Journal: Investigative Ophthalmology & Visual Science

Article Title: Mitochondrial ROS Induced Lysosomal Dysfunction and Autophagy Impairment in an Animal Model of Congenital Hereditary Endothelial Dystrophy

doi: 10.1167/iovs.62.12.15

Figure Lengend Snippet: Autophagy flux is aberrant in Slc4a11 KO corneal endothelial cells. ( A ) In basal autophagy, autophagosome formation occurs followed by fusion with lysosomes and the degradation of substrates. In BafilomycinA1 (BafA1) treated cells (1), the lysosomal proton pump (v-ATPase), which is responsible for its acidic environment is inactivated. This leads to the accumulation of non-degraded autophagy substrates in the cell (2), which further increases the levels of proteins involved in autophagosome (3), and phagophore formation (4), which eventually increases pmTOR/mTOR ratio (5). ( B ) Wes immunoassay for p-mTOR (Ser 2448), mTOR, Atg5, P62 in Slc4a11 WT and KO MCEC (± 50 nM BafA1). Western Blot of the same lysates were probed for LC3b and β-actin. ( C ) Quantification of panel B data, mean ± SD. *** P < 0.001, ** P < 0.01, * P < 0.05, ns = not significant (1-way ANOVA with Tukey's multiple comparisons test). ( D ) Immunofluorescence of corneal endothelial tissue from Slc4a11 WT and KO mice for LC3b ( red ) counterstained with DAPI ( blue ). Scale bar – 5 µm. ( E ) Quantification of LC3b puncta from panel D data, mean ± SD, *** P < 0.001 (Student's t -test). ( F ) Wes immunoassay of mTOR, pmTOR (Ser 2448) and P62 from Slc4a11 WT and KO corneal endothelial tissues. ( G ) Quantification of data from panel F , mean ± SD. *** P < 0.001 (Student's t -test, n = 3).

Article Snippet: Immortalized cell lines and mouse corneal endothelia were used to measure autophagy and lysosome associated protein expressions using Protein Simple Wes immunoassay.

Techniques: Western Blot, Immunofluorescence

Journal: Investigative Ophthalmology & Visual Science

Article Title: Mitochondrial ROS Induced Lysosomal Dysfunction and Autophagy Impairment in an Animal Model of Congenital Hereditary Endothelial Dystrophy

doi: 10.1167/iovs.62.12.15

Figure Lengend Snippet: Lysosomal dysfunction is evident in Slc4a11 KO corneal endothelial cells. ( A ) Wes immunoassay of v-ATPase, and Cathepsin B using Slc4a11 WT and KO MCEC whole cell lysates (± 50 nM BafA1). ( B ) Quantification of data from panel A , n = 3, mean ± SD. ** P < 0.01, *** P < 0.001, **** P < 0.0001, ns = not significant (1-way ANOVA with Tukey's multiple comparisons test). ( C ) Wes immunoassay of v-ATPase, and Cathepsin B from corneal endothelium of Slc4a11 WT and KO mice. ( D ) Quantification of panel C data, n = 3, mean ± SD. *** P < 0.0001, ** P < 0.01 (Student's t -test). ( E ) Magic Red assessment of Cathepsin L activity in Slc4a11 WT and KO corneal endothelial tissue. Scale bar – 50 µm. ( F ) Quantification of Magic Red fluorescence, n = 5, mean ± SD. **** P < 0.0001 (Student's t -test).

Article Snippet: Immortalized cell lines and mouse corneal endothelia were used to measure autophagy and lysosome associated protein expressions using Protein Simple Wes immunoassay.

Techniques: Activity Assay, Fluorescence

Journal: Investigative Ophthalmology & Visual Science

Article Title: Mitochondrial ROS Induced Lysosomal Dysfunction and Autophagy Impairment in an Animal Model of Congenital Hereditary Endothelial Dystrophy

doi: 10.1167/iovs.62.12.15

Figure Lengend Snippet: Deficient TFEB nuclear translocation in Slc4a11 KO cells. ( A ) Wes immunoassay of TFEB, Lamin A/C, and GAPDH in nuclear and cytosolic extracts from Slc4a11 WT and KO MCEC. Nuclear membrane proteins, Lamin A/C, and cytoplasmic protein, Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were used as loading control for the respective fractions and also to rule out cross-contamination between the fractions. ( B ) Quantification of Wes results from panel A . Mean ± SD. *** P < 0.001. ns = not significant (Student's t -test). ( C ) Q-PCR of a subset of TFEB regulated CLEAR network genes in Slc4a11 WT and KO MCEC. Blocs1 – Biogenesis of Lysosomal Organelles Complex 1 Subunit 1, Gabarap - GABAA receptor-associated protein 1, Atpv0b – vATPase subunit b, Atpv0c – vATPase subunit C, Gla –Alpha galactosidase A, Ctsd – Cathepsin D, Ctsb- Cathepsin B, and Lamp1- Lysosome-associated membrane glycoprotein 1. Expression normalized to β-actin, * P < 0.05, *** P < 0.001 (Student's t -test). ( D ) Wes immunoassay of TFEB from Slc4a11 WT and KO corneal endothelial tissue. ( E ) Quantification of panel D data, mean ± SD, *** P < 0.0001, n = 3 (Student's t -test).

Article Snippet: Immortalized cell lines and mouse corneal endothelia were used to measure autophagy and lysosome associated protein expressions using Protein Simple Wes immunoassay.

Techniques: Translocation Assay, Membrane, Control, Expressing

Journal: Investigative Ophthalmology & Visual Science

Article Title: Mitochondrial ROS Induced Lysosomal Dysfunction and Autophagy Impairment in an Animal Model of Congenital Hereditary Endothelial Dystrophy

doi: 10.1167/iovs.62.12.15

Figure Lengend Snippet: MitoQ improves autophagy Slc4a11 KO cells. ( A ) Wes immunoassay of pmTOR, mTOR, and P62 from Slc4a11 WT and KO MCEC ± MitoQ. ( B ) Quantification of panel A data, n = 3. ( C ) Western Blots of LC3b and β-actin in Slc4a11 WT and KO MCEC ± MitoQ. ( D ) Quantification of panel C data, n = 3. ( E , F ) Detection of GFP and RFP fluorescence ratio in Slc4a11 WT, KO (± MitoQ) MCEC transfected with GFP-LC3-RFP-LC3ΔG and Quantification. Scale bar – 10 µm, n = 7. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, mean ± SD (1-way ANOVA with Tukey's multiple comparisons test).

Article Snippet: Immortalized cell lines and mouse corneal endothelia were used to measure autophagy and lysosome associated protein expressions using Protein Simple Wes immunoassay.

Techniques: Western Blot, Fluorescence, Transfection

Journal: Investigative Ophthalmology & Visual Science

Article Title: Mitochondrial ROS Induced Lysosomal Dysfunction and Autophagy Impairment in an Animal Model of Congenital Hereditary Endothelial Dystrophy

doi: 10.1167/iovs.62.12.15

Figure Lengend Snippet: MitoQ improves lysosomal function in MCEC KO cells. ( A ) Wes immunoassay of v-ATPase from WT and KO Slc4a11 MCEC ± MitoQ treatment. ( B ) Quantification of panel A data, n = 3. ( C ) Wes immunoassay of TFEB, Lamin A/C, and GAPDH from Slc4a11 WT and KO MCEC cytosolic and nuclear fractions. ( D ) Electropherograms of Wes immunoassay from panel C showing the size and intensities of the bands in C . ( E ) Quantification of panel C data, n = 3. ( F ) Comparison of lysosome numbers (Lysotracker Red-DND 99) in Slc4a11 WT and KO MCEC, scale bar – 10 µm. ( G ) Quantification of panel F data, n = 7. ( H ) Comparison of lysosomal pH (Lysosensor Green -DND 189) in Slc4a11 WT and KO ± MitoQ, Scale bar – 10 µm. ( I ) Quantification of panel H data, n = 7. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, mean ± SD (1-way ANOVA with Tukey's multiple comparisons).

Article Snippet: Immortalized cell lines and mouse corneal endothelia were used to measure autophagy and lysosome associated protein expressions using Protein Simple Wes immunoassay.

Techniques: Comparison

Journal: Investigative Ophthalmology & Visual Science

Article Title: Mitochondrial ROS Induced Lysosomal Dysfunction and Autophagy Impairment in an Animal Model of Congenital Hereditary Endothelial Dystrophy

doi: 10.1167/iovs.62.12.15

Figure Lengend Snippet: MitoQ in vivo reduces mitoROS, decreases cell loss, and improves lysosome protein expression in CHED mice. Intra-peritoneal injections of MitoQ (68 µg) on alternate days starting at 8 weeks of age for 4 weeks. ( A ) MitoSox fluorescence intensity in corneal endothelium of WT, KO, KO + MitoQ animals, n = 3. Two females and one male mice for each group was used for this assessment. ( B ) Corneal endothelial density, n = 3, two female mice and one male mouse from each group. ( C ) Wes immunoassay of P62, Cathepsin B, and TFEB from Slc4a11 WT and KO animals, two female mice of each group, repeated three times ( n = 2 animals per group for each of the 3 trials). ( D ) Quantification of panel C data, mean ± SD, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 (1-way ANOVA with Tukey's multiple comparisons test).

Article Snippet: Immortalized cell lines and mouse corneal endothelia were used to measure autophagy and lysosome associated protein expressions using Protein Simple Wes immunoassay.

Techniques: In Vivo, Expressing, Fluorescence