Journal: Scientific Reports

Article Title: Autophagy–lysosome pathway alterations and alpha-synuclein up-regulation in the subtype of neuronal ceroid lipofuscinosis, CLN5 disease

doi: 10.1038/s41598-018-36379-z

Figure Lengend Snippet: Autophagy is enhanced in CLN5-deficient cells. ( A ) Total lysates of two healthy control fibroblasts (C1 and C2) and two CLN5 disease patient fibroblasts (P1 and P2) were analyzed by immunoblotting. Basal levels of autophagic markers LC3-II and P62 were shown. The CLN5 was absent in patient cells. ( B ) Total lysates of WT and stable CLN5 KD HeLa cells were analyzed by immunoblotting. The CLN5 was greatly reduced in CLN5 KD cells. Basal levels of autophagic markers LC3-II and P62 were shown. ( C ) WT and Stable CLN5 KD HeLa cells were treated with CQ, HBSS, or HBSS + CQ for 4 h. Samples were analyzed by immunoblotting. The relative amounts of LC3-II and P62 after normalization with GAPDH are indicated. ( D ) WT and Stable CLN5 KD HeLa cells were incubated with HBSS for 0, 0.5, 1, 1.5 h. Samples were analyzed by immunoblotting. The relative amounts of LC3-II and P62 after normalization with GAPDH are indicated. ( E ) WT and Stable CLN5 KD HeLa cells were incubated with HBSS for 0, 2, 4, 8 h in the presence of cycloheximide and bortezomib. Samples were analyzed by immunoblotting. For degradation quantification on the right (N = 3), P62 was normalized with GAPDH signal in each lane. 0 h in each cell line was set as 1. Error bar represents SEM. β-actin and GAPDH were blotted as loading controls. All experiments were repeated at least three times.

Article Snippet: Mouse monoclonal antibodies used in this study were against β-actin (GenScript, A00702), GAPDH (Developmental Studies Hybridoma Bank, DSHB-hGAPDH-4B7, deposited by DSHB), α-synuclein (Abcam, 4D6), P62 (Abcam, ab56416), GBA (Novus, MAB7410), and Lamp1 (DSHB, G1/139/5, deposited by Hauri, H.-P.) .

Techniques: Control, Western Blot, Incubation

Journal: Scientific Reports

Article Title: Autophagy–lysosome pathway alterations and alpha-synuclein up-regulation in the subtype of neuronal ceroid lipofuscinosis, CLN5 disease

doi: 10.1038/s41598-018-36379-z

Figure Lengend Snippet: Knockdown of α-syn reverses the perinuclear lysosome clustering phenotype found in CLN5 KD HeLa cells. ( A ) HeLa or CLN5 KD cells were transfected with control or SNCA small interfering RNA (siRNA) for 48 hours prior to fixation. Lysosomes were visualized by immunostaining lamp1. Scale bar: 20 μm. ( B ) Immunoblot shows efficient knockdown of α-syn expression in CLN5 KD cells. GAPDH was blotted as a loading control. ( C ) The cellular distribution area of lysosomes was quantified in the conditions indicated. An outline of the cells was marked by tracing the cell border in phase contrast images. The total area of the cell and the area with lamp1 signal in the cell were measured using ImageJ “measure” function. One-way ANOVA followed by the Tukey’s post hoc test was performed *P < 0.05, n = 25, error bar represents SEM. ( D ) Immunoblotting of WT, stable SNCA knock-out cell (SNCA KO), and siRNA of stable CLN5 KD as indicated. β-actin was blotted as a loading control. All experiments were repeated at least three times.

Article Snippet: Mouse monoclonal antibodies used in this study were against β-actin (GenScript, A00702), GAPDH (Developmental Studies Hybridoma Bank, DSHB-hGAPDH-4B7, deposited by DSHB), α-synuclein (Abcam, 4D6), P62 (Abcam, ab56416), GBA (Novus, MAB7410), and Lamp1 (DSHB, G1/139/5, deposited by Hauri, H.-P.) .

Techniques: Knockdown, Transfection, Control, Small Interfering RNA, Immunostaining, Western Blot, Expressing, Knock-Out