Journal: Breast cancer research : BCR

Article Title: A hybrid epithelial-mesenchymal transition program enables basal epithelial cells to bypass stress-induced stasis and contributes to a metaplastic breast cancer progenitor state.

doi: 10.1186/s13058-024-01920-8

Figure Lengend Snippet: Fig. 6 PRC2-mediated epigenetic regulation governs access to EMT states in basal epithelial variants. A Sorted basal epithelial cells (P3) were transduced with lentiviral CRISPR constructs containing CAS9 and sgRNA targeting EZH2 or non-targeting controls (Cont.). These constructs included an IRES-GFP sequence, which facilitated the selection of cells incorporating the vector using FACS. EZH2 and control cells were expanded in MEGM; at each passage, cells were counted, and a growth curve of the cumulative population doubling over time was plotted. B Same as in A, starting with P8 (variant) basal epithelial cells. Cells were collected at P8 + 3, as indicated by the arrows. C Western blot analysis of lysates from P8 EZH2 KO and control cells using antibodies against Zeb, N-cadherin, EZH2, H3K27me3, total Histone H3, and Actin ( loading control). D P-cadherin, N-cadherin, Slug, Snail, Twist, and Zeb were analyzed by qPCR using P8 + 3 EZH2 KO and control cells. E Sorted basal epithelial cells (P3) were continuously treated with 10 μM GSK126, an inhibitor of EZH2, or DMSO (vehicle control) as cells were expanded in MEGM; at each passage, cells were counted, and a growth curve of the cumulative population doubling over time was plotted. F Same as in E, starting with P8 (variant) basal epithelial cells. Cells were collected at P8 + 3, as indicated by the arrows. G P-cadherin, N-cadherin, Slug, Snail, Twist, and Zeb were analyzed by qPCR in P8 + 3 cells treated with GSK126 or DMSO. H In the EMT continuum, cells exist in various states ranging from epithelial to mesenchymal phenotypes, including metastable hybrid transition states. Epigenetic processes involving PRC2 and likely other mechanisms are altered during adaptation to unfavorable cell culture conditions. These alterations influence the transitions between states and contribute to the stabilization of cells within hybrid EMT states

Article Snippet: The EZH2 inhibitor GSK-126 (MedChemExpress) was dissolved in DMSO at 10 mM and used at a concentration of 10 μM.

Techniques: Transduction, CRISPR, Construct, Sequencing, Selection, Plasmid Preparation, Control, Variant Assay, Western Blot, Cell Culture

Journal: Scientific reports

Article Title: SIX1 aggravates the progression of spinal cord injury in mice by promoting M1 polarization of microglia.

doi: 10.1038/s41598-024-82121-3

Figure Lengend Snippet: Fig. 6. SIX1 enhances the transcription of VEGF-C in LPS-stimulated BV2 cells by downregulating EZH2. BV2 cells were divided into the normal, LPS, LPS + lenti-scrambled shRNA, LPS + lenti-SIX1 shRNA, and LPS + lenti-SIX1 shRNA + GSK126 groups. (A) qRT-PCR was performed to assess the mRNA expression of VEGF-C. (B) Western blotting was performed to assess the protein expression of VEGF-C. (C) VEGF-C protein levels relative to GAPDH protein levels. (D) SIX1-binding sites in the VEGF-C promoter. (E) Luciferase reporter assay was performed to detect the activity of the VEGF-C promoter. (F) CHIP was performed to detect the binding of SIX1 to the VEGF-C promoter. qPCR was performed with primers specific for the VEGF-C promoter (*P < 0.05 compared with the normal group; #P < 0.05 compared with the LPS group; %P < 0.05 compared with the LPS + SIX1 shRNA group; n = 4 per condition).

Article Snippet: The cells were treated with LPS (derived from Escherichia coli 0111:B4; L4391, Merck;100 ng/mL for 24 h), Cas9 plasmid (100091, Addgene, USA; 500 nM for 24 h), full-length EZH2-dCas9 (100086, Addgen; 500 nM for 24 h), lenti-scrambled shRNA, lenti-SIX1 shRNA, GSK126 (EZH2 inhibitor; 6790, Tocris, USA; dissolved in DMSO at the concentration of 10 mM; 20 μM for 24 h), rabbit IgG (02-6102, Thermo Fisher Scientific; 2 μg/mL for 24 h), 0.01% DMSO (C6164, Merck, USA), VEGF-C neutralizing antibody (GTX52393, GeneTex, USA; 2 μg/mL for 24 h), or MAZ51 (VEGFR3 inhibitor; S0765, Selleckchem, USA; 100 μM for 24 h).

Techniques: shRNA, Quantitative RT-PCR, Expressing, Western Blot, Binding Assay, Luciferase, Reporter Assay, Activity Assay