Journal: Molecular & Cellular Proteomics : MCP

Article Title: Identification of Cathepsin L as the Molecular Target of Hydroxychloroquine With Chemical Proteomics

doi: 10.1016/j.mcpro.2025.101314

Figure Lengend Snippet: HCQ targets endosomal virus entry mediated by CTSL. A , inhibition of CTSL activity by HCQ (50 μM), HCQ-P (50 μM), CQ (50 μM), quinine (50 μM), and mefloquine (4 μM). The known CTSL inhibitors, MG101 (4 μM) and E64 (30 μM), served as positive controls. Each group was calculated from three independent biological replicates. Statistical significance was measured by t test compared with dimethyl sulfoxide (DMSO) group. ∗∗ p < 0.01, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001. B , CTSL-mediated hydrolysis of the substrate AC-FR was quantified by monitoring the fluorescence of released 7-amino-4-trifluoromethylcoumarin (AFC). Reactions were conducted in pH 5.5 reaction buffer following overnight preincubation with HCQ at 4 °C. Each point was calculated from three independent biological replicates. C , inhibition of pseudovirus entry by HCQ. Huh7 CTSL-OE cells were treated with gradient dose of HCQ for 1 h before transduction with Omicron BA.1-S pseudoviruses. Pseudovirus entry was quantified by measuring the luciferase signal in the cell lysates 24 h after transduction. The inhibition (%) was normalized with 0 HCQ treatment group. D , inhibition of authentic Omicron BA.1 entry and replication by HCQ. Huh7 CTSL-OE cells were infected with authentic Omicron BA.1 at a multiplicity of infection (MOI) of 0.5. Viral gene copies in the cell lysate were determined by real-time quantitative PCR analysis at 24 hpi. The inhibition (%) was normalized to that of control group (0 HCQ). E and F , Huh7 cells were treated with CTSL gene knockdown (si-CTSL). The siRNA-treated cells were pretreated with HCQ for 1 h and then transduced with Omicron BA.1-S ( E ) or SARS-CoV-1-S ( F ) pseudoviruses. Pseudovirus entry was quantified by measuring the luciferase signal in cell lysates 24 h after transduction. The inhibition (%) was normalized to that of control group. The mean ± SEM from at least three independent experiments with technical triplicates is shown. Statistical significance was measured by t test between EV and CTSL-OE groups with the same HCQ concentration. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001. CQ, chloroquine; CTSL, cathepsin L; CTSL-OE, cathepsin L overexpression; EV, empty vector; HCQ, hydroxychloroquine; SARS-CoV, severe acute respiratory syndrome coronavirus.

Article Snippet: HCQ (HY-B1370), CQ (HY-17589A), E64 (HY-15282), quinine, mefloquine (HY-17437), promethazine (HY-B0781), azithromycin (HY-17506), and clemastine fumarate (HY-B0298A) are supplied by MCE.

Techniques: Virus, Inhibition, Activity Assay, Fluorescence, Transduction, Luciferase, Infection, Real-time Polymerase Chain Reaction, Control, Knockdown, Concentration Assay, Over Expression, Plasmid Preparation

Journal: Advanced Science

Article Title: A Nanomodulator Enhances Radiotherapy‐Induced In Situ Cancer Vaccine by Promoting Antigen‐Presenting of Tumor‐Associated Macrophage

doi: 10.1002/advs.202502876

Figure Lengend Snippet: Schematic of Ft‐E64/Hf@Lipo enhanced radio‐immunotherapy. A) Schematic illustration of the synthetic procedure for Ft‐E64/Hf@Lipo. B) Schematic illustration of Ft‐E64/Hf@Lipo to boost the in situ vaccine effect of cancer radiotherapy. Ft‐E64/Hf@Lipo can selectively target tumor cells, radiosensitizer Hf augments DNA damage to generate abundant tumor antigens; and then Ft‐E64, along with the tumor antigens, is phagocytosed by tumor‐associated macrophage (TAMs) through efferocytosis. The released E64 inhibits the lysosomal function of TAMs, enabling them to effectively present tumor antigens and activate antigen‐specific CD8 + T cells for enhanced radio‐immunotherapy.

Article Snippet: Proteinase inhibitor E64, DOPC (1, 2‐Dioleoyl‐sn‐glycero‐3‐phosphocholine), DOPE (Dioleoyl phosphatidylethanolamine), SM (N‐acetyl‐D‐erythro‐sphingosylphosphorylcholine), and CH (3β‐Hydroxy‐5‐cholestene, 5‐Cholesten‐3β‐ol) were purchased from Shanghai Yuanye.

Techniques: In Situ

Journal: Advanced Science

Article Title: A Nanomodulator Enhances Radiotherapy‐Induced In Situ Cancer Vaccine by Promoting Antigen‐Presenting of Tumor‐Associated Macrophage

doi: 10.1002/advs.202502876

Figure Lengend Snippet: Preparation of Ft‐E64/Hf@Lipo that enhances RT‐induced DNA damage and tumor apoptosis. A) TEM images of Ft‐E64/Hf and Ft‐E64/Hf@Lipo. Scale bars: 100 nm. B) CLSM images of Ft‐E64/Hf@Lipo (DiI‐labeled Lipo, red; FITC‐labeled Ft‐E64/Hf, green). Scale bar: 5 µm. C) Zeta potentials of various preparations ( n = 3). D) Loading amounts of Ft‐E64 and Hf in Ft‐E64/Hf ( n = 3). E) ICP‐MS analysis of Fe content across different cell populations within the TME after Ft‐E64/Hf@Lipo‐combined RT ( n = 3). F) Flow cytometry analysis of the uptake capacity of Ft‐F/Hf@Lipo by different cells. G) Study on the fusion process between Lipo (labeled with DIO) and CT26 cell membrane (labeled with DiI). Scale bar: 10 µm. H) Representative immunofluorescent images (Scale bar: 10 µm) and the quantitative analysis of γ‐H2AX in CT26 cells after different treatments ( n = 3). I) Flow cytometer analysis of the CT26 cells' apoptotic ratios. “+” represented RT. Data are means ± SD. *** p < 0.001 determined by Student's t‐test.

Article Snippet: Proteinase inhibitor E64, DOPC (1, 2‐Dioleoyl‐sn‐glycero‐3‐phosphocholine), DOPE (Dioleoyl phosphatidylethanolamine), SM (N‐acetyl‐D‐erythro‐sphingosylphosphorylcholine), and CH (3β‐Hydroxy‐5‐cholestene, 5‐Cholesten‐3β‐ol) were purchased from Shanghai Yuanye.

Techniques: Labeling, Flow Cytometry, Membrane

Journal: Advanced Science

Article Title: A Nanomodulator Enhances Radiotherapy‐Induced In Situ Cancer Vaccine by Promoting Antigen‐Presenting of Tumor‐Associated Macrophage

doi: 10.1002/advs.202502876

Figure Lengend Snippet: Ft‐E64/Hf@Lipo modulates the antigen presentation by M2 TAMs. A) The experimental design involves the efferocytosis of M2 TAMs to apoptotic CT26 cells. B) Flow cytometry analysis of M2 TAMs engulfing CT26 cells pre‐treated with Ft‐E64/Hf@Lipo (+). C) Cysteine protease activity of M2 TAMs after different treatments ( n = 3). D) The result of the DQ‐OVA antigen degradation assay after different treatments ( n = 3). E) Illustration of Ft‐E64/Hf@Lipo (+) treatment restoring the antigen presentation and CD8 + T cell activation capacity of M2 TAMs. Representative flow cytometry images and the quantification analysis of (F) MHC‐I expression on M2 TAMs, and (G) CD8 + T cell activation ( n = 3). “+” represented RT. Data are means ± SD. ** p < 0.01, *** p < 0.001, **** p < 0.0001 determined by Student's t‐test.

Article Snippet: Proteinase inhibitor E64, DOPC (1, 2‐Dioleoyl‐sn‐glycero‐3‐phosphocholine), DOPE (Dioleoyl phosphatidylethanolamine), SM (N‐acetyl‐D‐erythro‐sphingosylphosphorylcholine), and CH (3β‐Hydroxy‐5‐cholestene, 5‐Cholesten‐3β‐ol) were purchased from Shanghai Yuanye.

Techniques: Immunopeptidomics, Flow Cytometry, Activity Assay, Degradation Assay, Activation Assay, Expressing

Journal: Advanced Science

Article Title: A Nanomodulator Enhances Radiotherapy‐Induced In Situ Cancer Vaccine by Promoting Antigen‐Presenting of Tumor‐Associated Macrophage

doi: 10.1002/advs.202502876

Figure Lengend Snippet: In vivo antitumor effect of Ft‐E64/Hf@Lipo‐enhanced RT on mice with a CT26 bilateral tumor model. A) Schematic illustration of the tumor inoculation and therapeutic schedule. Representative photos of the (B) primary and (E) distant tumor after various treatments ( n = 6). Weights of the sacrificed (C) primary and (F) distant tumor on the 21st day ( n = 6). TUNEL staining of the (D) primary and (G) distant tumor section after different treatments. Scale bars: 100 µm. “+” represented RT. Data are means ± SD. * p < 0.1, ** p < 0.01 determined by Student's t‐test.

Article Snippet: Proteinase inhibitor E64, DOPC (1, 2‐Dioleoyl‐sn‐glycero‐3‐phosphocholine), DOPE (Dioleoyl phosphatidylethanolamine), SM (N‐acetyl‐D‐erythro‐sphingosylphosphorylcholine), and CH (3β‐Hydroxy‐5‐cholestene, 5‐Cholesten‐3β‐ol) were purchased from Shanghai Yuanye.

Techniques: In Vivo, TUNEL Assay, Staining

Journal: Advanced Science

Article Title: A Nanomodulator Enhances Radiotherapy‐Induced In Situ Cancer Vaccine by Promoting Antigen‐Presenting of Tumor‐Associated Macrophage

doi: 10.1002/advs.202502876

Figure Lengend Snippet: Systemic immune activation of Ft‐E64/Hf@Lipo enhanced radioimmunotherapy. A) The immunofluorescence analysis of γH2AX (Scale bar: 100 µm), CRT (Scale bar: 25 µm), and HMGB1 (Scale bar: 25 µm) in the primary tumor tissue extracted from mice. B) Representative flow cytometry images and the quantification analysis of MHC‐I molecule expression on M2 TAMs ( n = 3). C) Flow cytometry analysis of the IFN‐γ + CD8 + T cells in the primary tumor ( n = 3). D) Effector memory CD8 + T cells in the spleen of mice after various treatments. Data are means ± SD. ( n = 3). * p < 0.1, *** p < 0.001 determined by Student's t‐test.

Article Snippet: Proteinase inhibitor E64, DOPC (1, 2‐Dioleoyl‐sn‐glycero‐3‐phosphocholine), DOPE (Dioleoyl phosphatidylethanolamine), SM (N‐acetyl‐D‐erythro‐sphingosylphosphorylcholine), and CH (3β‐Hydroxy‐5‐cholestene, 5‐Cholesten‐3β‐ol) were purchased from Shanghai Yuanye.

Techniques: Activation Assay, Immunofluorescence, Flow Cytometry, Expressing

Journal: Advanced Science

Article Title: A Nanomodulator Enhances Radiotherapy‐Induced In Situ Cancer Vaccine by Promoting Antigen‐Presenting of Tumor‐Associated Macrophage

doi: 10.1002/advs.202502876

Figure Lengend Snippet: Synergistic antitumor effect of Ft‐E64/Hf@Lipo‐enhanced radioimmunotherapy and aPD‐L1 treatment in a large established CT26 tumor model. A) Schematic diagram illustrating the construction and therapeutic procedure of a CT26 large‐volume bilateral tumor‐bearing mouse model. B) Primary and F) distant tumor growth curves of CT26 large tumor‐bearing mice after various treatments ( n = 6). Representative photos of the sacrificed C) primary and G) distant tumor on the 28th day ( n = 6). The sacrificed D) primary and H) distant tumor weights ( n = 6). Individual E) primary and I) distant tumor growth curves of mice after different treatments ( n = 6). Data are means ± SD. *** p < 0.001, **** p < 0.0001 determined by Student's t‐test.

Article Snippet: Proteinase inhibitor E64, DOPC (1, 2‐Dioleoyl‐sn‐glycero‐3‐phosphocholine), DOPE (Dioleoyl phosphatidylethanolamine), SM (N‐acetyl‐D‐erythro‐sphingosylphosphorylcholine), and CH (3β‐Hydroxy‐5‐cholestene, 5‐Cholesten‐3β‐ol) were purchased from Shanghai Yuanye.

Techniques:

Journal: Nature Communications

Article Title: Integrin beta 1 facilitates non-enveloped hepatitis E virus cell entry through the recycling endosome

doi: 10.1038/s41467-025-61071-y

Figure Lengend Snippet: ( A ) S10-3 LAMP-1-GFP cells were inoculated with eHEV (MOI = 20 GE/cell) or nHEV (MOI = 30 GE/cell) for 6 h or 2 h on ice and incubated at 37 °C for 10 h or 7 h, respectively. Genomes (magenta) were detected by RNA-FISH using the ORF1 probe. Scale bar = 5 μm. ( B ) and ( C ) S10-3 cells were treated with cathepsin inhibitor E64 or DMSO and infected with ( B ) nHEV (MOI = 30 GE/cell, n = 7) or ( C ) eHEV (MOI = 20 GE/cell, n = 6). E64 was added with virus for 24 h (“during”), or 24 h post-infection and throughout the course of infection (“after”). Infectivity was assessed 5 days post-infection. ( D ) and ( E ) S10-3 cells were treated with E64 or DMSO and infected with ( D ) nHEV (MOI = 30 GE/cell, n = 9) or ( E ) eHEV (MOI = 20 GE/cell, n = 7). 8 h later, the inoculum was replaced with fresh media containing drugs. 24 h post-inoculation, HEV capsid were detected by staining and genomes as in ( A ) and quantified using CellProfiler. ( F ) Maximum projections of E64-treated S10-3 LAMP-1-GFP cells inoculated with nHEV (MOI = 30 GE/cell). 24 h later, capsids (magenta) and genomes (yellow) were detected as in ( C ). Representative of n = 6 microscope fields. ( G ) S10-3 LAMP-1-GFP cells were treated with E64 or DMSO 30 min prior to inoculation with nHEV (MOI = 30 GE/cell). After 8 h at 37 °C, inoculum was replaced with fresh media containing drugs. Genomes were detected and analysed as in ( A ). ( H ) Proposed working model on HEV cell entry. The interaction of nHEV with ITGB1 triggers internalisation through Rab11+ recycling endosomes, while eHEV is routed into Rab5a+ early endosomes. Both particles traffic through Rab7+ late endosomes and reach Lamp1+ lysosomes. The capsid and envelope are degraded by lysosomal cathepsins, allowing the release of viral genomes into the cytosol through an unknown penetration mechanism. This figure was created in BioRender. Dao Thi, V. (2025). https://BioRender.com/f6k0uqw . All replicates are from three independent experiments. Statistical analysis was performed by unpaired two-tailed Student’s t test ( A , D , E ) or one-way ANOVA ( B , C , G ). ****: p < 0.0001; ns, non-significant.

Article Snippet: Bafilomycin A (Sigma), Concanamycin A (Biomol) and E64 (Selleckchem, 25 μM) were used at the indicated concentrations.

Techniques: Incubation, Infection, Virus, Staining, Microscopy, Two Tailed Test