+ signaling patterns can vary in different regions of the cell and increases in nuclear Ca2+ levels have specific biological effects that differ from those of Ca2+ increase in the cytoplasm. There are PLCs in the cytoplasm and nucleus, but little is known about the functions of nuclear PLCs. This work aimed to characterize phenotypically the human PLC4 (hPLC4) in mesenchymal stem cells. This nuclear isoform of PLC is present in different cell types and has a Rabbit Polyclonal to Cofilin possible role in proliferative processes. In this work, hPLC4 was found to be mainly nuclear in human adipose-derived mesenchymal stem cells (hASC). PLC4 knockdown demonstrated that it is essential for hASC proliferation, without inducing cell death. An increase of cells in G1, and a reduction of cells on interphase and G2/M in knockdown cells were seen. Furthermore, PLC4 knockdown increased the percentage of senescent cells, and mRNAs expression, which could explain the impaired cell proliferation. The results show that hPLC4 is in involved in cellular proliferation and senescence in hASC. gene (and mRNAs expression. Our results indicate that PLC4 is a nuclear protein that is involved in hASC proliferation and senescence. 2. Materials and Methods 2.1. hASC isolation and culture Human adipose-derived mesenchymal stem cells (hASC) were obtained from the subcutaneous adipose tissue removed during liposuction surgeries or abdominoplasties. The samples were donated freely by lipoplasty surgery patients according to the regulations approved by the for 10 minutes at 25C, the precipitate was resuspended in complete (DMEM medium, Sigma-Aldrich) containing 10% fetal bovine serum (FBS; Thermo Fisher Scientific, Waltham, MA, USA) and 1% penicillin/streptomycin (PS; Sigma-Aldrich) and transferred to T25 culture flasks (Sarstedt, Nmbrecht, Germany), kept in a humidified atmosphere at 37C and 5% CO2. Cell media was replaced every 3 days. Passages 3 and 4 were used for performing the assays. 2.2. Immunofluorescence Stem cells were plated at a concentration of 2 105 cells per plate, in 6-well plates containing coverslips. After a 24 hour cultivation, cells were fixed, incubated with rabbit polyclonal primary antibody anti-PLC4 antibody (1:200, Santa Cruz Biotechnology, Dallas, TX, USA), and anti-rabbit IgG secondary antibody conjugated to Alexa Fluor? 488 (1:500, Thermo-Fisher Scientific). CAL-130 Coverslips were assembled using with DAPI (Thermo-Fisher Scientific). Fluorescence images were obtained using confocal microscope Zeiss 5 LIVE (Carl Zeiss, Jena, Germany), at or software (Carl Zeiss). At least three assays were performed. 2.3. siRNA transfection The knockdown of human PLC4 was achieved using siRNA (84812), (Dharmacon/Thermo Scientific; target sequences: CAAGAAGUUCAGCGGUUAU, GCUCAAUCCCAUACCGACA, GACCAAUGGCUGAGCGAUU, CAACAAGGUUACCGCCACA). Scrambled siRNA (Dharmacon/Thermo Scientific; target sequences: UAAGGCUAUGAAGAGAUAC, AUGUAUUGGCCUGUAUUAG, AUGAACGUGAAUUGCUCAA, UGGUUUACAUGUCGACUAA) was used as a control. Cells were transfected using Lipofectamine RNAiMAX (Thermo-Fisher Scientific). Lipofectamine alone, or 25 nM siRNA were diluted in the minimal medium (Thermo-Fisher Scientific), incubated for 5 minutes, then combined 1:1 CAL-130 and incubated for 20 minutes. The lipofectamine-siRNA complex was added to resuspended cells (the number of cells and the medium volume varied according to each assay). The cells were kept at 37C, 5% CO2, in for 5 hours for cell adhesion after which the medium was replaced by complete DMEM, and the cells maintained at 37C, 5% CO2, for the duration for each assay. 2.4. Quantitative Real-Time PCR (qPCR) RNA was extracted with TRIzol reagent (Thermo-Fisher Scientific), according to the manufacturer recommendations. RNA samples were treated CAL-130 using DNAse RQ1 (Promega, Madison, WI, USA) and reverse transcription reactions were performed using (Thermo-Fisher Scientific) with random primers. Primers were designed for (accession number on GenBank: NM_032726; hPLCd4 forward (F): 5-AGGTGGATGTATGGGATGGACC-3; hPLCd4 reverse (R): 5-GGGTAGTCTGATGTCTGGAAGG-3), (GenBank accession number: AB082924; RPL13A F: 5-TATGAGTGAAAGGGAGCC-3; RPL13A R: 5-ATGACCAGGTGGAAAGTC-3), (GenBank accession number: NM_000389.4; p21 CDKN1A (1) CAL-130 Fw: 5-CTGTCTTGTACCCTTGTGCCT-3; p21 CDKN1A (1) Rv: 5-AATCTGTCATGCTGGTCTGCC-3) and (GenBank accession number: NM_000077.4; p16 CDKN2A (1) Fw: 5-GAGCAGCATGGAGCCTTCG-3; p16 CDKN2A (1) Rv: 5-CGTAACTATTCGGTGCGTTGG-3) genes, using [30]. Primers for (GenBank accession number: NM_001256799) were designed by Thibeaux and colleagues (2014) [31]. Primers were chemically synthesized by Integrated DNA Technologies (IDT, Coralville, IA, USA). For qPCR, primers were added to cDNAs and to (Promega), which.