mcherry egfp lc3b sequences  (Addgene inc)

Journal: Autophagy

Article Title: Treatments targeting autophagy ameliorate the age-related macular degeneration phenotype in mice lacking APOE (apolipoprotein E)

doi: 10.1080/15548627.2022.2034131

Figure Lengend Snippet: Autophagy-Lysosomal pathways are impaired in the RPE and retina of APOE-mice and treatment with trehalose and metformin ameliorate aspects of this deficit. Autophagy–lysosomal pathways in RPE and photoreceptors were investigated using transverse sections labeled for autophagosomes with an LC3 antibody (red) and lysosomes with a LAMP1 antibody (green). (A-D) Representative images of RPE from (A) WT-control mice, with magnified view of (Ai) LC3-puncta, (Aii) LAMP1-puncta and (Aiii) colocalization (white arrowheads); (B) APOE-control mice; and APOE mice treated with (C) trehalose and (D) metformin are shown. (E) The number of LC3-puncta in the RPE was reduced in APOE-control mice relative to WT-control mice. This loss of LC3-puncta was not apparent in APOE-mice treated with trehalose or metformin relative to their treated WT counterparts. (F) LAMP1-puncta were also reduced in the RPE of APOE-control relative to WT-control mice and this was not altered by treatment with trehalose or metformin. (G) Both trehalose and metformin were found to enhance the number of colocalized LC3- and LAMP1-puncta, suggesting active autophagy was increased in the RPE of both WT and APOE mice relative to their genetic controls. (H-K) Representative images of the photoreceptor nuclei layer in the retina from (H) WT-control mice, (I) APOE-control mice, and APOE mice treated with (J) trehalose and (K) metformin are shown. (L) The number of LC3-puncta in the photoreceptors were reduced in APOE-control mice relative to WT-control mice but not in APOE-mice treated with trehalose or metformin. (M) LAMP1-puncta number and (N) colocalized LC3- and LAMP1-puncta were not altered by either genotype or treatment. For all groups n = >6; Two way ANOVA with post-hoc significance of p < 0.05 shown for genotype (*) and treatment (#). A-D, scale: 5 µm; Ai-Aiii, scale: 5 µm; H-K, scale: 10 µm; RPE, retinal pigment epithelium; ONL, outer nuclear layer.

Article Snippet: To label autophagosomes, a polyclonal rabbit antibody against MAP1LC3B/LC3 generated against a synthetic peptide to the N-terminal portion of the human LC3 protein sequence was used (1:300; Novus Biologicals, NB100-2220).

Techniques: Labeling, Control

Journal: Autophagy

Article Title: Treatments targeting autophagy ameliorate the age-related macular degeneration phenotype in mice lacking APOE (apolipoprotein E)

doi: 10.1080/15548627.2022.2034131

Figure Lengend Snippet: Analysis of LC3 expression shows slowing of autophagy in the RPE and retina of APOE-mice, which is enhanced by treatment with trehalose and metformin. LC3 expression was assessed using simple western and the ratio of LC3-II:LC3-I used as a measure of autophagy. At 5 months of age, WT- and APOE-mice were treated with metformin (0.4 g/kg/day) or trehalose (3 g/kg/day) provided in the drinking water, while sham controls received standard drinking water. At 13 months of age, (A-B) RPE and (C-D) retina samples were analyzed for LC3B. GAPDH was used as a protein loading control. Representative lane results for (A) RPE and (C) retina are presented: MW, molecular weight marker; 1, WT-control; 2, APOE-control; 3, WT-trehalose; 4, APOE-trehalose; 5, WT-metformin; and 6, APOE-metformin. In both (B) RPE and (D) retina, the LC3-II:LC3-I ratio was higher in APOE-mice suggesting a slowing of autophagy. In APOE animals treated with trehalose or metformin the ratio of LC3-II:LC3-I was restored to WT levels. (E-H) To assess autophagy flux, experiments to block autophagy-lysosomal degradation were completed using chloroquine. At 5 months of age, WT- and APOE-mice were treated for 2 weeks with metformin or trehalose and then retinae and RPE/choroid/sclera complex were incubated in culture media with or without 50 µM chloroquine for 24 h. Example results for E) RPE and (G) retina from WT and APOE mice are presented: Lanes 1, WT-control; 2, WT-control + chloroquine, 3, APOE-control; 4, APOE-control + chloroquine; 5, APOE-trehalose; 6, APOE-trehalose + chloroquine; 7, APOE-metformin; 8, APOE-metformin + chloroquine. The LC3-II:LC3-I ratio in (F) RPE and (H) retinal samples increased significantly in all samples treated with chloroquine to block autophagy-lysosomal function. For all groups, n = 5; Two way ANOVA with post-hoc significance of p < 0.05 (*).

Article Snippet: To label autophagosomes, a polyclonal rabbit antibody against MAP1LC3B/LC3 generated against a synthetic peptide to the N-terminal portion of the human LC3 protein sequence was used (1:300; Novus Biologicals, NB100-2220).

Techniques: Expressing, Simple Western, Control, Molecular Weight, Marker, Blocking Assay, Incubation