Journal: Investigative Ophthalmology & Visual Science

Article Title: Targeted Activation of OGG1 Inhibits Paraptosis in Lens Epithelial Cells of Early Age-Related Cortical Cataract

doi: 10.1167/iovs.66.1.29

Figure Lengend Snippet: Activation of OGG1 inhibits ROS-induced LEC paraptosis. ( A ) Representative immunofluorescence images of indicated LECs against 8-oxoG. ( B ) Quantification and statistical analysis of 8-oxoG fluorescence intensity. ( C , D ) The Western blot images and statistical analysis display the relative protein expression levels of OGG1, normalized to β-actin, after treatment with 50 µM H 2 O 2 for 24 hours. ( E ) Representative confocal images of OGG1-mCherry LECs following treatment at the indicated time points after laser micro-irradiation sites pointed by white arrows . ( F ) Connecting lines of mean values representing OGG1-mCherry recruitment kinetics. ( G , H ) The Western blot images and statistical analysis display the relative protein expression levels of ALIX, GRP78, p-ERK, and p-P38, normalized to β-actin. ( I ) ER and mitochondria were labeled with ER-Tracker green and Mito-Tracker red, respectively, after LECs were treated as indicated. Red arrowheads indicate the dilated ER. ( K ) Quantification and statistical analysis of intracellular vesicles. ( J , L ) Representative images of ROS generation and statistical results of indicated LECs treated with 50 µM H 2 O 2 and 10 µM TH10785. Data are displayed as mean ± SEM from three independent experiments. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: TH10785 (MCE), an OGG1 activator, was used at 10 μM for 8 hours.

Techniques: Activation Assay, Immunofluorescence, Fluorescence, Western Blot, Expressing, Irradiation, Labeling

Journal: Investigative Ophthalmology & Visual Science

Article Title: Targeted Activation of OGG1 Inhibits Paraptosis in Lens Epithelial Cells of Early Age-Related Cortical Cataract

doi: 10.1167/iovs.66.1.29

Figure Lengend Snippet: Schematic illustrating the mechanism of ROS-induced paraptosis in LECs of early ARCC patients. This comprehensive mechanism reveals that the elevated ROS orchestrates a cascade of cellular events, including ER stress induction, IGFIR activation, and subsequent mitogen-activated protein kinase pathway signaling. These molecular events result in ER dilation-formed intracellular vesicles and, ultimately, paraptosis in LECs. Notably, TH10785-mediated enhancement of OGG1 activity effectively suppresses ROS-induced paraptosis and vacuolar degeneration in the superficial lens cortex. AC, anterior chamber; C, cornea; CB, ciliary body; I, iris; N, nucleus; PC, posterior chamber; V, vacuoles.

Article Snippet: TH10785 (MCE), an OGG1 activator, was used at 10 μM for 8 hours.

Techniques: Activation Assay, Activity Assay

Journal: Journal of Biological Chemistry

Article Title: Myostatin Induces DNA Damage in Skeletal Muscle of Streptozotocin-induced Type 1 Diabetic Mice

doi: 10.1074/jbc.m113.483115

Figure Lengend Snippet: FIGURE 3. Absence of Mstn abrogated STZ-induced changes in p63 and REDD1 signaling. A, Western blot analysis (i) and densitometric analysis (ii) of p-Akt1/2/3,Akt1/2/3,IRS-1andIGF-1inWT-C(lane1),WT-STZ(lane2),Mstn/-C(lane3)andMstn/-STZ(lane4)Gastrocnemiusmuscleproteinlysates.*,p 0.05, **, p 0.01 when compared with WT-C; ˆˆ, p 0.01 when compared with Mstn/-C. GAPDH was used as an internal control for equal protein loading on the gel (n 7). B, Western blot analysis (i) and densitometric analysis (ii) of REDD1, OGG1, p53, and p63 protein levels in WT-C (lane 1), WT-STZ (lane 2), Mstn/-C (lane 3), and Mstn/-STZ (lane 4) Gastrocnemius muscle. GAPDH was used as an internal control for equal protein loading on the gel (n 5). *, p 0.05, ***, p 0.001 when compared with WT-C. Immunohistochemistry for REDD1 (C) and OGG1 (D) was performed on cryosections of Tibialis anterior muscles from WT-C and WT-STZ (i) and Mstn/-C and Mstn/-STZ (ii) mice. The fluorescence was viewed under a Leica upright microscope and images were taken at 5 magnification. Increased or decreased fluorescence (green) indicates changes in expression of REDD1 (C) or OGG1 (D); scale bar represents 100 m (n 3). E, ROS production was measured in proliferating C2C12 myoblasts treated with STZ1 for 48 h in Permanox chamber slides using the CM-H2DCFDA fluorescent probe. The fluorescence was viewed under a Leica upright microscope and images were taken at 10 magnification. Increased fluorescence (green) intensity is directly proportional to increased ROS production; scale bar represents 100 m (n 2).

Article Snippet: The immunohistochemistry protocol for REDD1 or OGG1 primary antibodies was followed as per manufacturer’s instructions (Proteintech).

Techniques: Western Blot, Control, Immunohistochemistry, Muscles, Fluorescence, Microscopy, Expressing

Journal: Journal of Biological Chemistry

Article Title: Myostatin Induces DNA Damage in Skeletal Muscle of Streptozotocin-induced Type 1 Diabetic Mice

doi: 10.1074/jbc.m113.483115

Figure Lengend Snippet: FIGURE 4. STZ-induced Mstn signaling in vitro via Foxa2 leads to oxidative stress-induced DNA damage, which was attenuated by Ant1. A, represent- ative Western blot (i) and densitometric analysis (ii) showing Foxa2 levels in protein lysates obtained from proliferating C2C12 cells treated with STZ1 for 48 h (lane 1-Untreated; lane 2-STZ1 treated). GAPDH was used as an internal control for equal protein loading on the gel (*, p 0.05; n 3). B, (i) Western blot and (ii) densitometric analysis showing protein levels of Mstn, p-Smad2/3, Smad2/3, p-Akt1/2/3, and Akt1/2/3 in proliferating C2C12 cells untreated (lane 1), treated with STZ1 (lane 2) or STZ2 (lane 3) or Ant1 (lane 4) for 48 h, pretreated with Ant1 for 1 h followed by either STZ1 (lane 5) or STZ2 (lane 6) treatment for 48 h. GAPDH was used as an internal control for equal protein loading on the gel (n 3) (*, p 0.05, **, p 0.01 when compared with untreated cells; ˆ, p 0.05 when compared with Ant1-treated cells). Western blot analysis (i) and densitometric analysis (ii) of p63, REDD1, and OGG1 in protein lysates obtained from proliferating C2C12 cells transfected with either (C) p-FLAG-CMV2 or p-FLAG-Foxa2 or (D) scrambled -ve control siRNA or Foxa2-siRNA. (Lanes 1 and 2-untreated; lanes 3 and 4-STZ1 treated; lanes 5 and 6-STZ2 treated). GAPDH was used as an internal control for equal protein loading on the gel (*, p 0.05). (n 3).

Article Snippet: The immunohistochemistry protocol for REDD1 or OGG1 primary antibodies was followed as per manufacturer’s instructions (Proteintech).

Techniques: In Vitro, Western Blot, Control, Transfection