Journal: Arteriosclerosis, thrombosis, and vascular biology

Article Title: Diet-induced aortic valve disease in mice haploinsufficient for the Notch pathway effector RBPJK/CSL.

doi: 10.1161/ATVBAHA.111.227561

Figure Lengend Snippet: Figure 2. Greater leaflet thickness and increased macrophage deposition in RBPKO/ mice fed an HCVD diet. Shown are Masson Trichrome staining (A to F) of aortic valves indicating collagen deposition (arrows) and Mac3 immuno- staining (G to L) of aortic valves demon- strating macrophage infiltration (red; arrows indicate positive immunostaining) in WT mice fed the CC diet (A and G) or the HCVD diet (D and J), Notch1KO/ mice fed the CC diet (B and H) or the HCVD diet (E and K), and RBPKO/ mice fed the CC diet (C and I) or the HCVD diet (F and L). M and N, Mean data of cusp area (M) and percentage of positively stained sections for Mac3 (N) for all groups (n4 per group). One-way ANOVA and LSD post hoc analysis were used to compare differences between groups. Bars with different letters indi- cate significant differences (P0.05) vs all other groups (LSD post hoc analysis). Scale bar0.1 mm.

Article Snippet: Inflammation was detected with anti-Mac3 rat monoclonal antibody (SC19991, Santa Cruz Technol, 1:200).

Techniques: Staining, Immunostaining

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Activation and Purification of ß-Glucocerebrosidase by Exploiting its Transporter LIMP-2 - Implications for Novel Treatment Strategies in Gaucher's and Parkinson's Disease.

doi: 10.1002/advs.202401641

Figure Lengend Snippet: Figure 1. Interaction of GCase variants with their lysosomal transporter LIMP-2. A) Crystal structure of GCase (PDB: 5LVX).[44] The domains and motifs are colored as follows: pink: antiparallel beta-sheet (domain 1); grey: TIM barrel (domain 2) containing active site (green); blue: beta-barrel (domain 3);[45] orange: proposed LIMP-2-binding motif.[42] Single amino acids comprising the active site, as well as amino acids exchanged in common disease- associated GCase variants E326K, N370S, and L444P are labeled and highlighted. B) GCase activity in cell lysates of HEK 293T cells overexpressing GCase variants (n = 3-8; mock: 8; wt: 8; E326K: 5; N370S: 3; L444P: 5; individual transfections). All disease-associated variants show significantly reduced activity. E326K shows a residual activity of 40.0 ± 2.2%, N370S, and L444P do not differ significantly from the mock. C) Illustration of the lysosomal transport of GCase and its interaction with LIMP-2. GCase binds to LIMP-2 in the ER to form a transporting complex. After trafficking through the Golgi, the LIMP-2/GCase complex is sorted to lysosomal compartments where low pH triggers complex dissociation. D) Western blot analyses of whole-cell lysates and LE fractions of HEK 293T cells expressing GCase variants and FL-LIMP-2 or FL-LIMP-2-3xD as a control. LE fractions are higher in GCase, LIMP-2, and the lysosomal protein LAMP-2A. Enriched fractions of cells overexpressing FL-LIMP-2 show increased levels of GCase. Blots containing a dashed line were spliced for a more comprehensive data presentation (both parts are from the same image). E) Quantification of western blot signal from lysate and LE HEK 293T samples (Figure 1D) (n = 3-6; mock: 6; wt: 6; E326K: 3; N370S: 3; L444P: 3; individual cell harvests). The abundance of wt, E326K, and N370S GCase was significantly increased in LE fractions when FL-LIMP-2 was co-expressed compared to the co-expression of the non- binding 3xD control. In general, the GCase signal was increased in LE fractions but did not reach statistical significance for FL-LIMP-2-3xD samples and FL-LIMP-2 + L444P. F) GCase activity in cell lysates and LE fractions of HEK 293T cells overexpressing GCase variants and FL-LIMP-2 or FL-LIMP-2-3xD (n = 3-7; mock: 7; wt: 7; E326K: 4; N370S: 3; L444P: 4; individual cell harvests). Overexpression of FL-LIMP-2 resulted in an increase of GCase activity in the lysate itself, but also in the LE fraction for wt GCase and the E326K variant, while no significant differences were observed for the N370S and

Article Snippet: Following antibodies and dilution factors were used for western blot analysis: Primary: goat anti LIMP2 (polyclonal, ThermoFisher Scientific Inc., Waltham, MA, United States, #PA5-19111; dilution: 1:1000), mouse anti GCase (monoclonal, clone E2E, Abnova, Taipeh, Taiwan,#H00002629-M01; dilution: 1:1000), rabbit anti GCase (polyclonal, Sigma-Aldrich, St. Louis, MO, United States, #G4171; dilution: 1:1000), rabbit anti LAMP-2A (monoclonal, clone SA4601, Novus Biologicals, Centennial, CO, United States, #NBP2-67298; dilution: 1:1000), goat anti GAPDH (polyclonal, R&D Systems, Minneapolis, MN, United States, #AF5718; dilution: 1:5000), rabbit anti calnexin (polyclonal, Cell Signaling Technology, Danvers, MA, United States, #2433, dilution: 1:1000).

Techniques: Binding Assay, Labeling, Activity Assay, Transfection, Western Blot, Expressing, Control, Over Expression, Variant Assay

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Activation and Purification of ß-Glucocerebrosidase by Exploiting its Transporter LIMP-2 - Implications for Novel Treatment Strategies in Gaucher's and Parkinson's Disease.

doi: 10.1002/advs.202401641

Figure Lengend Snippet: Figure 2. Characterization of primary human fibroblasts of controls and patients with PD and GD. A) Western blot of whole cell lysates from control (CTRL-1,2,3), PD patient-derived (PD-1,2), and GD patient-derived (GD) primary human fibroblasts. Quantitative analysis of signals can be found in Figure S3 (Supporting Information). GCase levels were comparable between control and PD but diminished in GD (Figure S3A, Supporting Information). LIMP-2 levels were increased in PD compared to controls (Figure S3B, Supporting Information). LAMP-2A and calnexin levels varied between cell lines and did not show any significant trend between groups (Figure S3C,D, Supporting Information). GAPDH and CBB staining of the gel are presented as loading controls. B) GCase activity in whole-cell lysates of control, PD, and GD fibroblast cell lines (n = 3-7; CTRL-1,2,3: 7; PD-1,2: 7; GD: 3; individual cell harvests). The E326K lines PD-1 and PD-2 showed significantly lower GCase activity (66.41±2.69% and 73.56±6.50%) compared to the control lines. In contrast, activity in the GD line (GBA1L444P/L444P) was almost fully abolished (3.11±0.11% residual activity). C) Colocalization of LIMP-2 and GCase in primary human fibroblasts determined via Pearson’s correlation coefficient (n = CTRL-1: 30; CTRL-2: 38; CTRL-3: 33; PD-1: 44; PD-2: 51; GD: 27; individual cells from multiple images). PD patient fibroblasts PD-1 and PD-2 show mildly reduced colocalization of LIMP-2 and GCase compared to control lines. In the GD patient line, colocalization was diminished even further. D) Representative immunofluorescence images of primary human fibroblast lines. Objective magnification: 40x. Green: LIMP-2; red: GCase, blue: DAPI. All lines show the vesicular distribution of LIMP-2, indicating lysosomes. Control and PD lines show visible colocalization of GCase with the LIMP-2 signal. In GD fibroblasts, the GCase signal was less intense and less granular, representing lower expression and lysosomal localization. Statistics: replicates (dots) with mean (column) ± SEM (B); violin plot with median (dashed line) and quartiles (dotted line) (C). Tests: Nested one-way ANOVA with Tukey’s multiple comparison test (B,C). * p < 0.05; ** p < 0.01; **** p < 0.0001.

Article Snippet: Following antibodies and dilution factors were used for western blot analysis: Primary: goat anti LIMP2 (polyclonal, ThermoFisher Scientific Inc., Waltham, MA, United States, #PA5-19111; dilution: 1:1000), mouse anti GCase (monoclonal, clone E2E, Abnova, Taipeh, Taiwan,#H00002629-M01; dilution: 1:1000), rabbit anti GCase (polyclonal, Sigma-Aldrich, St. Louis, MO, United States, #G4171; dilution: 1:1000), rabbit anti LAMP-2A (monoclonal, clone SA4601, Novus Biologicals, Centennial, CO, United States, #NBP2-67298; dilution: 1:1000), goat anti GAPDH (polyclonal, R&D Systems, Minneapolis, MN, United States, #AF5718; dilution: 1:5000), rabbit anti calnexin (polyclonal, Cell Signaling Technology, Danvers, MA, United States, #2433, dilution: 1:1000).

Techniques: Western Blot, Control, Derivative Assay, Staining, Activity Assay, Expressing, Comparison

Journal: Autophagy

Article Title: Pharmacological inhibition of lysosomes activates the MTORC1 signaling pathway in chondrocytes in an autophagy-independent manner

doi: 10.1080/15548627.2015.1068489

Figure Lengend Snippet: Baf does not affect localization of FLCN on lysosomes. C5.18 cells were differentiated into chondrocytes and treated with Baf for 48 h. Colocalization between lysosomes (visualized by LAMP2 antibody) and FLCN was explored by confocal microscopy ( A ). Quantification revealed no differences in lysosomal localization of FCLN upon Baf exposure ( B ). p = 0.7489, n = 3 independent experiments with approximately 400 cells analyzed in each experiment.

Article Snippet: The following antibodies were used: anti-p-RPS6 (Cell Signaling Technology, 4858), anti-AKT (Cell Signaling Technology, 9272), anti-p-AKT (Ser473; Cell Signaling Technology, 4060), anti-RPS6 (5G10; Cell Signaling Technology, 2217), anti-GAPDH (Cell Signaling Technology, 2118), anti-EIF4EBP1 (53H11; Cell Signaling Technology, 9644), anti-phospho-EIF4EBP1 (Thr37/46; Cell Signaling Technology, 2855), anti-MAP1LC3A (MBL, PM036), LAMP2 (Santa Cruz Biotechnology, sc-8100), FLCN (Cell Signaling Technology, 3697), anti-ATG5 (Cell Signaling Technology, 8540), anti-SQSTM1 (Progen Bioteknik, GP62-C), anti-COL10A1 (Quartett; GMBH, 1-CO097-05), biotin-conjugated anti-guinea pig, anti-rabbit and anti-mouse secondary antibodies (Jackson ImmunoResearch,106-065-003, 111-066-003 and 115-066-003, respectively), mouse anti-BrdU antibody (Sigma, B8434), biotinylated anti-mouse antibody (Jackson, 115-066-003), streptavidin-conjugated Alexa Fluor 546 (Life Technologies, S11225), fluorescently labeled anti-goat antibody (Jackson, 705165147) and fluorescently labeled anti-rabbit antibody (Jackson, 711545152).

Techniques: Confocal Microscopy

Journal: Cancers

Article Title: HSP90α Mediates Sorafenib Resistance in Human Hepatocellular Carcinoma by Necroptosis Inhibition under Hypoxia

doi: 10.3390/cancers13020243

Figure Lengend Snippet: HSP90α Promotes Chaperone-Mediated Autophagy (CMA) Degradation by directly binding to MLKL in hypoxia. ( A ) The degradation stability of MLKL detected by Western blot. ( B – D ) The expressions of LAMP2, HSP90α and HSP70 located in lysosomal observed by immunofluorescence. Scale bar = 40 μm. ( E ) The expression of LAMP2, HSP90α and HSP70 in hypoxia. ( F ) HSP90α binding with necroptosis protein. ( G , H ) The expression of LAMP2/HSP90α/HSP70/MLKL complex detected by Western blot in HepG2 and primary tumor cells. ( I ) Silenced HIF-1α and HSP90α affected necroptosis in hypoxia. ( J ) Necroptosis pathway in primary resistant cells detected by Western blot.

Article Snippet: Antibodies against AFP (4448) and LAMP2 (49067) were products of Cell Signaling Technology (Beverly, MA, USA).

Techniques: Binding Assay, Western Blot, Immunofluorescence, Expressing