Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: The lnc-CTSLP8 upregulates CTSL1 as a competitive endogenous RNA and promotes ovarian cancer metastasis

doi: 10.1186/s13046-021-01957-z

Figure Lengend Snippet: CTSL1 was upregulated in ovarian cancer. a Single-cell RNA sequencing (scRNA-seq) analysis of ovarian cancer. The heatmap shows cell marker (EPCAM for epithelial cells, CD14 for monocytes/macrophages, CD4 for CD4 + T cells) expression patterns in CTSL1 + cells. b scRNA-seq in EPCAM + cells from three ovarian cancer para-tumor tissues (used as control), ovarian cancer tissue, and the matched peritoneal metastasis tissues. c Quantification of CTSL1 expression in EPCAM + cells from benign ovarian cyst, ovarian cancer, and peritoneal metastasis tissues with different CTSL1 staining. e and f Quantification of CTSL1 IHC score in different tissues. g Overall survival of ovarian cancer patients with low/high CTSL1 IHC score. h Immunoblotting of CTSL1 in different tissues. i ELISA of CTSL1 in serum and ascites of ovarian cancer patients

Article Snippet: Soluble CTSL1 protein levels in the serum and ascites of ovarian cancer patients were detected via ELISA using the Human Cathepsin L DuoSet ELISA Kit (R&D Systems, USA).

Techniques: RNA Sequencing, Marker, Expressing, Control, Staining, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: Molecular Neurodegeneration

Article Title: Processing of progranulin into granulins involves multiple lysosomal proteases and is affected in frontotemporal lobar degeneration

doi: 10.1186/s13024-021-00472-1

Figure Lengend Snippet: PGRN undergoes pH-dependent processing into multi-granulin fragments and individual granulins in vitro . a , Schematic representation of the antibodies used. Anti-paragranulin (p-Gran) in green, anti- granulin F (Gran-F) in red and anti-granulin E (Gran-E) in blue. b-d, For in vitro protease assays, 400 ng of PGRN was incubated for 20 min with or without 1 μM of each enzyme at the indicated pH. Western blotting analysis was performed using antibodies as indicated. pH-dependent cleavage products are indicated with red asterisks (*). Progranulin (PGRN), multi-granulin fragments (MGFs), cathepsin B (CTSB), cathepsin L (CTSL), cathepsin K (CTSK), cathepsin S (CTSS), asparagine endopeptidase (AEP), cathepsin V (CTSV), cathepsin G (CTSG), cathepsin E (CTSE). A representative of n = 3 replicates is shown

Article Snippet: For overexpression studies, pCMV6 mammalian expressing vectors carrying the cDNA of AEP and CTSL were ordered from Origene (#RC224975 and #RC203143).

Techniques: In Vitro, Incubation, Western Blot

Journal: Molecular Neurodegeneration

Article Title: Processing of progranulin into granulins involves multiple lysosomal proteases and is affected in frontotemporal lobar degeneration

doi: 10.1186/s13024-021-00472-1

Figure Lengend Snippet: Asparagine endopeptidase (AEP) is a PGRN protease that liberates granulin F. a-b, Differentiated SH-SY5Y cells were treated with siRNA against AEP or scramble control for 72 h. Cells were lysed and western blotting performed for endogenous AEP, PGRN or Gran-F as indicated. Mature AEP, PGRN and Gran-F were significantly decreased in AEP siRNA treated cells (* p < 0.04, ** p = 0.0010) relative to actin. Unpaired student’s t-test, error bars represent mean with standard deviation, n = 3. c, HEK293FT cells were transiently transfected with FLAG-tagged AEP (AEP FLAG or AEP OE) or FLAG alone (mock) for 24 h and cells lysates were probed for endogenous PGRN and Gran-F. AEP expression was confirmed using an anti-FLAG antibody. Each biological replicate was run on a separate western blot and normalized to actin. d-e, Quantification of c showing that overexpression of AEP decreased endogenous PGRN and increased Gran-F levels. Paired t-test analysis, p values are indicated, error bars represent mean with standard deviation, n = 4. f, HEK293FT cells were transiently transfected with FLAG-tagged CTSL (CTSL FLAG) or FLAG alone (mock) for 24 h and cells lysates were probed for endogenous PGRN and Gran-F. CTSL expression was confirmed using an anti-FLAG antibody. Each biological replicate was run on a separate western blot and normalized to actin. g-h, Quantification of f showing that overexpression of CTSL decreased endogenous PGRN but did not alter Gran-F levels. Paired t-test analysis, p values are indicated, error bars represent mean with standard deviation, n = 4

Article Snippet: For overexpression studies, pCMV6 mammalian expressing vectors carrying the cDNA of AEP and CTSL were ordered from Origene (#RC224975 and #RC203143).

Techniques: Control, Western Blot, Standard Deviation, Transfection, Expressing, Over Expression

Journal: Molecular Neurodegeneration

Article Title: Processing of progranulin into granulins involves multiple lysosomal proteases and is affected in frontotemporal lobar degeneration

doi: 10.1186/s13024-021-00472-1

Figure Lengend Snippet: AEP activity in FTLD-TDP- Pgrn subjects versus controls. AEP ( a ) and CTSL ( b ) activity are compared between the degenerating middle frontal gyrus (MFG) and the non-degenerating inferior occipital cortex (IOC) regions in control versus FTLD-TDP- Pgrn subject neuropathological samples (*, p = 0.013). Unpaired two-tailed student t-test was used to compare. Error bars represent mean with standard deviation

Article Snippet: For overexpression studies, pCMV6 mammalian expressing vectors carrying the cDNA of AEP and CTSL were ordered from Origene (#RC224975 and #RC203143).

Techniques: Activity Assay, Control, Two Tailed Test, Standard Deviation

Journal: Frontiers in pharmacology

Article Title: Cathepsin L promotes chemresistance to neuroblastoma by modulating serglycin.

doi: 10.3389/fphar.2022.920022

Figure Lengend Snippet: FIGURE 4 The expression of multi-drug resistance proteins and autophagy level of NB cells after knockdown of CTSL (A) Western blot showing multi-drug resistance proteins expression in SK-N-BE (2) cells after CTSL silencing (B) Lysosome staining assay showing the quantity and intracellular location of lysosome in SK-N-BE (2) cells with different treatments (red bar: 20 μm) (C) The expression of LC3-I and LC3-II in SK-N-BE (2) cells were analyzed using Western blot. NS: not statistically significant, **, p < 0.01, ***, p < 0.001.

Article Snippet: CTSL, ABCB1, ABCG2, LC3, Bax, Bcl-2, and GAPDH primary anti-human antibodies were all purchased from Cell Signaling Technology.

Techniques: Expressing, Knockdown, Western Blot, Staining

Journal: Frontiers in pharmacology

Article Title: Cathepsin L promotes chemresistance to neuroblastoma by modulating serglycin.

doi: 10.3389/fphar.2022.920022

Figure Lengend Snippet: FIGURE 5 The apoptosis level of NB cells after knockdown of CTSL (A) Western blot showing apoptosis proteins expression in SK-N-BE (2) cells with different treatments (B) Immunofluorescence assay showing the expression and intracellular location of CTSL and Bcl-2 in SK-N-BE (2) cells (red bar: 10 μm). NS: not statistically significant, *, p < 0.05, **, p < 0.01, ***, p < 0.001.

Article Snippet: CTSL, ABCB1, ABCG2, LC3, Bax, Bcl-2, and GAPDH primary anti-human antibodies were all purchased from Cell Signaling Technology.

Techniques: Knockdown, Western Blot, Expressing

Journal: Human Genomics

Article Title: APM⁺ macrophages associated with plaque vulnerability via MIF–CD74 signaling: a multi-omics study

doi: 10.1186/s40246-025-00869-9

Figure Lengend Snippet: The expression and co-localization of APM-related biomarkers with macrophage markers in AS mice tissues. A qPCR results showed that biomarkers expression was high in AS mice tissues. B WB results showed that three biomarkers (GPNMB; LILRB4 and CTSL) relative protein levels were high in AS mice tissues. The reference is GAPDH. C – E Relative expression levels of GPNMB ( C ), LILRB4 ( D ), and CTSL ( E ) in NC and AS groups, assessed by WB. Data are shown as medians with interquartile ranges. Statistical significance was determined using Mann–Whitney U tests. * p < 0.05 and “ns” indicates not significant ( p > 0.05). F mIF staining showing the spatial distribution and co-localization of Gpnmb (red), Spp1 (green), and Cd68 (yellow) in AS mouse plaques. G mIF staining showing the spatial distribution and co-localization of Ctsl (red), Spp1 (green), and Cd68 (yellow) in AS mouse plaques. H mIF staining showing the spatial distribution and co-localization of Lilrb4 (red), Spp1 (green), and Cd68 (yellow) in AS mouse plaques. DAPI (blue) labels nuclei. Dashed white lines indicate the plaque area in the aortic sinus. Scale bars: 200 μm

Article Snippet: The following primary antibodies are used: GAPDH (GB15004, Servicebio, China); GPNMB (66926-1-Ig, proteintech, China); CTSL (66914-1-Ig, proteintech, China); and LILRB4 (28422-1-AP, proteintech, China).

Techniques: Expressing, MANN-WHITNEY, Staining

Journal: Nature Communications

Article Title: Cathepsin L as a dual-target to mitigate muscle wasting while enhancing anti-tumor efficacy of anti-PD-L1

doi: 10.1038/s41467-025-64500-0

Figure Lengend Snippet: A Human PPI network of CTSL-BNIP3 neighbors colored by log 2 FC (tumor versus matched normal lung) in TCGA LUAD. B Immunofluorescence of CTSL (green) and BNIP3 (red) in LUAD tissue microarray cores: normal adjacent, n = 22; tumor cores, n = 128. Bar graph shows proportions of BNIP3-high/-low within the CTSL-high and CTSL-low tumors. C Kaplan–Meier overall survival for TCGA LUAD stratified by combined CTSL and BNIP3 expression ( CTSL-BNIP3 -high, red, n = 183; CTSL-BNIP3 -low, black, n = 156). D GSEA plots of ALONSO_METASTASIS_UP in CTSL-BNIP3 high versus low groups in TCGA LUAD (left) and in TB mice (right). E Representative WB of indicated proteins in TB muscle. Two mice per group are shown ( n = 4 per group); quantification of all four mice in Supplementary Fig. . Samples derive from the same experiment; different gels for Ctsl, N-Cad, Gapdh, and another for Bnip3 were processed in parallel. F WB of indicated proteins in A549 cells treated with or without CTSLi; GAPDH as loading control; quantification from four independent biological replicates in Supplementary Fig. . Samples derive from the same experiment; different gels for CTSL and another for N-Cad, Bnip3, GAPDH, were processed in parallel. Migration ( G ) and invasion ( H ) assays for A549 cells treated with or without CTSLi. Bar graphs represent quantified gap closure ( G ) and invaded cells ( H ); biological triplicates per group. WB of indicated proteins in A549 cells transfected with si-CTSL ( I ) or si-BNIP3 ( J ); quantification from four independent biological replicates in Supplementary Fig. . For I , samples derive from the same experiment; different gels for CTSL and another for N-Cad, Bnip3, GAPDH, were processed in parallel. For J , samples derive from the same experiment; different gels for CTSL, N-cad, GAPDH, and another for BNIP3 were processed in parallel. Quantified migration after si-CTSL ( K ) or si-BNIP3 ( L ) transfection; biological triplicates per group. M Working model: combined αPD-L1 and CTSLi therapy suppresses tumorigenesis. For B , one-sided Fisher’s exact test; for C , log-rank test; for D , GSEA; for G , H , K , L , data are mean ± SEM, two-sided unpaired t -test. Source data are provided as a Source Data file.

Article Snippet: For gene silencing, A549 cells were seeded and then transfected with 20 nM siRNAs against CTSL (SR319840, OriGene Technologies) or BNIP3 (SR319494, OriGene Technologies) using Lipofectamine RNAiMax for 48 h following the manufacturer’s protocol.

Techniques: Immunofluorescence, Microarray, Expressing, Control, Migration, Transfection

Journal: Nature Communications

Article Title: Effective extracellular payload release and immunomodulatory interactions govern the therapeutic effect of trastuzumab deruxtecan (T-DXd)

doi: 10.1038/s41467-025-58266-8

Figure Lengend Snippet: a MDA-MB-231 tumors treated with T-DXd (4 mg/kg) were co-injected with fivefold higher dose of trastuzumab (20 mg/kg) to assess T-DXd efficacy after HER2 binding competition. Mixed-effects analysis (REML) with Tukey’s multiple comparisons ( n = 10). Arrows indicate treatment administered. b Liquid chromatography-mass spectrometry (LC-MS) quantification of DXd molecules in tumors treated with T-DXd (10 mg/kg) for 3 days. DXd levels per tumor mass were quantified. One-way ANOVA with Tukey’s multiple comparisons test ( n = 13). c Systemic DXd payload levels in plasma from same treated animals in ( b ). DXd levels (ng) per plasma volume (mL) were quantified. No statistical significance between plasma groups was observed ( n = 13). d T-DXd was incubated with recombinant human CTSL or CTSB for 6 h in low pH assay buffer as described in Methods. Released DXd levels were quantified by LC-MS. e Cytotoxicity of cleaved T-DXd was assessed on MDA-MB-468 cells. f Immunohistochemistry analysis of primary core needle biopsies from BC patients. Representative HER2 expression (top row) is shown. CTSL expression and localization in the same patient’s biopsy are shown (bottom row). Positive staining in tumor beds (red arrows) and stromal compartments (black arrows) are indicated. g – i Tissue microarray comprising 321 invasive BC, 20 DCIS, and 49 normal breast samples were assessed for CTSL expression via IHC. Pixelwise H-scores were used for quantification by QuPath. One-way ANOVA with Tukey’s multiple comparisons test. g CTSL expression differences in invasive BC, DCIS, and normal breast. h CTSL expression among lymph node-negative, lymph node-positive, or metastatic BC. i CTSL expression among BC with different HER2 IHC scores.

Article Snippet: When indicated, 3 μM of CTSL inhibitor Z-Phe-Phe-FMK (MCE, #HY-115733) or 3 μM of chloroquine (MCE, #HY-17589A) were added before ADC treatment.

Techniques: Injection, Binding Assay, Liquid Chromatography, Mass Spectrometry, Liquid Chromatography with Mass Spectroscopy, Clinical Proteomics, Incubation, Recombinant, Immunohistochemistry, Expressing, Staining, Microarray

Journal: Nature Communications

Article Title: Effective extracellular payload release and immunomodulatory interactions govern the therapeutic effect of trastuzumab deruxtecan (T-DXd)

doi: 10.1038/s41467-025-58266-8

Figure Lengend Snippet: a , b Au565 cells were assessed for T-DXd ( a ) or T-DM1 ( b ) cytotoxicity in vitro in the presence of CTSL inhibitor (3 µM Z-Phe-Phe-FMK). c Au565 cells overexpressing CTSL or lacking CTSL were assessed for T-DXd cytotoxicity in vitro. d Extracellular CTSL secretion by MDA-MB-231 lines was quantified with ELISA analysis of conditioned media. e Secreted proteins in conditioned media of indicated cell lines were concentrated and quantified for CTSL enzymatic activity in assay buffer. a – e Two-way ANOVA with Tukey’s multiple comparisons test ( n = 3 per group). Nonlinear regression curve fit to calculate IC50 values. f Parental and CTSL-overexpressed MDA-MB-231 cells were implanted in SCID mice, and treated with T-DXd (5 mg/kg) or control PBS. g Control-KO and CTSL-KO MDA-MB-231 cells were implanted in SCID mice, and treated with T-DXd (10 mg/kg) or control PBS. f – h Mixed-effects analysis (REML) with Tukey’s multiple comparisons ( n = 10). Arrows indicate time of treatments administered. h Tumor sizes (mm 3 ) measured at end point for studies using MDA-MB-231 CTSL-KO lines. i Immunohistochemistry verifications of CTSL expression in parental or CTSL-modified MDA-MB-231 tumors. j LC-MS quantification of DXd in MDA-MB-231 parental ( n = 15) versus CTSL-overexpressed tumors ( n = 10), treated with T-DXd (10 mg/kg) for 5 days. k LC-MS quantification of DXd in MDA-MB-231 control-KO versus three different CTSL-KO pools, treated with T-DXd (10 mg/kg) for 3 days ( n = 10 for all groups, except n = 6 for CTSL-KO #2). h , k One-way ANOVA with Tukey’s multiple comparisons. j Two-sided Mann–Whitney test. All data is presented as mean ± SEM with p values indicated.

Article Snippet: When indicated, 3 μM of CTSL inhibitor Z-Phe-Phe-FMK (MCE, #HY-115733) or 3 μM of chloroquine (MCE, #HY-17589A) were added before ADC treatment.

Techniques: In Vitro, Enzyme-linked Immunosorbent Assay, Activity Assay, Control, Immunohistochemistry, Expressing, Modification, Liquid Chromatography with Mass Spectroscopy, MANN-WHITNEY