Journal: Nature Communications

Article Title: DSS1 inhibits autophagy to activate epithelial-mesenchymal transition in a pro-metastatic niche of renal cell carcinoma

doi: 10.1038/s41467-025-62135-9

Figure Lengend Snippet: a Co-Immunoprecipitation (Co-IP) with antibody to the FLAG epitope, followed by SDS-PAGE and immunoblotting with antibody to LC3 (endogenous, IgG: isotype control; Input: 5% lysate). Short/long: exposure time of the same membrane. b Co-IP with antibody to the LC3 epitope, followed by SDS-PAGE and immunoblotting showing endogenous DSS1. a , b IgG light chain-specific secondary antibody was used. c Glutathione-S-Transferase (GST)-pulldown analysis for DSS1-LC3B interaction using 50 ng recombinantly expressed human full-length LC3B and 1 mg GST/GST-DSS1 (GST: negative control; Purified LC3B: positive control; Glutathione agarose beads: 50 µL). d Cells were lysed with EDTA (10 mM) or not, followed by metal bath at 95 °C for 5 min and immunoblotting with antibody to DSS1. e GST-pulldown analysis showing the interaction of LC3 and DSS1 mutant using 50 ng recombinantly expressed human LC3 and 1 mg GST-DSS1 W27GW39GW43GF52A (Glutathione agarose beads: 50 µL). f Caki-1 cells were transfected with plasmids encoding HA-DSS1 W27GW39GW43GF52A . Cell lysates and cell immunoprecipitants were immunoprecipitated with antibody to the LC3 epitope and HA epitope, respectively. g Knockdown of DSS1 or treatment with MG-132 (20 μM, 6 h before harvest) elevates LC3 protein levels in HEK293T cells (control: siNC and/or 0.02% DMSO, error bar: mean ± SD, two-tailed Welch’s t-test). h Immunoblotting showing the indicated protein levels in HEK293T cells transfected with control or DSS1 siRNA, and/or treated with 0.02% DMSO or MG-132 (20 μM, 6 h before harvest). i Immunoblotting of cycloheximide (CHX, 50 μg/mL) chase assay in sh DSS1 cells compared to sh NC cells (error bar: mean ± SD, two-tailed Welch’s t-test). c –e , g–i The samples derived from the same experiment were run on parallel gels, with each gel probed for a different antibody. a – i n = 3 independent experiments. Statistics are provided in the source data. Source data are provided as a Source Data file.

Article Snippet: Purified human LC3B protein was purchased from MedChemExpress ® (HY- P70909 ).

Techniques: Immunoprecipitation, Co-Immunoprecipitation Assay, FLAG-tag, SDS Page, Western Blot, Control, Membrane, Negative Control, Purification, Positive Control, Mutagenesis, Transfection, Knockdown, Two Tailed Test, Derivative Assay

Journal: Nature Communications

Article Title: DSS1 inhibits autophagy to activate epithelial-mesenchymal transition in a pro-metastatic niche of renal cell carcinoma

doi: 10.1038/s41467-025-62135-9

Figure Lengend Snippet: a Representative images of immunofluorescence showing the subcellular localization of DSS1 (magenta), LC3 (yellow), and PSMD3 (azure) in ACHN cells ( n = 3 independent experiments). Nuclei: DAPI (blue). Scale bar: 5 μm. b LC-MS/MS analysis of LC3 interactomes identifies E3 ligases potentially associated with LC3 ( n = 3 independent experiments; Proteome Discoverer 2.4, Thermo Scientific; False discovery rate <0.01). c The mRNA expression of TRIM25 in ccRCC patients from the TCGA-KIRC dataset ( n = 595 distinct samples, tumor vs. normal, two-tailed Welch’s test). Boxplot: Center line = median; box = 25th to 75th percentiles; whiskers = minima to maxima. d Representative images of immunohistochemistry analysis showing TRIM25 protein levels upregulated in ccRCC tissues vs. normal ( n = 66 distinct pairs; scale bar: 50 μm; error bar: mean (centre) ± SD; two-tailed paired t-test). e , f Immunoblotting showing TRIM25 knockdown increases LC3 levels, while TRIM25 overexpression reduces LC3 levels ( n = 3 independent experiments, error bar: mean ± SD, two-tailed Welch’s t-test). g Co-IP analysis in Caki-1 cells showing the interaction between endogenous TRIM25 and LC3 ( n = 3 independent experiments). h GST-pulldown analysis for TRIM25-LC3B interaction using 50 ng recombinantly expressed human full-length LC3B and 1 mg GST-TRIM25 ( n = 3 independent experiments; Glutathione agarose beads: 50 µL). Asterisk: GST-TRIM25. i Immunoblotting of HEK293T cells transfected with HA-Ub , pcDNA3.1, and Myc-LC3B (wildtype) or Myc-LC3B mutant plasmids ( n = 3 independent experiments). j Immunoblotting of HEK293T cells transfected with Myc-LC3B and HA-Ub -wildtype or HA-Ub -K48R/K63R mutant plasmids ( n = 3 independent experiments). k Recombinantly expressed human GST-TRIM25 was incubated with recombinant Ub, LC3B, UbcH5a/UbcH5c, and His-UBE1 in a reaction system containing MgATP at 37 °C for 4 h. Samples were analyzed by SDS-PAGE and immunoblotting with antibody to Ub epitope ( n = 3 independent experiments). As shown in the result of GST-TRIM25, the polyubiquitination of LC3B (LC3B-Ub) is shown only when all the components are present in the mix. e , f , h –j The samples derived from the same experiment were run on parallel gels, with each gel probed for a different antibody. Statistics are provided in the source data. Source data are provided as a Source Data file.

Article Snippet: Purified human LC3B protein was purchased from MedChemExpress ® (HY- P70909 ).

Techniques: Immunofluorescence, Liquid Chromatography with Mass Spectroscopy, Expressing, Two Tailed Test, Immunohistochemistry, Western Blot, Knockdown, Over Expression, Co-Immunoprecipitation Assay, Transfection, Mutagenesis, Incubation, Recombinant, SDS Page, Derivative Assay

Journal: Nature Communications

Article Title: DSS1 inhibits autophagy to activate epithelial-mesenchymal transition in a pro-metastatic niche of renal cell carcinoma

doi: 10.1038/s41467-025-62135-9

Figure Lengend Snippet: a Multiplex immunohistochemistry showing spatial distribution of DSS1-driven cells with a high epithelial-mesenchymal transition (EMT) feature (E-cad low , Vimentin high ) around vascular cells (CD31 + , arrows, n = 4 distinct patients). Scale bar: 40 μm. b SPP1 RNA and SPP1 protein levels in tissues of diverse cancer types (kidney cancer: RNA, n = 35 distinct cell lines, protein, n = 110 distinct samples) from the Human Protein Atlas (HPA, www.proteinatlas.org ). c SPP1 Protein levels in normal tissues from the HPA database (kidney, high expression in proximal tubules, n = 3 slides). d Pseudo-bulk RNA levels of SPP1 in epithelial and immune cell types. AvgExp, Average expression. e Protein abundance of SPP1 between tumor and normal tissues in Clinical Proteomic Tumor Analysis Consortium (CPTAC)-ccRCC dataset ( n = 194 distinct samples, two-tailed Mann-Whitney U test). Boxplot: Center line = median; box = 25th to 75th percentiles; whiskers = minima to maxima. f Spearman correlation between SPP1 and DSS1 mRNA expression (log 2 Count Per Million, two-tailed spearman’s rank correlation test). g Immunoblotting showing SPP1 protein levels in ccRCC cells (sh DSS1 vs. sh NC , n = 3 independent experiments, error bar: mean ± standard deviation [SD], two-tailed Welch’s t-test). The samples derived from the same experiment were run on parallel gels, with each gel probed for a different antibody. h Spearman correlation between CD68 expression and coexpression of SPP1 - ITGB1 in spots of sample sections from either tumor core or tumor-stromal interface (Li2022 spatial dataset, n = 13 sections from 10 distinct patients; error bar: mean ± SD). i Schematic diagram (by Figdraw.com) illustrates the proposed mechanistic model of DSS1-driven cells in ccRCC metastasis: DSS1 (oligomer) interacts with pro-LC3B or LC3B-I, promoting LC3B degradation via E3 ubiquitin ligase TRIM25-mediated Lys-63 (K63)-linked polyubiquitination at LC3B-K51, leading to impaired macroautophagic flux and p62 accumulation, TWIST1 stabilization and increased TWIST1 nuclear transport, promoting EMT activation. DSS1 highly expressed (DSS1 hi ) tumor cells were increased in late-stage tumors and linked to microvascular invasion within a vascularized invasive niche at the tumor-stromal interface, mediated by SPP1-ITGB1 interactions. Statistics are provided in the source data. Source data are provided as a Source Data file.

Article Snippet: Purified human LC3B protein was purchased from MedChemExpress ® (HY- P70909 ).

Techniques: Multiplex Assay, Immunohistochemistry, Expressing, Quantitative Proteomics, Two Tailed Test, MANN-WHITNEY, Western Blot, Standard Deviation, Derivative Assay, Ubiquitin Proteomics, Activation Assay

Journal: Oncotarget

Article Title: Berberine-induced autophagic cell death by elevating GRP78 levels in cancer cells

doi: 10.18632/oncotarget.14959

Figure Lengend Snippet: A and B . Western blots of LC3, p62, Beclin1 and GAPDH were performed on HCT-116 cell lysates treated with berberine at the indicated concentrations for 24 h. The relative protein expression was calculated by Image J. C-D . HCT-116 cells were treated with the indicated concentrations of berberine for 24 h with or without CQ (50 μM). The levels of LC3, p62 and Beclin1 were monitored by western blot of the cell lysates (C) and the relative protein expression was calculated by Image J (D). E-F . Western blotting analysis of LC3-II/LC3-I, p62 and Beclin1 levels (E) and the relative protein expression were quantified by Image J (F) in HCT-116 cells treated with berberine at the indicated concentrations for 24 h, in the absence or presence of 37.5 μM BAF (treated in combination with berberine). G . Protein expression levels of LC3, p62 and Beclin1 were analyzed by western blot in HCT-116 cells after treatement with berberine at the indicated concentrations for 24 h, in the absence or presence of 3-MA at different concentrations (treated in combination with berberine). Three independent experiments were performed, and the data were expressed as the mean ± SD. *p<0.05, **p<0.01, ***p<0.001 were compared to the untreated group.

Article Snippet: Antibodies for LC3, p62, AMPK, p-AMPK T183/172 and ATF6 were obtained from Proteintech (Chicago, USA).

Techniques: Western Blot, Expressing

Journal: Oncotarget

Article Title: Berberine-induced autophagic cell death by elevating GRP78 levels in cancer cells

doi: 10.18632/oncotarget.14959

Figure Lengend Snippet: A. Quantification of the relative protein expression is shown in Figure of three independent experiments using Image J software. B and C. Western blot analysis of LC3, Beclin1 and p62 expression (B) and the relative protein expression were evaluated by Image J (C) in HL-7702 cells treated with various concentrations of berberine. D. HCT-116 cells incubated with 0.05 mM monodansylcadaverine (MDC) for 10 min after treatment with the indicated concentration of berberine 24 h (left panel) or transfected with mCherry-hLC3B treated with 120 μM berberine for 24 h (right panel). The cells were then analyzed using fluorescence microscopy. Scale bar, 5 μm. E-F. HepG2 and DLD1 cells were treated with the indicated concentrations of berberine for 24 h with or without BAF co-incubation. LC3, Beclin1 and p62 were analyzed by western blotting analyses (E) and relative protein expression levels were calculated by Image J (F). Three independent experiments were performed, and the data were expressed as the mean ± SD. *p<0.05, **p<0.01, ***p<0.001 compared to the untreated group.

Article Snippet: Antibodies for LC3, p62, AMPK, p-AMPK T183/172 and ATF6 were obtained from Proteintech (Chicago, USA).

Techniques: Expressing, Software, Western Blot, Incubation, Concentration Assay, Transfection, Fluorescence, Microscopy

Journal: Oncotarget

Article Title: Berberine-induced autophagic cell death by elevating GRP78 levels in cancer cells

doi: 10.18632/oncotarget.14959

Figure Lengend Snippet: A-F. HCT-116 and DLD1 cells were transfected with control siRNA or siRNA targeting GRP78, respectively. After 48 h, the cells were treated with indicated concentrations of berberine for 24 h, then GRP78 was measured by qRT-PCR (A and C) and GRP78, LC3 were examined by western blotting analyses (B and E). The LC3-II/LC3-I relative protein expression levels were calculated by Image J (C and F). Data were expressed as the mean ± SEM of three different experiments. *p<0.05 and **p<0.01 vs. respective control.

Article Snippet: Antibodies for LC3, p62, AMPK, p-AMPK T183/172 and ATF6 were obtained from Proteintech (Chicago, USA).

Techniques: Transfection, Control, Quantitative RT-PCR, Western Blot, Expressing

Journal: Oncotarget

Article Title: Berberine-induced autophagic cell death by elevating GRP78 levels in cancer cells

doi: 10.18632/oncotarget.14959

Figure Lengend Snippet: A-C. HepG2 cells were transfected with control siRNA or siRNA targeting GRP78, respectively. After 48 h, the cells were treated with indicated concentrations of berberine for 24 h, then GRP78 was measured by qRT-PCR (A) and GRP78, LC3 were examined by western blotting analyses (B). The LC3-II/LC3-I relative protein expression levels were calculated by Image J (C). D. The cytotoxicity of berberine can be attenuated by introducing siRNA against GRP78 into DLD1 cells. Data were expressed as the mean ± SEM of three different experiments. *p<0.05 vs. respective control.

Article Snippet: Antibodies for LC3, p62, AMPK, p-AMPK T183/172 and ATF6 were obtained from Proteintech (Chicago, USA).

Techniques: Transfection, Control, Quantitative RT-PCR, Western Blot, Expressing

Journal: Scientific Reports

Article Title: Direct binding to GABARAP family members is essential for HIV-1 Nef plasma membrane localization

doi: 10.1038/s41598-017-06319-4

Figure Lengend Snippet: Co-immunoprecipitation (IP) of the Nef-ATG8 complex. Co-IP studies were carried out with lysates prepared from HEK293 cells stably expressing Nef-DsRed ( A–C ) and cotransfected with plasmids encoding for the indicated YFP-ATG8 constructs ( D–F ). ( A ) and ( D ) show samples before immunoprecipitation. In ( A ) an anti-GABARAPL2 (a), an anti-GABARAP (b), an anti-GABARAPL1 (c), and an anti-LC3B antibody (d) were used to detect the different ATG8s. ( B ) Immunoprecipitates with anti-DsRed antibody followed by SDS-PAGE and immunoblotting with various ATG8 antibodies (a: anti-GABARAPL2, b: anti-GABARAP, c: anti-GABARAPL1 and d: anti-LC3B). ( C ) In a reciprocal set of experiments, immunoprecipitates were carried out with various ATG8 (a: anti-GABARAPL2, b: anti-GABARAP, c: anti-GABARAPL1 and d: anti-LC3B) antibodies followed by SDS-PAGE and immunoblotting with an anti-DsRed antibody. In ( D ) an anti-DsRed antibody was used to detect Nef fused to DsRed and an anti-YFP antibody to detect YFP and the ATG8s fused to YFP. ( E ) Immunoprecipitates with an anti-DsRed antibody or ( F ) with an anti-GFP antibody followed by SDS-PAGE and immunoblotting with the antibodies are indicated. Data are representative of three independent experiments (U: unbound material; E: eluate fraction). Full-length western blots are presented in Supplementary Figs and .

Article Snippet: Rabbit polyclonal antibodies raised against GABARAP (18723-1-AP), GABARAPL2 (18724-1-AP), GABARAPL1 (18721- 1-AP), and LC3B (18725-1-AP) were obtained from Proteintech Group, Inc. (Chicago, IL, USA).

Techniques: Immunoprecipitation, Co-Immunoprecipitation Assay, Stable Transfection, Expressing, Construct, SDS Page, Western Blot

Journal: Scientific Reports

Article Title: Direct binding to GABARAP family members is essential for HIV-1 Nef plasma membrane localization

doi: 10.1038/s41598-017-06319-4

Figure Lengend Snippet: GABARAP residues S53 and S62 are essential for Nef binding. ( A ) Human ATG8s sequence alignment indicates putative key residues for Nef binding specificity. Protein names of ATG8s showing Nef binding during pull-down and immunoprecipitation analysis are given in different shades of yellow, and include all members of the GABARAP subfamily. Protein names of LC3B and of the other LC3s are given in different shades of brown. Purple arrows indicate residues of GABARAP showing chemical shift changes higher than 0.05 ppm (see Fig. ) upon Nef titration. Residues forming HP1 and HP2 are shaded in light and dark blue, respectively. Red asterisks highlight putative key positions for determining Nef-binding specificity. The corresponding amino acids found in GABARAP and LC3B at these positions are highlighted in red. ( B ) Visualization of HP1 and HP2 on the surface of the GABARAP structure [PDB ID: 1KOT]. ( C ) Structural overlay of GABARAP and LC3B with the putative key residues necessary for Nef-binding highlighted. Cartoon representations of GABARAP (yellow) and LC3B (brown) [PDB ID: 1V49] showing their regular secondary structure elements demonstrate that S53 and F62 of GABARAP as well as the corresponding D56 and K65 of LC3B are surface exposed, and therefore are available for ligand binding. Details highlight the side chain orientations of the key residues, for this the structures have been rotated appropriately. ( D ) Nef-conjugated or free Sepharose beads (control) were incubated with GABARAP(S53D/F62K) or LC3B(D56S/K65F) mutants. The input, the unbound material of the flow through (U),the wash (W) fractions and the eluate (E) fractions were subjected to SDS-PAGE and visualized by CBB staining. Full-length gels are presented in Supplementary Fig. .

Article Snippet: Rabbit polyclonal antibodies raised against GABARAP (18723-1-AP), GABARAPL2 (18724-1-AP), GABARAPL1 (18721- 1-AP), and LC3B (18725-1-AP) were obtained from Proteintech Group, Inc. (Chicago, IL, USA).

Techniques: Binding Assay, Sequencing, Immunoprecipitation, Titration, Ligand Binding Assay, Control, Incubation, SDS Page, Staining

Journal: Endocrinology

Article Title: Deficiency of C1QL1 reduced murine ovarian follicle reserve through intraovarian and endocrine control.

doi: 10.1210/endocr/bqac048

Figure Lengend Snippet: Figure 5. Deficiency of C1QL1 promoted apoptosis and inhibited autophagy in the ovary. (A) Representative images of TUNEL staining of ovarian sections. The green signals indicate representative cells with positive TUNEL staining, which was mainly detected in the large atretic follicles. (B) The intensity of TUNEL-positive signals was shown in charts (n = 4 per group). (C) Representative images of immunofluorescence staining of ovarian sections with LC3B antibody. LC3B-positive signals were found in granulosa cells and oocytes and were weakened in C1QL1-deficient ovaries. (D) The intensity of LC3B-positive signals are shown (n = 4 per group). (E) Apoptosis- and autophagy-related proteins in the ovaries were analyzed by Western blotting. Representative blots are shown in the left panel, and the amount of protein normalized to β-tubulin, which is presented as mean ± SEM, is shown in the right panel. n = 6 per group. *P < 0.05, **P < 0.01, ***P < 0.001. NS, no significance.

Article Snippet: The sections and granulosa cells were stained with anti-rabbit LC3B antibody and Alexa Fluor 594-conjugated secondary antibody and mounted with DAPI (Boster).

Techniques: TUNEL Assay, Staining, Immunofluorescence, Western Blot

Journal: Human & experimental toxicology

Article Title: Hypoxia-induced autophagy attenuates ferredoxin 1-mediated cuproptosis in colorectal cancer cells.

doi: 10.1177/09603271251335393

Figure Lengend Snippet: Figure 4. Hypoxia induces autophagy in colorectal cancer cells. (a) Western blot analysis of LC3B, Beclin 1, and P62 expression in LoVo and RKO cells under hypoxic conditions. (b) Immunofluorescence staining of LC3B in LoVo and RKO cells under hypoxic conditions. *p < .05, **p < .01.

Article Snippet: Tissue sections were deparaffinized and incubated with the LC3B antibody (BM4827, 1:200; Boster, Shanghai, China) and secondary antibodies.

Techniques: Western Blot, Expressing, Staining

Journal: Human & experimental toxicology

Article Title: Hypoxia-induced autophagy attenuates ferredoxin 1-mediated cuproptosis in colorectal cancer cells.

doi: 10.1177/09603271251335393

Figure Lengend Snippet: Figure 5. Hypoxia-induced autophagy attenuates FDX1-mediated cuproptosis in colorectal cancer cells. (a) Western blot analysis of LC3B, Beclin 1, and P62 expression in LoVo and RKO cells treated with rapamycin under hypoxic conditions. (b) Immunofluorescence staining of LC3B in LoVo and RKO cells. (c) Transwell assay of cell migration and invasion in LoVo and RKO cells. (d) EdU staining and TUNEL assay of cell proliferation and damage in LoVo and RKO cells. (e) Western blot analysis of DLAT oligomerization in LoVo and RKO cells. *p < .05, **p < .01.

Article Snippet: Tissue sections were deparaffinized and incubated with the LC3B antibody (BM4827, 1:200; Boster, Shanghai, China) and secondary antibodies.

Techniques: Western Blot, Expressing, Staining, Transwell Assay, Migration, TUNEL Assay

Journal: Human & experimental toxicology

Article Title: Hypoxia-induced autophagy attenuates ferredoxin 1-mediated cuproptosis in colorectal cancer cells.

doi: 10.1177/09603271251335393

Figure Lengend Snippet: Figure 6. Autophagy inhibition enhances the tumor-suppressive effects of FDX1 overexpression in vivo. (a) Representative images of tumors from nude mice implanted with LoVo cells under different conditions. (b, c, d) Tumor volume (b, c) and weight of nude mice implanted with LoVo cells under different conditions. (e) H&E staining and immunohistochemical staining of Ki67 in tumor tissues from nude mice implanted with LoVo cells under different conditions. (f) TUNEL assay of cell damage in tumor tissues from nude mice implanted with LoVo cells under different conditions. (g) qPCR analysis of FDX1 expression in tumor tissues from nude mice implanted with LoVo cells under different conditions. (g) Western blot analysis of FDX1 and DLAT oligomerization in tumor tissues. (h) Western blotting of LC3B and Beclin 1 expression in tumor tissues from nude mice implanted with LoVo cells under different conditions. *p < .05, **p < .01, ns noted no significance.

Article Snippet: Tissue sections were deparaffinized and incubated with the LC3B antibody (BM4827, 1:200; Boster, Shanghai, China) and secondary antibodies.

Techniques: Inhibition, Over Expression, In Vivo, Staining, Immunohistochemical staining, TUNEL Assay, Expressing, Western Blot

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: 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.

Techniques: Expressing, Western Blot, Immunofluorescence, Ubiquitin Proteomics, 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: Lysosomal damage blocks the autophagy flux. (A) The Jurkat cells transfected with a plasmid encoding LC3-GFP-mCherry were treated with 8 μmol/L LW-218 for different time periods. The cells also treated with BAF A1 (7.5 nmol/L) and rapamycin (250 nmol/L). The colocalization of LC3-GFP and LC3-mCherry were detected by immunofluorescence. (B) The Jurkat and THP-1 cells were treated with 8 μmol/L of LW-218 for 0–12 h. The expressions of LC3 and P62 were determined by Western blot. (C) The Jurkat cells were cotreated with 8 μmol/L LW-218 and MG-132 (15 μmol/L, pre-treated for 1 h) for different time periods and the expression of ubiquitin were determined by Western blot. β -Actin was used as loading control. (D) Immunofluorescence analysis performed with anti-LC3B antibody (red) and galectin-3 (green) in Jurkat cells. The cells were with LW-218 for 12 h.

Article Snippet: 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.

Techniques: Transfection, Plasmid Preparation, Immunofluorescence, Western Blot, Expressing, Ubiquitin Proteomics, Control