Journal: Investigative Ophthalmology & Visual Science

Article Title: Ly93 Inhibits Sphingomyelin Synthesis and Attenuates Inflammation and Injury in Dry Eye Conjunctival Organoids

doi: 10.1167/iovs.67.2.58

Figure Lengend Snippet: Hyperosmolarity induced DED-associated injury and inflammation of conjunctival organoids. ( A ) Representative images showing hyperosmolarity-induced organoid disintegration ( scale bar , 100 µm). ( B ) Quantification of the percentage of intact organoids. ( C ) Western blotting analysis of proliferation markers (p63 and Ki67), inflammatory mediators (MMP-9 and IL-1β), signaling molecules (NF-κB p65, STAT1, and PI3K), ferroptosis markers (TFR and GPX4), and apoptosis marker (caspase-3). ( D – F ) Immunofluorescence staining of p63 + and Ki67 + cells in organoids (scale bar , 50 µm). ( G ) Immunofluorescence staining of caspase-9, NF-κB p65, STAT1, and IL-1β in organoids ( scale bar , 50 µm). * P < 0.05; *** P < 0.001; **** P < 0.0001; ns, not significant.

Article Snippet: Immunofluorescence staining was performed to evaluate the expression of K19 (1:200, Cell Signaling Technology, Danvers, MA, USA), K13 (1:200, Cell Signaling Technology), IL-1β (1:200, Abcam, Cambridge, UK), STAT1 (1:200, Proteintech, Rosemont, IL, USA), p63 (1:250, Abcam), Ki67 (1:200, Abcam), NF-κB p65 (1:200, Abcam), MUC1 (1:200, Abcam), MUC5AC(1:250, Abcam), caspase-9 (1:200, Cell Signaling Technology), ZO-1 (1:200, Abcam), and 4-hydroxynonenal (4-HNE) (1:100, Invitrogen, Carlsbad, CA, USA) in frozen sections of the conjunctival organoids.

Techniques: Western Blot, Marker, Immunofluorescence, Staining

Journal: Investigative Ophthalmology & Visual Science

Article Title: Ly93 Inhibits Sphingomyelin Synthesis and Attenuates Inflammation and Injury in Dry Eye Conjunctival Organoids

doi: 10.1167/iovs.67.2.58

Figure Lengend Snippet: Exogenous SM induced inflammation and injury of conjunctival organoids. ( A ) Effects of SM treatment on the viability of conjunctival cells and morphology of organoids ( scale bar , 100 µm for cells and 200 µm for organoids). ( B , C ) Western blotting analysis of inflammatory markers (phospho-STAT1, STAT1, IL-1β, and iNOS) in SM-treated organoids. ( D – F ) Relative mRNA expression of IL-1β, IL-6, and STAT1 after SM treatment. ( G ) Immunofluorescence staining of K13, K19, Ki67, MUC1, and MUC5AC in SM-treated organoids ( scale bar , 50 µm). ( H – L ) Comparative analysis of immunofluorescent results: K13 ( H ), K19 ( I ), Ki67 ( J ), MUC1 ( K ), and MUC5AC ( L ). * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001; ns, not significant.

Article Snippet: Immunofluorescence staining was performed to evaluate the expression of K19 (1:200, Cell Signaling Technology, Danvers, MA, USA), K13 (1:200, Cell Signaling Technology), IL-1β (1:200, Abcam, Cambridge, UK), STAT1 (1:200, Proteintech, Rosemont, IL, USA), p63 (1:250, Abcam), Ki67 (1:200, Abcam), NF-κB p65 (1:200, Abcam), MUC1 (1:200, Abcam), MUC5AC(1:250, Abcam), caspase-9 (1:200, Cell Signaling Technology), ZO-1 (1:200, Abcam), and 4-hydroxynonenal (4-HNE) (1:100, Invitrogen, Carlsbad, CA, USA) in frozen sections of the conjunctival organoids.

Techniques: Western Blot, Expressing, Immunofluorescence, Staining

Journal: Investigative Ophthalmology & Visual Science

Article Title: Ly93 Inhibits Sphingomyelin Synthesis and Attenuates Inflammation and Injury in Dry Eye Conjunctival Organoids

doi: 10.1167/iovs.67.2.58

Figure Lengend Snippet: Ly93 attenuated DED-associated inflammation in conjunctival organoids. ( A ) Immunofluorescence staining of caspase-9, ZO-1, IL-1β, STAT1, and NF-κB p65 between vehicle control, DED, and DED + Ly93 groups ( scale bar , 50 µm). ( B ) Intracellular Fe 2+ analysis ( scale bar , 50 µm). ( C ) Immunofluorescence staining of 4-HNE ( scale bar , 50 µm). ( D – H ) Western blotting analysis of GPX4 ( D ), TFR ( E ), AKT pathway ( F ), caspase-9 and NF-κB p65 pathway ( G ), and IL-1β and STAT1 pathway ( H ) protein expression.

Article Snippet: Immunofluorescence staining was performed to evaluate the expression of K19 (1:200, Cell Signaling Technology, Danvers, MA, USA), K13 (1:200, Cell Signaling Technology), IL-1β (1:200, Abcam, Cambridge, UK), STAT1 (1:200, Proteintech, Rosemont, IL, USA), p63 (1:250, Abcam), Ki67 (1:200, Abcam), NF-κB p65 (1:200, Abcam), MUC1 (1:200, Abcam), MUC5AC(1:250, Abcam), caspase-9 (1:200, Cell Signaling Technology), ZO-1 (1:200, Abcam), and 4-hydroxynonenal (4-HNE) (1:100, Invitrogen, Carlsbad, CA, USA) in frozen sections of the conjunctival organoids.

Techniques: Immunofluorescence, Staining, Control, Western Blot, Expressing

Journal: Aging Cell

Article Title: Rapamycin Reverses the Hepatic Response to Diet‐Induced Metabolic Stress That Is Amplified by Aging

doi: 10.1111/acel.70395

Figure Lengend Snippet: Rapamycin co‐treatment mitigates proinflammatory transcriptional hyperactivation in aged mice exposed to HFD. (A) Protocol: Mice received ND or eRapamycin‐containing diet from 4 to 18 months, followed by 9 weeks on HFD or ND. (B) Western blot of liver tissue. (C) Fold change of p‐S6/S6 compared to control diet. (D) PCA of RNA‐seq from isolated hepatocytes. (E) Number of DEGs across treatment groups. (F) Scatter plot comparing HFD‐induced gene expression changes (HFD + veh vs. ND + veh) to all gene expression changes with eRapamycin (eRapa+HFD vs. HFD + veh). (G) Heatmap of genes upregulated by HFD + vehicle treatment. (H) Venn diagram showing overlap between transcripts elevated by HFD versus ND and those decreased by rapamycin. (I) KEGG enrichment analysis of overlapping genes. (J) Heatmap of innate immune response genes. (K) Top transcription factor pathways predicted by IPA in the overlapping gene set. (L) Heatmap of Stat1 target genes. (M) Tumorigenic index score calculated per mouse. Statistical analyses for p‐S6/S6 ratios and tumorigenic index scores were performed using one‐way ANOVA with post hoc Tukey's test; p < 0.05 (*), p < 0.1 (**).

Article Snippet: Antibodies used: total Stat1 (Cell Signaling Technologies 9172S), S6 Ribosomal protein (Cell Signaling Technologies 2317), phospho‐S6 Ribosomal protein (Cell Signaling Technologies 5364).

Techniques: Western Blot, Control, RNA Sequencing, Isolation, Gene Expression