Journal: Acta Pharmaceutica Sinica. B

Article Title: Cholesterol-associated lysosomal disorder triggers cell death of hematological malignancy: Dynamic analysis on cytotoxic effects of LW-218

doi: 10.1016/j.apsb.2021.02.004

Figure Lengend Snippet: Damaged lysosomes are ubiquitinated and cleared via autophagy pathway. (A) The Jurkat cells were treated with LW-218 (8 μmol/L) for 0–12 h and expression of LAMP2 were determined by Western blot. (B) Immunofluorescence analysis performed with anti-ubiquitin (red) antibody and anti-LAMP1 antibody (blue) in Jurkat cells (N.C, autophagy related 7 ( ATG7 ) shRNA, and BAF A1 group) after treatment with LW-218 for 12 h. (C) The Jurkat and THP-1 cells were cotreated with LW-218 and BAF A1 for 12 h. The expression levels of LAMP2 were determined by Western blot. (D) The Jurkat cells stably-transfected with an ATG7 shRNA were treated with LW-218. The expression of LAMP2 was determined by Western blot, in which above β -actin was used as loading controls. (E) Immunofluorescence analysis performed with anti-microtubule-associated protein 1 light chain 3B (LC3B) antibody (green) in Jurkat cells transfected with a plasmid encoding mCherry-galectin-3 after treatment with LW-218, BAF A1 (7.5 nmol/L) and chloroquine (50 μmol/L) for 1 h or 6 h. (F) Immunofluorescence analysis performed with anti-LAMP1 antibody (blue) and anti-ubiquitin antibody (green) in Jurkat cells transfected with a plasmid encoding mCherry-galectin-3 after treatment with LW-218 for 6 h. (G) The Jurkat cells transfected with a plasmid encoding mCherry-galectin-3 were treated with LW-218 (6 μmol/L) for 1 h. After LW-218 washout (withdrawal group) or sustained action (sustain group), the positive for galectin-3 puncta cells ratio were calculated (total cells in each group >100). (H) The Jurkat cells stably-transfected with an ATG7 shRNA were transfected with a plasmid encoding mCherry-galectin-3. The N.C. cells and ATG7 shRNA cells were treated with LW-218 for 2 h and then washout. The positive for galectin-3 puncta were determined by fluorescence microscopy at 12 h after washed, and galectin-3 puncta-positive cells ratio was calculated (total cells in each group >100). Data are mean ± SEM for ≥3 independent experiments; P values are shown on the graph.

Article Snippet: Antibodies: active caspase 3 (A11021), caspase 3 (A2156), caspase 9 (A0281), caspase 8 (A0215), β -actin (AC026), BH3-interacting domain death agonist (BID; A0210), cathepsin B (CTSB; A19005), lysosomal-associated membrane proteins 2 (LAMP2; A1961), microtubule-associated protein 1 light chain 3 (LC3; A17424), lysine 48 (K48)-linkage (A18163), lysine 63 (K63)-linkage (A18164), sequestosome 1 (P62/SQSTM1; A19700), lamin A/C (A19524) and galectin-3 (A13506; ABclonal Technology, Wuhan, China), lysosomal-associated membrane proteins 1 (LAMP1; 15665, Cell Signaling Technology, Danvers, MA, USA), cathepsin D (CTSD; ab75852) and TFEB (ab267351; Abcam, UK), RAS-related protein RAB-7a (RAB7A; 55469-1-AP), ubiquitin (10201-2-AP) and LC3 (14600-1-AP; Proteintech Group, Chicago, IL, USA), ubiquitin (sc-271289; Santa Cruz Biotechnology, CA, USA).

Techniques: Expressing, Western Blot, Immunofluorescence, shRNA, Stable Transfection, Transfection, Plasmid Preparation, Fluorescence, Microscopy

Journal: Acta Pharmaceutica Sinica. B

Article Title: Cholesterol-associated lysosomal disorder triggers cell death of hematological malignancy: Dynamic analysis on cytotoxic effects of LW-218

doi: 10.1016/j.apsb.2021.02.004

Figure Lengend Snippet: LW-218 promotes autophagy-related gene expression via calcium–CaN–TFEB axis. (A) The Jurkat and THP-1 cells were cotreated with LW-218 and BAF A1 (7.5 nmol/L) for 12 h and the expression of LC3 and sequestosome 1 (P62/SQSTM1) were determined by Western blot. β -Actin was used as loading control. (B) The Jurkat cells were cotreated with LW-218 (8 μmol/L) and BAF A1 (7.5 nmol/L) or BAPTA-AM (10 μmol/L) for 6 h or 12 h. The inhibitors were pre-treated for 1 h. The total RNAs were extracted and messenger RNA levels were detected by real time quantitative PCR. Data are mean ± SEM for ≥3 independent experiments; P values are shown on the graph. (C) The Jurkat cells were cotreated with LW-218 and BAPTA-AM (5 or 10 μmol/L) for 12 h, and the expression of P62, LC3 and LAMP2 were determined by Western blot. β -Actin was used as loading control. (D) The Jurkat cells were treated with LW-218 for 12 h. The levels of transcription factor EB (TFEB) in cytoplasm/membrane and nuclei fractions were determined by Western blot. β -Actin and lamin A/C were used as cytoplasm/membrane and nuclei loading control, respectively. (E) Immunofluorescence analysis performed with anti-TFEB antibody (green) and 4′,6-diamidino-2-phenylindole, dihydrochloride (DAPI, blue) in Jurkat cells. The cells were cotreated with LW-218 and BAF A1 (7.5 nmol/L) or BAPTA-AM (10 μmol/L) for 12 h. The inhibitors were pre-treated for 1 h. (F) The Jurkat cells were cotreated with LW-218 and BAPTA-AM (10 μmol/L) or cotreated with LW-218 and CaN inhibitors for 12 h. The apoptosis cells were determined by Annexin V/PI staining and flow cytometry (apoptosis cells are positive for Annexin V). Data are mean ± SEM for ≥3 independent experiments; P values are shown on the graph.

Article Snippet: Antibodies: active caspase 3 (A11021), caspase 3 (A2156), caspase 9 (A0281), caspase 8 (A0215), β -actin (AC026), BH3-interacting domain death agonist (BID; A0210), cathepsin B (CTSB; A19005), lysosomal-associated membrane proteins 2 (LAMP2; A1961), microtubule-associated protein 1 light chain 3 (LC3; A17424), lysine 48 (K48)-linkage (A18163), lysine 63 (K63)-linkage (A18164), sequestosome 1 (P62/SQSTM1; A19700), lamin A/C (A19524) and galectin-3 (A13506; ABclonal Technology, Wuhan, China), lysosomal-associated membrane proteins 1 (LAMP1; 15665, Cell Signaling Technology, Danvers, MA, USA), cathepsin D (CTSD; ab75852) and TFEB (ab267351; Abcam, UK), RAS-related protein RAB-7a (RAB7A; 55469-1-AP), ubiquitin (10201-2-AP) and LC3 (14600-1-AP; Proteintech Group, Chicago, IL, USA), ubiquitin (sc-271289; Santa Cruz Biotechnology, CA, USA).

Techniques: Expressing, Western Blot, Real-time Polymerase Chain Reaction, Immunofluorescence, Staining, Flow Cytometry