Supplementary MaterialsSupplementary material 1 (PDF 47 kb) 12195_2019_602_MOESM1_ESM. but has an unclear impact on phenotypic changes in melanoma. Recent data using cells edited a double-nickase CRISPR/Cas9 approach suggest that CCN4/WISP1 stimulates invasion and metastasis of melanoma cells. While these data also suggest that loss of CCN4/WISP1 increases cell proliferative, the CRISPR approach used may Microtubule inhibitor 1 be an alternative explanation rather than the loss of gene function. Methods To test whether CCN4/WISP1 also influences the proliferative phenotype of melanoma cells, we used mouse melanoma models and knocked out using a homology-directed repair CRISPR/Cas9 system Rabbit Polyclonal to Involucrin to generate pools of and assays. Results assays using knockout pools supported previous findings that CCN4/WISP1 promoted an epithelialCmesenchymal-like transition in melanoma cells and stimulated invasion and metastasis. While knockout also enhanced cell growth in optimal 2D culture conditions, the knockout suppressed certain cell survival signaling pathways and rendered cells less resistant to stress conditions. Tumor cell growth assays at sub-optimal conditions overexpressed CCN4 driven by a CMV promoter to conclude that CCN4 represses the growth and metastasis of a highly metastatic mouse melanoma line.15 In addition, Shao and CCN4 secretion by adjacent fibroblasts represses melanoma growth.31 In contrast, our work using double nickase-based CRISPR/Cas9 systems to modify mouse and human melanoma cells demonstrate that CCN4 stimulates invasion and metastasis by promoting an EpithelialCMesenchymal Transition (EMT)-like process.6 In addition to invasion and metastasis as phenotypic attributes of CCN4 stimulation, CCN4 knock-out also seemed to promote the proliferation of melanoma cells. Yet, challenging a single edited clone to generate a clonal populace of millions is usually a strong selective pressure for retaining highly proliferative variants that pre-exist within the parental cell line.13,18 Thus the association of changes in proliferative phenotype upon CCN4 knockout may be attributed to the double nickase-based CRISPR/Cas9 approach rather than the loss of gene function. To test this hypothesis directly, the objective of this study was to clarify the role of CCN4 in the context of melanoma using a homology directed repair-based CRISPR/Cas9 approach that generates a pool of edited cells. Materials and Methods Cell Culture, Conditioned Media and CCN4 ELISA Mouse melanoma lines B16F0, B16F10, mouse fibroblast line NIH3T3, human metastatic melanoma lines RPMI-7951, SH-4, Microtubule inhibitor 1 SK-MEL-3 and SK-MEL-24 were from ATCC and produced as recommended. NIH3T3-derived cells with knockout (NIH3T3-KO), mouse CCN4 overexpression (NIH3T3-mCCN4, formerly NIH3T3-mWisp1) and control retrovirus contamination (NIH3T3-pBabe) were described before.6 Media conditioned for 48-h (DMEM with 0.1% FBS) from the indicated cells were Microtubule inhibitor 1 prepared for transwell assays, and conditioned media with 10% FBS were used for gene expression stimulation. CCN4 concentration in conditioned medium was determined by ELISA using Human WISP-1/CCN4 DuoSet ELISA Development Kit (R&D Systems, Minneapolis, MN). Creation of CRISPR/Cas9 KO Plasmid (sc-423705) and Homology-Directed Repair (HDR) plasmids (sc-423705-HDR) were from Santa Cruz Biotechnology (Dallas, Texas). B16F0 cells were transfected with a mix of CRISPR/Cas9 KO plasmid and HDR plasmids, followed by puromycin selection (1.0?knockout cells, B16F0-KO. The cells were expanded, frozen down and passage 3C6 cells were used in this work. To remove the LoxP-flanked puromycin-resistant cassette, B16F0-KO were transfected by a mix of Cre recombinase expression (sc-418923, Santa Cruz Biotechnology) and GFP plasmids. The second knockout cells, B16F0-KO, were created by flow sorting of GFP-positive cells. Only Microtubule inhibitor 1 passage 4 cells were used in this work. A control cell, B16F0-Ctr, was also made using pBabe-puro retrovirus. The same strategy was used to create B16F10 knockout cells. A double-nickase-based CRISPR/Cas9 approach was used to generate KO variants of B16F10 and YUMM1.7 cell lines, as described previously.6 Protein and RNA Analysis Western blotting was performed as described.6 Mouse monoclonal anti–actin (C4) Microtubule inhibitor 1 was from Santa Cruz Biotechnology, and the following rabbit monoclonal antibodies were purchased from Cell Signaling Technology (Danvers, MA): anti-Snail (C15D3), anti-Slug (C19G7), anti-Vimentin (D21H3), anti-N-Cadherin (D4R1H). RNA isolation and qRT-PCR was performed as described. 6 Samples for RNA analysis were prepared in biological triplicates and cells were plated on 6-well plates for 48?h before harvested for gene expression analysis. To induce knockout cells for.