Journal: Protein & Cell

Article Title: Single-cell transcriptomic Atlas of aging macaque ocular outflow tissues

doi: 10.1093/procel/pwad067

Figure Lengend Snippet: Aging increases the expression level of APOE, CLU, and VEGFA in TMCs. (A) Morphologic changes of trabecular meshwork cells (TMCs) after senescence induction. (B and C) SA-β-gal staining exhibits the increase of aging cells with H 2 O 2 . (D) QRT–PCR was used to detect the mRNA level of three DEGs. (E) Immunostaining of p21 and SA-β-gal in aging TMCs. Scale bars: 20 μm (left). (F) Western blot was performed to detect the protein levels of three DEGs, senescence markers, and β-actin. (G) Quantification of the relative expression level of proteins in (F). (H) Immunostaining of APOE , CLU, and VEGFA in normal and aging TMCs. Scale bars: 20 μm. (I) Quantification of relative fluorescence intensity in (E and H). (J) Flow cytometry was used to detect cell cycle of TMCs with different treatments. (K) Proportion of each cell cycle phase in TMCs at different stage. Data are presented as mean ± SEM values; * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001; n = 3 ( n = 5 for immunofluorescence).

Article Snippet: Primary human trabecular meshwork cells (TMCs), isolated from the juxtacanalicular and corneoscleral regions of human eye, were obtained from the Sciencell corporation (ScienCell, Carlsbad, CA, USA).

Techniques: Expressing, Staining, Quantitative RT-PCR, Immunostaining, Western Blot, Fluorescence, Flow Cytometry, Immunofluorescence

Journal: Protein & Cell

Article Title: Single-cell transcriptomic Atlas of aging macaque ocular outflow tissues

doi: 10.1093/procel/pwad067

Figure Lengend Snippet: Knockdown of APOE partially rescues TMCs degeneration with aging. Functional enrichment analyses in biological process, cellular component, and molecular function of cluster 0. Schematic diagram illustrating possible mechanisms of APOE in trabecular meshwork degeneration with aging. TMCs were transfected with 50 nmol/L APOE siRNA or negative control (NC) siRNA for 24 h, followed by stimulation with H 2 O 2 for 2 h and replacing fresh medium for more than 24 h. Transwell assay was used to investigate the migration ability of TMCs. (D) Quantification of cell migration in (C). (E) Western blot was performed to detect the protein levels of extracellular matrix (ECM) components including fibronectin, laminin, CD44, and α-SMA under different cell treatments. (F) Quantification of relative protein levels in (E). (G) Western blot was performed to detect the key molecular levels of PI3K-AKT pathway and the downstream caspase 3/9 under different cell treatments. (H) Quantification of relative protein levels in (G). (I) The apoptosis rate of TMCs in four groups was measured by flow cytometry. (J) Quantification of apoptotic proportion in all cells from (I). Data are presented as mean ± SEM values; * P < 0.05, ** P < 0.01, *** P < 0.001; n = 3. ns: no significance.

Article Snippet: Primary human trabecular meshwork cells (TMCs), isolated from the juxtacanalicular and corneoscleral regions of human eye, were obtained from the Sciencell corporation (ScienCell, Carlsbad, CA, USA).

Techniques: Knockdown, Functional Assay, Transfection, Negative Control, Transwell Assay, Migration, Western Blot, Flow Cytometry