Journal: Brain, behavior, and immunity

Article Title: Single-cell RNA sequencing of peripheral blood reveals that monocytes with high cathepsin S expression aggravate cerebral ischemia-reperfusion injury.

doi: 10.1016/j.bbi.2022.11.001

Figure Lengend Snippet: Fig. 3. Gene functional analysis of cell clusters. (A) Biological process and molecular function analyses for cluster 2. (B) Combined analysis of the genes in the aforementioned significant enrichment pathways identified two representative genes, S100a8 and Saa3. EX stimulus, response to external stimulus; SR binding, signaling receptor binding. (C) Molecular distribution of S100a8 and Saa3. (D) Biological process and molecular function analyses, and functional enrichment analysis for group cluster 10. (E) Combined analysis of the genes in the aforementioned significant enrichment pathways identified the representative genes for Ctss. (F) Molecular distribution of Ctss.

Article Snippet: Inflammatory factors of IL-1, IL-6 and TNF in brain tissues are detected also by Mouse CTSS Quantikine ELISA Kit (CUSABIO, Wuhan, China) according to the manufacturer’s instructions.

Techniques: Functional Assay, Binding Assay

Journal: Brain, behavior, and immunity

Article Title: Single-cell RNA sequencing of peripheral blood reveals that monocytes with high cathepsin S expression aggravate cerebral ischemia-reperfusion injury.

doi: 10.1016/j.bbi.2022.11.001

Figure Lengend Snippet: Fig. 4. Peripheral and central cathepsin S are increased in mice after tMCAO. C57BL/6N mice were subjected to ischemia for 90 min or sham operation followed by 0,1, 3, 7, and 14 days of reperfusion. (A) ELISA showed that peripheral CTSS levels were significantly increased after tMCAO (n = 4). (B) Representative immunoblots and quantification of CTSS (relative to β-Tublin) in the ischemic ipsilateral brain at various time points after tMCAO (n = 4). (C) The mRNA abundance of Ctss in the ischemic ipsilateral brain normalized to that of the Gapdh gene (n = 5). (D and E) Representative images and quantitation of in situ hybridization results for Ctss and C1q in the sham group 3, 7 days after tMCAO (n = 4, magnification, 100 or 200; scale bar, 100 or 50 μm; yellow triangle indicates C1q + Ctss + cells; the dotted line depicts the edge of the infarct). (F) Peripheral CTSS levels 24 h after stroke in humans from the GEO analysis database. Data are presented as mean ± SEM, *p < 0.05; **p < 0.01; ***p < 0.001; n.s. not significant.

Article Snippet: Inflammatory factors of IL-1, IL-6 and TNF in brain tissues are detected also by Mouse CTSS Quantikine ELISA Kit (CUSABIO, Wuhan, China) according to the manufacturer’s instructions.

Techniques: Enzyme-linked Immunosorbent Assay, Western Blot, Quantitation Assay, In Situ Hybridization

Journal: Brain, behavior, and immunity

Article Title: Single-cell RNA sequencing of peripheral blood reveals that monocytes with high cathepsin S expression aggravate cerebral ischemia-reperfusion injury.

doi: 10.1016/j.bbi.2022.11.001

Figure Lengend Snippet: Fig. 5. Ctss KO alleviates mice neurological impairments. (A) Ctss KO efficiency by qRT-PCR (n = 3). (B and C) Representative images and relative quantification of cortical cerebral blood flow (CBF) by two-dimensional laser speckle imaging (n = 6). (D) Representative photographs of coronal brain sections of Ctss KO and WT mice stained with TTC after tMCAO and relative infarct volume (n = 6). (E) Neurological Longa scores of Ctss KO and WT mice 3 days after tMCAO (n = 6). (F) Rotarod test of Ctss KO and WT mice after tMCAO (n = 8). (G) Pole test of Ctss KO and WT mice after tMCAO (n = 8).

Article Snippet: Inflammatory factors of IL-1, IL-6 and TNF in brain tissues are detected also by Mouse CTSS Quantikine ELISA Kit (CUSABIO, Wuhan, China) according to the manufacturer’s instructions.

Techniques: Quantitative RT-PCR, Quantitative Proteomics, Imaging, Staining

Journal: Brain, behavior, and immunity

Article Title: Single-cell RNA sequencing of peripheral blood reveals that monocytes with high cathepsin S expression aggravate cerebral ischemia-reperfusion injury.

doi: 10.1016/j.bbi.2022.11.001

Figure Lengend Snippet: Fig. 6. Ctss KO protects mice from cerebral I/R injury. Ctss KO and WT mice (6–8 weeks old) were subjected to ischemia for 90 min or sham surgery, followed by reperfusion for 3 days. (A) Representative confocal images of vascular permeability and quantitative analyses of extravascular leakage in WT and Ctss KO mice cortex (green: thiocarbamoyl dextrans; red: CD31; n = 6, magnification, 100 or 400, scale bar, 100 or 25 μm. White boxes indicate vascular leakage, and the dotted line depicts the edge of the infarct). (B) The number of total infiltrating CD45hi leukocytes in the brain according to flow cytometry (n = 6). (C–E) Brain tissue expression of IL-1β, IL-6, and TNF-α as determined by ELISA kit after tMCAO (n = 6). Data are presented as mean ± SEM; *p < 0.05; **p < 0.01; ***p < 0.001; n.s. not significant.

Article Snippet: Inflammatory factors of IL-1, IL-6 and TNF in brain tissues are detected also by Mouse CTSS Quantikine ELISA Kit (CUSABIO, Wuhan, China) according to the manufacturer’s instructions.

Techniques: Permeability, Flow Cytometry, Expressing, Enzyme-linked Immunosorbent Assay

Journal: Brain, behavior, and immunity

Article Title: Single-cell RNA sequencing of peripheral blood reveals that monocytes with high cathepsin S expression aggravate cerebral ischemia-reperfusion injury.

doi: 10.1016/j.bbi.2022.11.001

Figure Lengend Snippet: Fig. 7. CTSS promotes the in vitro cleavage of vascular tight junction proteins. (A) Interaction prediction between JAMs and CTSS. (B) Verification of the interaction between JAM-A and CTSS by co-immunoprecipitation experiment. (C) Silver staining analysis of CTSS-mediated cleavage of recombinant tight junction proteins (JAM-A, JAM-B, and JAM-C) at pH 4.5, 7.2, and 9.8, respectively, in the presence or absence of the CTSS inhibitor; the CTSS inhibitor was added at pH of 7.2.

Article Snippet: Inflammatory factors of IL-1, IL-6 and TNF in brain tissues are detected also by Mouse CTSS Quantikine ELISA Kit (CUSABIO, Wuhan, China) according to the manufacturer’s instructions.

Techniques: In Vitro, Immunoprecipitation, Silver Staining, Recombinant

Journal: Brain, behavior, and immunity

Article Title: Single-cell RNA sequencing of peripheral blood reveals that monocytes with high cathepsin S expression aggravate cerebral ischemia-reperfusion injury.

doi: 10.1016/j.bbi.2022.11.001

Figure Lengend Snippet: Fig. 8. CTSS inhibitor protects mice from cerebral I/R injury. (A) Representative photographs of coronal brain sections and relative infarct volume in mice brain stained with TTC 3 days after tMCAO in the CTSS inhibitor and control mice (n = 8). (B) Neurological scores of inhibitor-treated and control-treated mice 3 days after tMCAO (n = 8). (C) Representative confocal images and fluorescence quantification of vascular permeability inhibitor-treated and control mice (n = 6). Data are presented as mean ± SEM; *p < 0.05; **p < 0.01; ***p < 0.001; n.s. not significant.

Article Snippet: Inflammatory factors of IL-1, IL-6 and TNF in brain tissues are detected also by Mouse CTSS Quantikine ELISA Kit (CUSABIO, Wuhan, China) according to the manufacturer’s instructions.

Techniques: Staining, Control, Fluorescence, Permeability

Journal: Journal of Oncology

Article Title: A Novel Role for Cathepsin S as a Potential Biomarker in Triple Negative Breast Cancer

doi: 10.1155/2019/3980273

Figure Lengend Snippet: Representative images of CTSS expression in patient samples. CTSS-specific expression is indicated by brown staining versus blue nuclear counter staining. Samples represent either epithelial or stromal CTSS staining. Black arrows indicate areas of CTSS expression, which was separated according to high (3), moderate (2), low (1), or no expression (0).

Article Snippet: Sections were cut from the TMA blocks to a diameter of 4 μ m using a rotary microtome, dried at 37°C overnight, and then used for immunohistochemical staining with rabbit anti-human CTSS antibody (1:250) (HPA002988, Atlas Antibodies, UK) using an automated immuno-stainer (Leica Bond-Max, UK).

Techniques: Expressing, Staining

Journal: Journal of Oncology

Article Title: A Novel Role for Cathepsin S as a Potential Biomarker in Triple Negative Breast Cancer

doi: 10.1155/2019/3980273

Figure Lengend Snippet: Epithelial cell CTSS expression in TNBC patients is associated with an M1 macrophage phenotype. Immunohistochemical epithelial CTSS scores were matched with (a) macrophage marker CD68, (b) M1 polarisation marker CD14 and (c) M2 polarisation marker CD163. Shading indicates proportion of IHC score for each marker. Statistical significance determined by Chi-Square analysis. Macrophage polarisation was analysed using gene expression algorithms and correlated with CTSS IHC expression generating (d) M2/M1 and (e) CD68/CD8 signature scores. Statistical significance determined by one-way ANOVA. N=number of patients. ∗ p<0.05, ∗∗ p<0.01, and ∗∗∗ p<0.001.

Article Snippet: Sections were cut from the TMA blocks to a diameter of 4 μ m using a rotary microtome, dried at 37°C overnight, and then used for immunohistochemical staining with rabbit anti-human CTSS antibody (1:250) (HPA002988, Atlas Antibodies, UK) using an automated immuno-stainer (Leica Bond-Max, UK).

Techniques: Expressing, Immunohistochemical staining, Marker, Gene Expression

Journal: Genes & Development

Article Title: Combined deletion of cathepsin protease family members reveals compensatory mechanisms in cancer

doi: 10.1101/gad.270439.115

Figure Lengend Snippet: Simultaneous deletion of CtsB and CtsS reduces angiogenic switching and tumor growth but does not affect tumor invasion. ( A ) Angiogenic switching was assessed in 10.5-wk-old wild-type RT2 or CtsB −/− S −/− RT2 mice ( n = 10 mice for both genotypes) by manually counting the number of angiogenic islets in the pancreas. The graph shows the average number of angiogenic islets per mouse. ( B ) Cumulative tumor volume, represented as the sum of the volumes of all tumors per mouse, was calculated for 13.5-wk-old wild-type RT2 ( n = 57) and CtsB −/− S −/− RT2 ( n = 46) mice. ( C ) Graph depicting the average number of tumors per mouse in wild-type and CtsB −/− S −/− RT2 animals at the 13.5-wk endpoint. The following numbers of animals were analyzed per group: wild-type RT2, n = 52; CtsB −/− S −/− RT2, n = 37. ( D ) Quantitation of Ki67 + cells in wild-type and CtsB −/− S −/− RT2 tumors relative to the total number of DAPI + cells showed an 85% decrease in cell proliferation in tumors deficient for both CtsB and CtsS . All tumors from five wild-type RT2 and 11 CtsB −/− S −/− RT2 mice were analyzed. ( E ) Graph showing the proportions of encapsulated, microinvasive (IC1), and invasive (IC2) carcinomas in wild-type RT2 and CtsB −/− S −/− RT2 mice at 13.5 wk. The following numbers of samples were analyzed: wild-type RT2, 18 mice, 97 tumors; CtsB −/− S −/− RT2, 14 mice, 68 tumors. The graphs show mean + SEM. Statistical significance was calculated by unpaired two-tailed Student's t -test ( A – D ) or using a cumulative logit model with generalized estimating equations to correct for correlations within individual mice ( E ). (n.s.) Nonsignificant; (***) P < 0.001.

Article Snippet: TaqMan probes (Applied Biosystems) were used for quantifying expression of CtsB (Mm00514439_m1), CtsZ (Mm00517697_m1), CtsC (Mm00515580_m1), CtsH (Mm00514455_m1), CtsS (Mm00457902_m1), CtsL (Mm00515597_m1), CstC (Mm00438347_m1), Mmp2 (Mm00439502_m1), Mmp3 (Mm00440295_m1), Mmp9 (Mm00600163_m1), Mmp12 (Mm00500554_m1), Mmp13 (Mm00439491_m1), Mmp14 (Mm00485054_m1), Mmp15 (Mm00485062_m1), Csf-1 (Mm00432688_m1), Ccl2 (Mm00441242_m1), Ccl5 (Mm01302428_m1), Cxcl2 (Mm00436450_m1), Cxcl14 (Mm00444699_m1), Cxcl15 (Mm00469712_m1), and Vegf-a (Mm00437304_m1).

Techniques: Quantitation Assay, Two Tailed Test

Journal: Genes & Development

Article Title: Combined deletion of cathepsin protease family members reveals compensatory mechanisms in cancer

doi: 10.1101/gad.270439.115

Figure Lengend Snippet: Analysis of TFMs in the CtsZ promoter reveals a unique NFκB motif and elevated nuclear p65 levels in CtsB −/− S −/− RT2 macrophages. ( A ) Venn diagram demonstrating the overlap of predicted TFMs present in the CtsZ promoter compared with the CtsB , CtsC , CtsH , CtsL , and CtsS promoters. There are nine TFMs present in the CtsZ promoter that were not identified in any of these other cathepsin family members. Meanwhile, 83 TFMs were present in at least one of the other cathepsins but absent in the CtsZ promoter (see Supplemental Table 1 for a full list of TFMs). ( B ) The CtsZ promoter is shown with exons and the 5′ untranslated region (UTR) on the top bar and nine TFMs listed below . An NFκB consensus site is highlighted in red upstream of the 5′ UTR. ( C ) Wild-type RT2 or CtsB −/− S −/− RT2 BMDMs were subjected to subcellular fractionation, and lysates of the nuclear fraction ( top two panels) or cytoplasmic fraction ( bottom two panels) were isolated for immunoblotting of the NFκB subunit p65 and lamin A/C or p65 and actin. Results are representative of n = 3 independent biological replicates. ( D ) Quantification of p65—normalized to lamin A/C or actin for the nuclear and cytoplasmic fractions, respectively, using ImageJ software—showed a significant increase in p65 expression in the nucleus of CtsB −/− S −/− RT2 BMDMs. n = 3 replicate experiments. Statistical significance was calculated by unpaired two-tailed Student's t -test. (*) P < 0.05; (**) P < 0.01.

Article Snippet: TaqMan probes (Applied Biosystems) were used for quantifying expression of CtsB (Mm00514439_m1), CtsZ (Mm00517697_m1), CtsC (Mm00515580_m1), CtsH (Mm00514455_m1), CtsS (Mm00457902_m1), CtsL (Mm00515597_m1), CstC (Mm00438347_m1), Mmp2 (Mm00439502_m1), Mmp3 (Mm00440295_m1), Mmp9 (Mm00600163_m1), Mmp12 (Mm00500554_m1), Mmp13 (Mm00439491_m1), Mmp14 (Mm00485054_m1), Mmp15 (Mm00485062_m1), Csf-1 (Mm00432688_m1), Ccl2 (Mm00441242_m1), Ccl5 (Mm01302428_m1), Cxcl2 (Mm00436450_m1), Cxcl14 (Mm00444699_m1), Cxcl15 (Mm00469712_m1), and Vegf-a (Mm00437304_m1).

Techniques: Fractionation, Isolation, Western Blot, Software, Expressing, Two Tailed Test

Journal: Genes & Development

Article Title: Combined deletion of cathepsin protease family members reveals compensatory mechanisms in cancer

doi: 10.1101/gad.270439.115

Figure Lengend Snippet: Deletion of CtsZ in CtsB −/− S −/− RT2 animals reduces macrophage infiltration and tumor invasion. ( A ) Graph depicting the cumulative tumor burden, represented as the sum of the volumes of all tumors per mouse, in wild-type RT2 and CtsB −/− S −/− Z −/− RT2 animals at the 13.5-wk endpoint. Numbers of mice per group were as follows: wild-type RT2, n = 57; CtsB −/− S −/− Z −/− RT2, n = 14. ( B ) Graph depicting the average number of tumors per mouse in wild-type RT2 and CtsB −/− S −/− Z −/− RT2 animals at 13.5 wk. The following numbers of animals were analyzed per group: wild-type RT2, n = 58; CtsB −/− S −/− Z −/− RT2, n = 18. ( C ) Graph showing the proportions of encapsulated, microinvasive, and invasive carcinomas in CtsB −/− S −/− RT2 and CtsB −/− S −/− Z −/− RT2 mice at 13.5 wk. The following number of mice were analyzed: CtsB −/− S −/− RT2, 14 mice, 68 tumors; CtsB −/− S −/− Z −/− RT2, 13 mice, 32 tumors. ( D ) Quantitation of cleaved caspase-3 (CC3) staining in wild-type RT2 and CtsB −/− S −/− Z −/− RT2 tumors relative to the total number of DAPI + cells revealed a significant 3.6-fold increase in apoptosis in tumors deficient for CtsB , CtsS , and CtsZ . Tumors from 11 wild-type RT2 and seven CtsB −/− S −/− Z −/− RT2 mice were analyzed. ( E ) Quantitation of Ki67 staining in wild-type RT2 ( n = 5) and CtsB −/− S −/− Z −/− RT2 ( n = 7) tumors relative to the total number of DAPI + cells. This revealed a 53% decrease in cell proliferation in tumors simultaneously deficient for CtsB , CtsS , and CtsZ . ( F ) Quantification of CD31 + endothelial cells in wild-type RT2 ( n = 4) and CtsB −/− S −/− Z −/− RT2 ( n = 10) tumors relative to the total number of DAPI + cells, as determined by immunostaining of tissue sections. This analysis revealed a significant decrease in tumor vascularization in CtsB −/− S −/− Z −/− RT2 mice. ( G ) Quantification of Iba + macrophages in wild-type RT2 ( n = 108), CtsB −/− S −/− RT2 ( n = 28), and CtsB −/− S −/− Z −/− RT2 ( n = 14) tumors relative to the total number of DAPI + cells. This analysis revealed that the increase in TAM numbers observed in CtsB −/− S −/− RT2 tumors is reversed when CtsZ is deleted in these tumors.

Article Snippet: TaqMan probes (Applied Biosystems) were used for quantifying expression of CtsB (Mm00514439_m1), CtsZ (Mm00517697_m1), CtsC (Mm00515580_m1), CtsH (Mm00514455_m1), CtsS (Mm00457902_m1), CtsL (Mm00515597_m1), CstC (Mm00438347_m1), Mmp2 (Mm00439502_m1), Mmp3 (Mm00440295_m1), Mmp9 (Mm00600163_m1), Mmp12 (Mm00500554_m1), Mmp13 (Mm00439491_m1), Mmp14 (Mm00485054_m1), Mmp15 (Mm00485062_m1), Csf-1 (Mm00432688_m1), Ccl2 (Mm00441242_m1), Ccl5 (Mm01302428_m1), Cxcl2 (Mm00436450_m1), Cxcl14 (Mm00444699_m1), Cxcl15 (Mm00469712_m1), and Vegf-a (Mm00437304_m1).

Techniques: Quantitation Assay, Staining, Immunostaining

Journal: Journal of Biomedical Science

Article Title: PPARγ activation improves the microenvironment of perivascular adipose tissue and attenuates aortic stiffening in obesity

doi: 10.1186/s12929-021-00720-y

Figure Lengend Snippet: Expression and location of elastolytic enzymes in the aorta and PVAT. Expression of genes for cathepsins and MMPs in the a aorta ( n = 7, 5, and 6 for control, ob/ob , and ob/ob + Piog groups respectively) and b PVAT ( n = 3, 5, and 6 for control, ob/ob , ob/ob + Piog groups respectively). mRNA amount is expressed relative to the average expression in control mice. * P < 0.05, ** P < 0.01, and *** P < 0.001. ND, not detectable. c Immunofluorescence staining for CTSS (green), MMP-12 (red) and F4/80 (blue) in the thoracic aorta and PVAT. The DAPI nuclear counterstain appears light blue. Magnification of the square in ( c ) is shown in ( d ). The while arrow indicates the location of elastic fiber break with increased signals of MMP-12 and F4/80. e Plasma levels of CTSS, MMP-12, and MCP-1 by ELISA (n = 7, 5, and 6 for control, ob/ob , and ob/ob + Piog groups respectively). All data are resulted from 3-month-old male mice. Original magnification × 400 for ( c ) and × 1200 for ( d )

Article Snippet: Plasma levels of cathepsin S, MMP-12, and MCP-1 in mice were measured using respective mouse ELISA kits (CSB-EL006204MO, CUSABIO; ab246540, Abcam; and MJE00B, R&D Systems).

Techniques: Expressing, Control, Immunofluorescence, Staining, Clinical Proteomics, Enzyme-linked Immunosorbent Assay

Journal: Aging (Albany NY)

Article Title: Amyotrophic lateral sclerosis, gene deregulation in the anterior horn of the spinal cord and frontal cortex area 8: implications in frontotemporal lobar degeneration

doi: 10.18632/aging.101195

Figure Lengend Snippet: Main significant clusters of altered genes in spinal cord of ALS samples

Article Snippet: Cathepsin S , CTSS , Hs00356423_m1.

Techniques: Activation Assay, Coagulation, Modification, Membrane, Binding Assay, Activity Assay, Chemotaxis Assay, Cell Differentiation

Journal: Aging (Albany NY)

Article Title: Amyotrophic lateral sclerosis, gene deregulation in the anterior horn of the spinal cord and frontal cortex area 8: implications in frontotemporal lobar degeneration

doi: 10.18632/aging.101195

Figure Lengend Snippet: Genes, gene symbols and TaqMan probes used for the study of gene expression in the anterior horn of the spinal cord and frontal cortex area 8 in ALS cases and controls including probes for normalization ( AARS , GUS-β , HPRT-1 and XPNPEP-1 )

Article Snippet: Cathepsin S , CTSS , Hs00356423_m1.

Techniques: Gene Expression, Immunopeptidomics