go 6976  (MedChemExpress)

Journal: Investigative ophthalmology & visual science

Article Title: Mechanisms Regulating Mitochondrial Transfer in Human Corneal Epithelial Cells.

doi: 10.1167/iovs.65.13.10

Figure Lengend Snippet: FIGURE 7. Inhibition of phagocytosis has no impact on mitochondrial content but does reduce cell adhesion and increases mitochondrial transfer. (A) Incubating HCLE cells in Gö6976 (G0) does not reduce mitochondrial content assessed using CMXROS. (B) Inhibition of phagocytosis using Gö6976 (G0) reduces phagocytosis in HCLE cells. (C) Pharmacological inhibition of phagocytosis using G0 reduces HCLE cell adhesion. (D) Gö6976 treatment of donor cells increase their transfer of mitochondria to both control and Gö6976-treated recipient cells. (A, B, D) n = 600 values per variable assessed; (C) n = 6 values per variable assessed. Experiments were repeated two or three times. Data were analyzed for statistical significance using Graphpad Prism. **P < 0.01; ***P < 0.001; ****P < 0.0001. ns above a graph indicates P ≥0.05.

Article Snippet: For phagocytosis inhibitor studies, Gö6976 (#2253; Tocris Bioscience, Bristol, UK) was used at a concentration of 10 μM (stock made in DMSO).

Techniques: Inhibition, Control

Journal: Science (New York, N.Y.)

Article Title: Vitamin A resolves lineage plasticity to orchestrate stem cell lineage choices

doi: 10.1126/science.adi7342

Figure Lengend Snippet: ( A ) Cultured HFSCs were stably transduced with a lentiviral Klf5-EGFP reporter (left). Flow cytometry quantification (center) showed that serum replacement reduced Klf5 reporter expression in 3D cultures. Colony diameter plateaued over time in serum replacement media, in contrast to unrestrained outgrowth in serum-replete media (right) (n=5 technical replicates across three purified HFSC lines). ( B ) Quantification of Klf5-EGFP + cells after screening a library of small molecules implicated in wound repair identified several small molecule ‘hits’ (n=5). The dashed line indicates baseline EGFP levels in B-27 media with vehicle-only controls. The significant hits that repressed Klf5-EGFP were: atRA, the PKCa/b inhibitor Gö6976, and FR180204, a competitive inhibitor of ERK1/2. ( C and D ) Immunofluorescence (C) and immunoblot (D) analyses show that serum replacement and treatment with atRA and PKCi (n=6) resolves lineage plasticity, as judged by elevated HFSC master regulator SOX9 and silenced EpSC transcriptional regulator KLF5. Keratin profiles reflect the loss of epidermal differentiation and wound-induced suprabasal markers (KRT10, KRT6), uniform expression of pan-skin progenitor marker KRT14, and gain of KRT15, a HFSC marker. The epidermal-dermal interface of sagittal skin section is delineated by a dashed line. epi, epidermis; sg, sweat gland; dp, dermal papilla. Scale bar, 20μm. ( E ) UMAP representation and unsupervised k-nearest-neighbor-based clustering of single cell transcriptomes from 3,757 cultured HFSCs reveal that RA-PKCi co-treatment groups cells in near-uniform clusters distinct from wound-like and epidermal cell types (left, center). Annotated cluster assignments made using gene signatures derived from independent bulk sequencing of in vivo HFSCs, EpSCs, and lineage traced HFSCs isolated at intermediate stages of wound repair confirmed that atRA+PKCi-treated cells had a strong HFSC signature and reduced EpSC and wound signatures (right).

Article Snippet: To resolve lineage plasticity, serum-replete culture media was washed with sterile PBS and changed to Dulbecco’s Modified Eagle Media/Ham’s F12 media (DMEM/F12) (3:1) containing 1x B-27 supplement (1:50; Gibco) , 125 mg/l cholesterol-cyclodextrin (Sigma), 300 μM calcium chloride, 10 μM Y-27632, 100 nM atRA (Sigma), and 1 μM Go6976 (PKCi; R&D Systems).

Techniques: Cell Culture, Stable Transfection, Transduction, Flow Cytometry, Expressing, Purification, Immunofluorescence, Western Blot, Marker, Derivative Assay, Sequencing, In Vivo, Isolation