Journal: Investigative Ophthalmology & Visual Science

Article Title: Hepatocyte Growth Factor Confers Trabecular Meshwork Stem Cell Resilience and Paracrine Protection of Trabecular Meshwork Cells in Glaucoma

doi: 10.1167/iovs.67.4.1

Figure Lengend Snippet: TMSCs exhibit attenuated fibrotic and oxidative stress responses compared with TM cells under TGF-β2 stimulation, and NAC mitigates these effects. ( A , B ) MTT assays in TMSCs ( A ) and TM cells ( B ) used to determine the NAC concentration applied in subsequent experiments. ( C–E ) Immunofluorescent staining of CTGF ( C ), α-SMA ( D ), and FN ( E ) in TMSCs and TM cells after a 5-day treatment with control, TGF-β2 (3 ng/mL), or TGF-β2 combined with NAC (40 µM). ( F–H ) Quantification of fluorescence intensities for CTGF ( F ), α-SMA ( G ), and FN ( H ). ( I ) Western blotting analyses of CTGF, α-SMA, and FN expression in TMSCs and TM cells across the same treatment groups. ( J–L ) Densitometric quantification of CTGF ( J ), α-SMA ( K ), and FN ( L ) protein levels. ( M–O ) Measurements of total ROS levels ( M ), lipid peroxidation assessed by C11-BODIPY ( N ), and mitochondrial membrane potential measured by TMRM ( O ) in TMSCs and TM cells across treatment groups. Data are presented as mean ± SD ( n ≥ 3). Statistical analyses were conducted using two-way ANOVA, followed by Tukey's multiple comparisons test. * P < 0.05; ** P < 0.01, *** P < 0.001, **** P < 0.0001; ns, not significant.

Article Snippet: After blocking with 1% bovine serum albumin (MilliporeSigma) for 30 minutes, samples were incubated overnight at 4°C with primary antibodies against CHI3L1 (R&D Systems, 1:100), MYOC (a gift from Dr. Stamer, 1:300), OCT4 (Cell Signaling Technology, 1:200), Ki67 (Abcam, 1:200), 8F1-3 (a kind gift from Dr. Nirmala SundarRaj, University of Pittsburgh, 1:200), CTGF (Santa Cruz Biotechnology, 1:200), α-SMA (R&D Systems, 1:200), and FN (Abcam, 1:200).

Techniques: Concentration Assay, Staining, Control, Fluorescence, Western Blot, Expressing, Membrane

Journal: Investigative Ophthalmology & Visual Science

Article Title: Hepatocyte Growth Factor Confers Trabecular Meshwork Stem Cell Resilience and Paracrine Protection of Trabecular Meshwork Cells in Glaucoma

doi: 10.1167/iovs.67.4.1

Figure Lengend Snippet: HGF and TMSC-derived secretome alleviate TGF-β2–induced fibrotic and oxidative stress responses in TM cells. ( A , B ) MTT assays in TM cells assessing the effects of HGF ( A ) and HGFI ( B ) on cell viability. ( C ) Bright-field images showing TM cell morphology after 5-day treatment with control, TGF-β2 (3 ng/mL), HGF (10 ng/mL), HGFI (100 nM), TMSC-derived secretome (TMSC-Scr), or TMSC-Scr combined with HGFI. ( D ) Immunofluorescent staining of CTGF, α-SMA, and FN. ( E–G ) Quantification of fluorescence intensities for CTGF ( E ), α-SMA ( F ), and FN ( G ). ( H ) Western blotting analyses of CTGF, α-SMA, and FN expression under the same treatment conditions. ( I–K ) Densitometric quantification of CTGF ( I ), α-SMA ( J ), and FN ( K ) protein levels. ( L–N ) Oxidative stress analyses including total ROS levels ( L ), lipid peroxidation measured by C11-BODIPY ( M ), and mitochondrial membrane potential assessed by TMRM ( N ). Data are presented as mean ± SD ( n ≥ 3). Statistical analyses were performed using one-way ANOVA, followed by Tukey's multiple comparisons test. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001; ns, not significant.

Article Snippet: After blocking with 1% bovine serum albumin (MilliporeSigma) for 30 minutes, samples were incubated overnight at 4°C with primary antibodies against CHI3L1 (R&D Systems, 1:100), MYOC (a gift from Dr. Stamer, 1:300), OCT4 (Cell Signaling Technology, 1:200), Ki67 (Abcam, 1:200), 8F1-3 (a kind gift from Dr. Nirmala SundarRaj, University of Pittsburgh, 1:200), CTGF (Santa Cruz Biotechnology, 1:200), α-SMA (R&D Systems, 1:200), and FN (Abcam, 1:200).

Techniques: Derivative Assay, Control, Staining, Fluorescence, Western Blot, Expressing, Membrane

Journal: Investigative Ophthalmology & Visual Science

Article Title: Hepatocyte Growth Factor Confers Trabecular Meshwork Stem Cell Resilience and Paracrine Protection of Trabecular Meshwork Cells in Glaucoma

doi: 10.1167/iovs.67.4.1

Figure Lengend Snippet: Proposed mechanism by which TMSCs and TMSC-derived factors protect TM cells from TGF-β2–induced injury. This schematic summarizes the integrated model derived from our experimental findings. In TM cells, TGF-β2 activates oxidative stress pathways, leading to increased ROS production, lipid peroxidation, mitochondrial depolarization, and induction of profibrotic markers, including CTGF, α-SMA, and FN, ultimately promoting cytoskeletal remodeling and extracellular matrix accumulation, thereby increasing aqueous humor outflow resistance and elevating IOP. TMSCs exhibit intrinsic resistance to these deleterious pathways, maintaining low oxidative burden, preserved mitochondrial membrane potential, and attenuated fibrotic activation. The TMSC-Scr, enriched in HGF and additional trophic factors, reduces ROS and lipid peroxidation, stabilizes mitochondrial function, and suppresses α-SMA and FN expression while partially modulating CTGF.

Article Snippet: After blocking with 1% bovine serum albumin (MilliporeSigma) for 30 minutes, samples were incubated overnight at 4°C with primary antibodies against CHI3L1 (R&D Systems, 1:100), MYOC (a gift from Dr. Stamer, 1:300), OCT4 (Cell Signaling Technology, 1:200), Ki67 (Abcam, 1:200), 8F1-3 (a kind gift from Dr. Nirmala SundarRaj, University of Pittsburgh, 1:200), CTGF (Santa Cruz Biotechnology, 1:200), α-SMA (R&D Systems, 1:200), and FN (Abcam, 1:200).

Techniques: Derivative Assay, Membrane, Activation Assay, Expressing

Journal: The Journal of Biological Chemistry

Article Title: Ganglioside GM2 induces epithelial–mesenchymal transition (EMT) in cancer cells in a MEK/ERK/Egr1-dependent transcriptional program

doi: 10.1016/j.jbc.2026.111306

Figure Lengend Snippet: GM2-mediated ERK-target gene expression is Egr1-dependent. A , screening of Egr1 KO clones from an array of Egr1 sgRNA transfected clones (CRISPR-Cas9) in HeLa cells. The panel depicting Egr1 expression in clones 1, 2, 3, 4, 6, and 7 in comparison to the HeLa wt, is also shown along with an array of total 17 clones selected as depicted in E . B , sequencing data depict 1 bp insertion in Egr1 KO3 clone ( B , top panel) and 52 bp deletion in Egr1 KO6 clone ( B , lower panel) resulting in complete Egr1 KO clones generation. Inserted and deleted bases are marked, and the boxed portion shows guide RNA targeted DNA sequence. RT-PCR data show diminished expression of GM2-induced ( C ) Tgfβ1 , ( D ) Pai-1 and ( E ) Ctgf genes in Egr1 KO clones in the presence or absence of exogenous GM2. All mRNA quantification data are normalized against 7SL and represented as relative fold changes with respect to untreated control. Data represents mean ± SEM of at least two independent experiments [Student's t test, two-tailed; unpaired ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001]. F , western blot show GM2-induced Ctgf protein expression in Egr1 KO clones 3 and 6 in the presence of exogenous GM2. G , bar diagram shows densitometric quantification data of Egr1 and Ctgf protein. Band intensities are quantified using ImageJ software and normalized to GAPDH (loading control). Data represent mean ± SEM of at least two independent experiments [Student's t test (one-tailed; unpaired); ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ns = nonsignificant]. gRNA, guide RNA; wt, wild type; SFM, serum-free media, KO clone, knock out clone. ERK, extracellular signal-regulated kinase; Egr1, early growth response protein 1; Tgfβ1, transforming growth factor beta 1; Pai-1, plasminogen activator 1; CRISPR-Cas9, clustered regularly interspaced short palindromic repeats and CRISPR associated protein; RT-PCR, real-time PCR; Ctgf, connective tissue growth factor.

Article Snippet: FosB (#2251), Ctgf (#86641S), total ERK (#4695S), GAPDH (#97166) pMEK (#9154), N-cadherin (#14215), and Vimentin (#5741) were purchased from Cell Signaling Technology and Hsp90 (#BB-AB012), β-actin (#BB-AB0024) from BioBharati Life Science Pvt Ltd. Adam10 (#ab124695) primary antibody was purchased from Abcam.

Techniques: Targeted Gene Expression, Clone Assay, Transfection, CRISPR, Expressing, Comparison, Sequencing, Reverse Transcription Polymerase Chain Reaction, Control, Two Tailed Test, Western Blot, Software, One-tailed Test, Knock-Out, Real-time Polymerase Chain Reaction

Journal: The Journal of Biological Chemistry

Article Title: Ganglioside GM2 induces epithelial–mesenchymal transition (EMT) in cancer cells in a MEK/ERK/Egr1-dependent transcriptional program

doi: 10.1016/j.jbc.2026.111306

Figure Lengend Snippet: Schematic diagram of the role of MEK-ERK-Egr1 axis GM2-mediated EMT process. In summary, ganglioside GM2 interacts with membrane bound integrin receptor and induces MEK activation which in turn phosphorylates and activates ERK. Activated ERK then activates immediate early genes like Egr1 which further activates the transcription of its downstream gene like Ctgf that may play a critical role in GM2-mediated tumor cell migration, invasion and EMT process. The figure was made by BioRender. ERK, extracellular signal-regulated kinase; Egr1, early growth response protein 1; EMT, epithelial–mesenchymal transition; Ctgf, connective tissue growth factor.

Article Snippet: FosB (#2251), Ctgf (#86641S), total ERK (#4695S), GAPDH (#97166) pMEK (#9154), N-cadherin (#14215), and Vimentin (#5741) were purchased from Cell Signaling Technology and Hsp90 (#BB-AB012), β-actin (#BB-AB0024) from BioBharati Life Science Pvt Ltd. Adam10 (#ab124695) primary antibody was purchased from Abcam.

Techniques: Membrane, Activation Assay, Migration