Journal: Journal of Translational Medicine

Article Title: M2-like GAMs secreting CSTA drive glioblastoma progression via the ITGB4-TGFB1 feedback axis

doi: 10.1186/s12967-026-08009-0

Figure Lengend Snippet: CSTA is a secretory signal of M2-like GAMs. ( A ) The neural network was constructed using the random forest method, comprising 16 immune signature genes as the input layer, 5 hidden layers, and an output layer. ( B ) UMAP illustrating the spatial distribution patterns of distinct cell subpopulations in GBM scRNA-seq data from GSE182109 . ( C ) Differential expression profiles of immune signature genes across various cell subpopulations, where bubble size denotes the positive rate within each subpopulation and color intensity indicates the expression level. ( D ) Peak plot showing the distribution of different GAMs subpopulations along the pseudotime axis. ( E ) Differential expression patterns of M1/M2 macrophage markers along the pseudotime axis. ( F ) Differences in basal CSTA protein levels between GAMs and glioma cell lines. G , H. Differences in RNA ( G ) and protein ( H ) levels of CSTA, CD206, and ARG1 in BV2 cells with or without IL-4 treatment. I , J. Immunofluorescence (IF) co-localization results of ARG1 ( I ) and CD206 ( J ) with CSTA in GBM patient tissue sections. K , L. Correlation analysis of fluorescence intensity in co-localized cells between ARG1 ( K ) and CD206 ( L ) with CSTA in GBM patient tissue sections. M , N. ELISA assay showed the changes in CSTA concentration in the supernatants of BV2 ( M ) and HMC3 ( N ) cells with IL-4 induction time. O . Western blot (WB) results showing the effect of CSTA knockdown on protein levels of M2-like polarization markers ARG1 and CD206 in BV2 cells. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001

Article Snippet: Six standard concentrations (0, 5, 10, 15, 20, 25 ng/mL) were prepared with recombinant CSTA protein (MCE, HY-P7868).

Techniques: Construct, Quantitative Proteomics, Expressing, Immunofluorescence, Fluorescence, Enzyme-linked Immunosorbent Assay, Concentration Assay, Western Blot, Knockdown

Journal: Journal of Translational Medicine

Article Title: M2-like GAMs secreting CSTA drive glioblastoma progression via the ITGB4-TGFB1 feedback axis

doi: 10.1186/s12967-026-08009-0

Figure Lengend Snippet: CSTA enhances GBM cells malignancy. A. The CCK-8 assay showing the correlation of CT2A cell proliferation rate with CSTA stimulation concentration and time. B , C , D. The EdU assay displaying the effect on CT2A proliferation in BV2-CT2A co-culture system under CSTA recombinant protein stimulation alone ( B ), CSTA intervention after BV2 M2-like polarization ( C ), and exogenous CSTA supplementation after BV2 M2-like polarization plus CSTA intervention ( D ). E. The RT-PCR results showing the difference in CSTA overexpression efficacy between two plasmids in BV2, OE.1 was selected to represent OE-CSTA. F. The schematic of in vivo animal model construction: Mice were divided into 3 subgroups based on CSTA interventions in mixed-implanted BV2 cells, including untreated control, CSTA-knockdown (sh-CSTA) and CSTA-overexpressing (OE-CSTA) groups. Each group was further divided into time-point analysis cohort ( N = 3, for tracking tumor volume) and survival analysis cohort ( N = 6, for survival information). G , H. In vivo imaging ( G ) and fluorescence readout statistics ( H ) at 7, 14 and 21 days in time-point analysis cohort in the CT2A-BV2 mixed tumor formation model. I , J. HE staining of mouse brain coronal sections ( I ) and maximum cross-sectional area statistics ( J ) at 21 days in time-point analysis cohort. K. Kaplan-Meier (K-M) curves showing survival time and status of different subgroups in survival analysis cohort of the CT2A-BV2 mixed tumor formation model. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001

Article Snippet: Six standard concentrations (0, 5, 10, 15, 20, 25 ng/mL) were prepared with recombinant CSTA protein (MCE, HY-P7868).

Techniques: CCK-8 Assay, Concentration Assay, EdU Assay, Co-Culture Assay, Recombinant, Reverse Transcription Polymerase Chain Reaction, Over Expression, In Vivo, Animal Model, Control, Knockdown, In Vivo Imaging, Fluorescence, Staining

Journal: Journal of Translational Medicine

Article Title: M2-like GAMs secreting CSTA drive glioblastoma progression via the ITGB4-TGFB1 feedback axis

doi: 10.1186/s12967-026-08009-0

Figure Lengend Snippet: ERK/AP-1 axis promotes the expression of CSTA in GAMs. ( A ) In the GSE242543 cohort, the distribution of differentially expressed genes (DEGs) between human peripheral blood macrophages with and without IL-4 stimulation, with screening criteria set as |LogFC| ≥ 1 and FDR < 0.05. ( B ) KEGG functional enrichment analysis results of the DEGs above. ( C ) The intersection of prediction results for potential transcription factors of CSTA from hTarget, FMO_JASPA, KnockTF, GTRD, and Chip_Atlas databases, retaining three potential transcription factors: TP53, FOS (c-FOS), and CTCF. D , E. Correlation tests between c-FOS and CSTA in TCGA pan-cancer ( D ) and GTEx normal human tissue ( E ) RNA-seq cohorts, where red dots indicate a significant association ( p < 0.05 and correlation coefficient ≥ 0.25). F. Motif diagram of the potential binding base sequence of c-FOS in the CSTA promoter region. G. Results of ChIP-PCR for c-FOS and CSTA promoter fragments in BV2 and HMC3 cells, statistically analyzed based on relative expression compared to the Input group. H. Co-localization of c-FOS and CSTA by immunofluorescence (IF) staining in BV2 and HMC3 cell climbing sheets after IL-4-induced polarization. I. Effect of c-FOS knockdown on CSTA protein levels in IL-4-induced BV2 cells. J. Changes in protein levels of CSTA, c-JUN, c-FOS, and phosphorylated signaling molecules in BV2 cells after separate activation of MAPK, JAK/STAT, and RAP1 signaling pathways. K , L. Changes in protein levels of CSTA, c-JUN, c-FOS, and phosphorylated signaling molecules in BV2 cells after simultaneous MAPK inhibition and IL-4 induction ( K ), as well as after simultaneous c-FOS knockdown and MAPK activation ( L ). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001

Article Snippet: Six standard concentrations (0, 5, 10, 15, 20, 25 ng/mL) were prepared with recombinant CSTA protein (MCE, HY-P7868).

Techniques: Expressing, Functional Assay, RNA Sequencing, Binding Assay, Sequencing, Immunofluorescence, Staining, Knockdown, Activation Assay, Protein-Protein interactions, Inhibition

Journal: Journal of Translational Medicine

Article Title: M2-like GAMs secreting CSTA drive glioblastoma progression via the ITGB4-TGFB1 feedback axis

doi: 10.1186/s12967-026-08009-0

Figure Lengend Snippet: ITGB4 is the functional receptor of CSTA. ( A ) Affinity IP-mass spectrometry to identify glioma plasma membrane proteins potentially binding to CSTA; performed in CT2A membrane protein extracts, including 6 datasets (3 IP vs. 2 IgG, one-to-one comparisons). After intersection, Robust Rank Aggregation (RRA) ranking was applied (top to bottom = high to low RRA rank), with color intensity indicating LogFC levels in corresponding comparisons. ( B ) Molecular docking prediction of murine CSTA and ITGB4, enlarged area shows potential binding sites and corresponding amino acid residues. ( C ) Alignment of ITGB4 amino acid residues 80–100 across species, red box = residues 87–91 co-predicted to interact with CSTA in humans and mice via molecular docking, blue = conserved residues. ( D ) IP validation of CSTA-ITGB4 interaction in CT2A and LN229 cells. ( E ) IP validation of CSTA-ITGB4 interaction in CT2A and LN229 cells with ITGA6 knockdown. ( F ) Brief summary of ITGB4 truncation mutant design: residues 1–683 aa encode the extracellular domain, and residues 684–1822 aa encode the transmembrane and intracellular domains. ( G ) Pull down assay results of His-tagged human and murine ITGB4 truncation mutants and Fc-CSTA recombinant protein. H , I. Binding efficiency of Fc-CSTA to different ITGB4 AAPM mutants in CT2A ( H ) and LN229 ( I ) cells. J. Phosphorylation levels of signaling molecules in ITGB4-related pathways (MAPK, PI3K/AKT, NF-κB) in CT2A cells after CSTA stimulation, and effects of AAPM #88 and sh-ITGB4. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001

Article Snippet: Six standard concentrations (0, 5, 10, 15, 20, 25 ng/mL) were prepared with recombinant CSTA protein (MCE, HY-P7868).

Techniques: Functional Assay, Mass Spectrometry, Clinical Proteomics, Membrane, Binding Assay, Biomarker Discovery, Knockdown, Mutagenesis, Pull Down Assay, Recombinant, Phospho-proteomics

Journal: Journal of Translational Medicine

Article Title: M2-like GAMs secreting CSTA drive glioblastoma progression via the ITGB4-TGFB1 feedback axis

doi: 10.1186/s12967-026-08009-0

Figure Lengend Snippet: ITGB4 intervention inhibits CSTA-related GBM malignancy. ( A ) Schematic of in vivo ITGB4 intervention experiments: Four groups including CT2A/GL261 (ITGB4 WT) + BV2 WT (negative control), CT2A/GL261 (ITGB4 WT) + BV2 OE-CSTA (positive control), and CT2A/GL261 (ITGB4 AAPM #88 or sh-ITGB4) co-cultured with BV2 OE-CSTA. Each group was further divided into time-point analysis cohort ( N = 3, for tracking tumor volume) and survival analysis cohort ( N = 6, for survival information). ( B ) Validation of ITGB4 knockdown in GL261 cells ( C ) In vivo imaging ( H ) and fluorescence readout statistics at 7, 14 and 21 days in time-point analysis cohort in the CT2A-BV2 mixed tumor formation model. D , E. HE staining of mouse brain coronal sections ( D ) and maximum cross-sectional area statistics ( E ) at 21 days in time-point analysis cohort. F. Kaplan-Meier curve showing survival time and status of different subgroups in survival analysis cohort of the CT2A-BV2 mixed tumor formation model. G. Protein levels of TGFB1 in CT2A cells after CSTA stimulation, and effects of AAPM #88 and sh-ITGB4. H , I. Promoter-binding motifs of RELA/p65 ( H ) and AP- 1 ( I ). J , K. ChIP-PCR validation results for c-FOS ( J ) and p65 ( K ) binding to the TGFB1 promoter region in CT2A and LN229 cells. L , M. Dual-luciferase reporter assay results demonstrated the differences in the activities of AP-1 and p65 transcription factors in CT2A ( L ) and LN229 ( M ) cells before and after CSTA induction. N. Differences in the phosphorylation of key proteins of the relevant signaling pathways and TGFB1 protein levels in CT2A cells following treatment with MAPK and NF-κB signaling pathway agonists. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001

Article Snippet: Six standard concentrations (0, 5, 10, 15, 20, 25 ng/mL) were prepared with recombinant CSTA protein (MCE, HY-P7868).

Techniques: In Vivo, Negative Control, Positive Control, Cell Culture, Biomarker Discovery, Knockdown, In Vivo Imaging, Fluorescence, Staining, Binding Assay, Luciferase, Reporter Assay, Phospho-proteomics, Protein-Protein interactions

Journal: Journal of Translational Medicine

Article Title: M2-like GAMs secreting CSTA drive glioblastoma progression via the ITGB4-TGFB1 feedback axis

doi: 10.1186/s12967-026-08009-0

Figure Lengend Snippet: The CSTA-ITGB4-TGFB1 loop and the biomarker potential of CSTA. A , B , C. IF staining of TGFB1 and macrophage/microglia marker IBA1 in brain sections from CT2A/BV2 mixed orthotopic GBM xenograft models ( A ), with statistics on the proportions of TGFB1+ ( B ) and IBA1+ ( C ) cells among total cells across groups. D , E. IF staining of IBA1 and M2 polarization marker ARG1 ( D ), and statistics on the proportion of IBA1 + ARG1+ cells relative to total cells in the visual field across groups ( E ). F. Correlation between TGFB1 + and IBA1 + ARG1+ cell proportions, with each point representing values from an entire field. G. CSTA concentrations in cerebrospinal fluid (CSF) samples from patients with different glioma grades. H. CSTA concentrations in preoperative CSF samples from primary vs. recurrent glioma patients. I , J. ROC analysis of CSF CSTA concentrations in glioma vs. normal patients ( I ) and in LGG (G2, G3) vs. GBM (G4) patients ( J ), with corresponding AUC values of 0.9891 and 0.9589. K. Paired t-test of CSF CSTA concentrations in patients with different glioma grades before and 1 week after surgery. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001

Article Snippet: Six standard concentrations (0, 5, 10, 15, 20, 25 ng/mL) were prepared with recombinant CSTA protein (MCE, HY-P7868).

Techniques: Biomarker Discovery, Staining, Marker

Journal: International Journal of Molecular Sciences

Article Title: Expression and Regulation of a Novel Decidual Cells-Derived Estrogen Target during Decidualization

doi: 10.3390/ijms24010302

Figure Lengend Snippet: Cstb expression is associated with angiogenesis in mouse uterine stromal cells Angptl7 . ( A ) Gene ontology (GO) functional classification of the DEGs. ( B ) The expressions of Cstb and ZO1 were detected by immunofluorescence in the uterus on day 6. Bar = 300 μm. ( C ) The expression of Angptl7 in stromal cells of the con NC group, dc NC group, and dc si Cstb group was detected by RT-qPCR. ( D ) The expression of Angptl7 in mouse uteri from days 5 to 8 of pregnancy was detected by in situ hybridization. Bar = 300 μm. * p < 0.05. NC, negative control; si Cstb , siRNA of Cstb ; dc, in vitro decidualization.

Article Snippet: Briefly, frozen sections were fixed in 4% paraformaldehyde solution for 10 min and then soaked in 0.1% Triton X-100 in PBS for 15 min. After blocking with 5% donkey serum (Zhongshan Jinqiao, Beijing, China) in a 37 °C oven for 60 min, the sections were incubated with mouse anti- Cstb (1:500, Santa Cruz, sc-166561), ZO1 (1:200, Cell Signaling Technology, Boston, MA, USA, #13663) diluted in PBS overnight in a 4 °C refrigerator.

Techniques: Expressing, Functional Assay, Immunofluorescence, Quantitative RT-PCR, In Situ Hybridization, Negative Control, In Vitro

Journal: The FASEB Journal

Article Title: Cathepsin B as a potential cystatin M/E target in the mouse hair follicle

doi: 10.1096/fj.201700267R

Figure Lengend Snippet: Regulation of epidermal protease activity by CST6. Model of the regulatory role of CST6 in processes that control epidermal cornification, desquamation and HF maintenance. 1 ) Inhibition of CTSL activity by CST6 is important in the cornification process, as CTSL is the elusive processing and activating enzyme for (TGM)-3. CTSL is also able to process CTSD, which in turn can activate TGM-1. 2 ) Inhibition of CTSV regulates desquamation, as CTSV is able to degrade desmosomal and corneodesmosomal proteins, such as desmoglein-1, desmocollin-1, and corneodesmosin. As CTSV is expressed only in humans, murine CTSL probably controls the specific functional enzymatic activities of both human CTSL and CTSV. 3 ) The findings in the present study suggest that inhibition of CtsB by Cst6 protects HF maintenance in mice. 4 ) Inhibition of human LGMN regulates the processing of (pro)-cathepsins; however mouse LGMN is not inhibited by mouse Cst6.

Article Snippet: Each well was measured for mouse Cst6 at an absorbance of 450 nm with an ELISA microplate reader (Bio-Rad, Hercules, CA, USA).

Techniques: Activity Assay, Control, Inhibition, Functional Assay

Journal: Molecular Metabolism

Article Title: Glucocorticoid induces human beta cell dysfunction by involving riborepressor GAS5 LincRNA

doi: 10.1016/j.molmet.2019.12.012

Figure Lengend Snippet: GAS5 KD with or without GC treatment affects the expression of key proteins in glucocorticoid signaling (GR and SGK1) and beta cell function (PDX1, NKX6-1, and SYT13). A. Expression of GAS5. B. Protein expression of GR. C. Protein expression of SGK1. D. Expression of PDX1. E. Protein expression of NKX6-1. F. Protein expression of SYT13. The data are presented as mean ± SEM. n = 4, *p < 0.05 vehicle vs Dexa; # p < 0.05 scramble vs GAS5 KD.

Article Snippet: Probe-based TaqMan Assays (Applied Biosystems) were used to measure the expression levels of human GAS5 (Hs03464472_m1), PDX1 (Hs00236830_m1), NKX6-1 (Hs00232355_m1), SYT13 (Hs00951871_m1), SGK1 (Hs00985033_g1), and GR (Hs00353740_m1).

Techniques: Expressing, Cell Function Assay

Journal: Molecular Metabolism

Article Title: Glucocorticoid induces human beta cell dysfunction by involving riborepressor GAS5 LincRNA

doi: 10.1016/j.molmet.2019.12.012

Figure Lengend Snippet: GAS5 and gene expression changes in islets from diabetic donors, GK islets, and beta cells under glucotoxic conditions. A. Expression of GAS5 in islets from control (ND) (n = 10) and T2D donors (n = 9) as measured by qPCR assay. B. GAS5 expression in islets vs HbA1c levels in all of the donors (n = 19). C. GAS5 expression in the islets of T2D model GK rats. D. Glucose regulation of GAS5 in EndoC-βH1 cells (n = 4) exposed to glucotoxic conditions. E. GR protein expression. F. Protein expression of PDX1. G. Protein expression of NKX6-1 and SYT13. The data are presented as mean ± SEM; *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: Probe-based TaqMan Assays (Applied Biosystems) were used to measure the expression levels of human GAS5 (Hs03464472_m1), PDX1 (Hs00236830_m1), NKX6-1 (Hs00232355_m1), SYT13 (Hs00951871_m1), SGK1 (Hs00985033_g1), and GR (Hs00353740_m1).

Techniques: Gene Expression, Expressing, Control

Journal: Journal of Virology

Article Title: NF-κB Activation Stimulates Transcription and Replication of Retrovirus XMRV in Human B-Lineage and Prostate Carcinoma Cells

doi: 10.1128/jvi.02333-10

Figure Lengend Snippet: FIG. 1. TNF- and LMP1 enhance LTR-mediated viral transcrip- tion. (A) XMRV-infected HEK293 cells were incubated with TNF- at the indicated concentrations. After 24 h, the conditioned media were harvested and ultracentrifuged (100,000 g for 1 h), and each pellet was subjected to Western blot analysis for Gag protein (p30CA) by using the rat MAb R187. The bar graph reflects quantitative analysis of duplicate band intensities; the results are expressed as relative band intensities, and the error bars reflect the ranges of measurements. (B) TNF- (5 ng/ml) was added to HEK293 cells transiently cotrans- fected with the XMRV LTR construct fused to firefly luciferase (FL) and with a renilla luciferase (RL) plasmid (pTK-RL). After a 16-h incubation, FL and RL activities were measured. FL activity was nor- malized to RL activity. The results are presented as fold increases ( standard deviations [SD] for triplicate cultures) in normalized FL in cultures with TNF- relative to that in medium only. (C) HEK293 cells were cotransfected with the pCMV-LMP1 plasmid at the indicated concentrations and the XMRV LTR FL reporter. For normalization, we also transfected with pTK-RL. FL and RL activities were measured from cells harvested 16 h after transfection. The results are presented as fold increases ( SD for triplicate wells) of normalized FL induced by LMP1 transfection relative to that without LMP1 transfection.

Article Snippet: Human embryonic kidney-derived HEK293 and HEK293T/17 cells (American Type Culture Collection [ATCC]) were propagated in Dulbecco’s modified Eagle’s medium (DMEM)-Glutamax medium (Invitrogen) with 10% fetal bovine serum (FBS) (Atlanta Biologicals).

Techniques: Infection, Incubation, Western Blot, Construct, Luciferase, Plasmid Preparation, Activity Assay, Transfection

Journal: Journal of Virology

Article Title: NF-κB Activation Stimulates Transcription and Replication of Retrovirus XMRV in Human B-Lineage and Prostate Carcinoma Cells

doi: 10.1128/jvi.02333-10

Figure Lengend Snippet: FIG. 2. Identification of NF-B binding sites B-1 and B-2 in the XMRV LTR and functional analysis of NF-B binding to the XMRV LTR. (A) DNA sequences of consensus and NF-B binding sites in the immunoglobulin (Ig) chain enhancer and in the XMRV LTR. Mutated (mut) sequences are listed below the WT sequences; complementary sequences (c) are noted. Lowercase letters represent mutations. (B) Representative immunoblotting results from DNA affinity binding assays. Individual biotinylated double-stranded DNA probes (sequences shown in panel A) were incubated with nuclear extracts from the EBV-infected lymphoblastoid IB4 cell line as a source of p65/RelA. After precipitation with streptavidin Sepharose, the binding of p65/RelA to biotinylated DNA was revealed by Western blotting (WB) with a specific antibody to p65/RelA. NE, nuclear extracts only. (C) Effects of TNF and LMP1 on WT and mutant XMRV LTR transcriptions as measured in luciferase reporter assays. Mutations in the B-1 and B-2 sites (shown in panel A) were introduced in the XMRV LTR reporter construct. Each firefly luciferase (FL) LTR reporter construct (the WT is XLTR, and mutants 1 and 2 are XLTR mut1 and mut2, respectively) was transiently transfected into HEK293 cells; the cells were cultured in medium alone or with 5 ng/ml of TNF-. The cells were also cotransfected with pCMV-LMP1 (0.1 g/well). After 16 h of incubation, FL activity was measured and normalized for renilla luciferase (from cotransfection with pTK-RL). The results are presented as relative luciferase activities ( SD for triplicate wells) in comparison to HEK293 cells transfected with XLTR and cultured in medium alone. R, repeat sequence.

Article Snippet: Human embryonic kidney-derived HEK293 and HEK293T/17 cells (American Type Culture Collection [ATCC]) were propagated in Dulbecco’s modified Eagle’s medium (DMEM)-Glutamax medium (Invitrogen) with 10% fetal bovine serum (FBS) (Atlanta Biologicals).

Techniques: Binding Assay, Functional Assay, Western Blot, Incubation, Infection, Mutagenesis, Luciferase, Construct, Transfection, Cell Culture, Activity Assay, Cotransfection, Comparison, Sequencing

Journal: Journal of Virology

Article Title: NF-κB Activation Stimulates Transcription and Replication of Retrovirus XMRV in Human B-Lineage and Prostate Carcinoma Cells

doi: 10.1128/jvi.02333-10

Figure Lengend Snippet: FIG. 3. Mutation of the B-1 site reduces XMRV replication. (A) Schematic representation of the XMRV provirus constructs [pcDNA3.1()VP62 and pcDNA3.1()VP62mB-1]; the WT B-1 site and the mutant mB-1 site are expanded. The mutated bases (G to C at position 7959 and C to G at position 7967) are shown. CMV, human cytomegalovirus immediate early promoter; , packaging signal for XMRV. Lowercase letters represent mutations. (B) Conditioned medium from 293T/17 cells transfected with pcDNA3.1()VP62 or pcDNA3.1()VP62mB-1 was spun at 100,000 g for 1 h, and virus-like particles were subjected to Western blotting for Gag protein by using the rat MAb R187. (C) Duplicate semiconfluent cultures of the prostate carcinoma cell lines LNCaP and DU145 were infected with equal amounts (500 l [top and bottom panels] and50 l [middle panel] viral preparation shown in panel B) of WT XMRV (VP62) or mutant virus (VP62mB-1). After culture for the indicated periods (2 to 6 days), individual conditioned media were ultracentrifuged and the pellets analyzed by Western blotting for Gag protein (p30CA) by using the rat MAb R187. The bar graphs reflect quantitative analysis of duplicate band intensities from the membranes shown above; the results are expressed as relative band intensities, and error bars reflect the ranges of measurements. U, sample from uninfected cells. Each experiment was repeated at least three times, and representative results are shown. (D) Reverse transcriptase (RT) activity in the culture supernatants of LNCaP cells left uninfected or infected (50 l) with VP62 or VP62mB-1. The results reflect RT activity (ng/ml; SD for triplicate cultures) in the precipitates from conditioned media (0.5 ml) tested in duplicate. Exp., experiment. (E) Relative luciferase activity after transfection of the pIL-6-Luc plasmid into the cell lines HEK293, LNCaP, and DU145, with or without the addition of TNF- (20 ng/ml) to the cultures. For normalization, each cell line was cotransfected with pTK-RL. FL and RL activities were measured from cells harvested 18 h after transfection.

Article Snippet: Human embryonic kidney-derived HEK293 and HEK293T/17 cells (American Type Culture Collection [ATCC]) were propagated in Dulbecco’s modified Eagle’s medium (DMEM)-Glutamax medium (Invitrogen) with 10% fetal bovine serum (FBS) (Atlanta Biologicals).

Techniques: Mutagenesis, Construct, Transfection, Virus, Western Blot, Infection, Reverse Transcription, Activity Assay, Luciferase, Plasmid Preparation

Journal: Journal of Virology

Article Title: NF-κB Activation Stimulates Transcription and Replication of Retrovirus XMRV in Human B-Lineage and Prostate Carcinoma Cells

doi: 10.1128/jvi.02333-10

Figure Lengend Snippet: FIG. 5. Relative effects of dexamethasone and NF-B on XMRV production. (A) HEK293 cells were infected with WT XMRV (pcDNA- VP62). Ten days after infection (75% of cells expressed Gag protein, per immunostaining), cells were harvested, washed, and seeded onto 6-well plates in fresh medium supplemented with dexamethasone (Dex) at the indicated concentrations. After 24 h, the conditioned media (0.5 ml) were harvested and ultracentrifuged (100,000 g for 1 h), and each pellet was analyzed by Western blotting for Gag protein (p30CA) by using the rat MAb R187. (B) HEK293 cells were infected with WT or B-1 mutant XMRV (VP62 or VP62mB-1) with or without Dex (1.5 M). After 6 days of incubation, the conditioned media (0.5 ml) were harvested and ultracentrifuged, and each pellet was analyzed by Western blotting for Gag protein (p30CA). The bar graphs below the nitrocellurose membrane images in panels A and B reflect quantitative analysis of duplicate band intensities; the results are expressed as relative band intensities, and the error bars reflect the ranges of measurements. The dotted line in panel B demarks the mean relative band intensity from culture supernatants of XMRV VP62-infected HEK293 cells cultured in medium. Each experiment was repeated three times, and representative results are shown. (C) Schematic representation of XMRV LTR-mediated replication regulated by the Dex-glucocorticoid receptor (GR)-GRE and TNF-/LMP1–NF-B–B-1 pathways. TNFR, tumor necrosis factor receptor; IKK, IB kinase.

Article Snippet: Human embryonic kidney-derived HEK293 and HEK293T/17 cells (American Type Culture Collection [ATCC]) were propagated in Dulbecco’s modified Eagle’s medium (DMEM)-Glutamax medium (Invitrogen) with 10% fetal bovine serum (FBS) (Atlanta Biologicals).

Techniques: Infection, Immunostaining, Western Blot, Mutagenesis, Incubation, Membrane, Cell Culture

Journal: The FASEB Journal

Article Title: Cathepsin B as a potential cystatin M/E target in the mouse hair follicle

doi: 10.1096/fj.201700267r

Figure Lengend Snippet: Figure 1. Phenotype of rescued Tg(INV-Cst6)Cst6ichq/ichq mice. A) Tg(INV-Cst6)Cst6ichq/ichq mice survived and showed periodic hair loss. After 4 mo the progenies became completely bald. Keratitis and thickening of the cornea were observed in Tg(INV-Cst6)Cst6ichq/ichq mice from 4 to 5 mo. The mice shown are 9 and 32 wk old. Inset: magnified view of an affected eye. B) Keratitis and metaplasia of the corneal epithelium in Tg(INV- Cst6)Cst6ichq/ichq mice. H&E staining of the eye and the cornea in WT and Tg(INV-Cst6)Cst6ichq/ichq mice. C) Immunofluores- cence staining for the expression of loricrin (LOR) and filaggrin (FLG) in the cornea. Scale bars, 100 mm.

Article Snippet: Protease inhibitor activity of recombinant mouse Cst6 (R&D Systems) against recombinant mouse CtsB (R&D Systems) was determined by measuring the hydrolysis of the fluorescent Z-Leu-Arg-AMC substrate (R&D Systems) (4).

Techniques: Staining, Expressing

Journal: The FASEB Journal

Article Title: Cathepsin B as a potential cystatin M/E target in the mouse hair follicle

doi: 10.1096/fj.201700267r

Figure Lengend Snippet: Figure 2. Destruction of the HFs in Tg(INV-Cst6)Cst6ichq/ichq mice. A) Immunofluorescence double labeling of INV and Cst6 in the epidermis and the HF of WT mice. Cst6 was expressed in the stratum granulosum and halfway up the HF, including the area around the bulge, whereas INV expression was also seen in the stratum granulosum, but remained only in the proximal part of the HF. B) Immunohistological labeling in WT mice to detect the location of the bulge area (arrows) using the stem cell markers CD34 and keratin 15 and the proliferation marker Ki67. C) H&E staining of Tg(INV-Cst6)Cst6ichq/ichq mice from 11, 13, and 16 wk showed disappearance of the HFs. Scale bar, 100 mm.

Article Snippet: Protease inhibitor activity of recombinant mouse Cst6 (R&D Systems) against recombinant mouse CtsB (R&D Systems) was determined by measuring the hydrolysis of the fluorescent Z-Leu-Arg-AMC substrate (R&D Systems) (4).

Techniques: Labeling, Expressing, Marker, Staining

Journal: The FASEB Journal

Article Title: Cathepsin B as a potential cystatin M/E target in the mouse hair follicle

doi: 10.1096/fj.201700267r

Figure Lengend Snippet: Figure 3. Inhibition of mouse CtsB by mouse Cst6. A) BMV109 labels active cysteine cathepsins, such as CTSB and CTSX (14) in RAW cell lysates (murine Mf cell line). Lane 1, no inhibitor; lane 2, JPM-OEt (pan-cathepsin inhibitor, 50 mM); lane 3, CA-074 (CtsB inhibitor, 10 mM); lane 4, Z-FY (t-BU)DMK (CtsL inhibitor, 10 mM); lane 5, mouse Cst6 (18 mM); and lane 6, heat inactivation. Note that CA-074 and Cst6 inhibited CtsB (lane 3 and 5). M = precision plus protein dual-color marker. B) The Ki for the inhibition of CtsB by Cst6 was determined by measuring the residual enzymatic activity of a fixed concentration of enzyme, incubated with increasing concentrations of the inhibitor. An Easson-Stedman plot (inset) was used to calculate the Ki, according to the following equation: [I]/1 2 a = (Ki/a) + E0, were I is the inhibitor concentration, E0 is the enzyme concentration at time 0, and a is the fractional activity. The plot yielded a straight line with a Ki slope of 0.98 nM.

Article Snippet: Protease inhibitor activity of recombinant mouse Cst6 (R&D Systems) against recombinant mouse CtsB (R&D Systems) was determined by measuring the hydrolysis of the fluorescent Z-Leu-Arg-AMC substrate (R&D Systems) (4).

Techniques: Inhibition, Chromosome Transmission Fidelity Colony Color Assay, Activity Assay, Concentration Assay, Incubation

Journal: The FASEB Journal

Article Title: Cathepsin B as a potential cystatin M/E target in the mouse hair follicle

doi: 10.1096/fj.201700267r

Figure Lengend Snippet: Figure 4. CtsB and Cst6 colocalize in the mouse HF. A) Immunofluorescence double staining for Cst6 (green) and CtsB (red) in WT mice revealed that colocalization of both proteins was found in the proximal part of the HF as well as the lower region where the bulge area resides. Cst6 is also expressed in the epidermis, whereas no CtsB is observed. Scale bar, 100 mm. B) Cst6 and CtsB expression in mouse epidermis and HFs of Tg(INV-Cst6)Cst6ichq/ichq mice. No immunofluorescence staining for Cst6 was observed in the lower region of the HF (close to the bulge, arrowheads). Scale bar, 100 mm. C) Schematic presentation of INV, Cst6, and CtsB localization in the HF and epidermis of WT mice and the situation in rescued transgenic Tg(INV-Cst6)Cst6ichq/ichq mice.

Article Snippet: Protease inhibitor activity of recombinant mouse Cst6 (R&D Systems) against recombinant mouse CtsB (R&D Systems) was determined by measuring the hydrolysis of the fluorescent Z-Leu-Arg-AMC substrate (R&D Systems) (4).

Techniques: Double Staining, Expressing, Staining, Transgenic Assay

Journal: The FASEB Journal

Article Title: Cathepsin B as a potential cystatin M/E target in the mouse hair follicle

doi: 10.1096/fj.201700267r

Figure Lengend Snippet: Figure 5. Regulation of epidermal protease activity by CST6. Model of the regulatory role of CST6 in processes that control epidermal cornification, desquamation and HF maintenance. 1) Inhibition of CTSL activity by CST6 is important in the cornification process, as CTSL is the elusive processing and activating enzyme for (TGM)-3. CTSL is also able to process CTSD, which in turn can activate TGM-1. 2) Inhibition of CTSV regulates desquamation, as CTSV is able to degrade (corneo)-desmosomal proteins, such as desmoglein-1, desmocollin-1, and corneodesmosin. As CTSV is expressed only in humans, murine CTSL probably controls the specific functional enzymatic activities of both human CTSL and CTSV. 3) The findings in the present study suggest that inhibition of CtsB by CST6 protects HF maintenance in mice. 4) Inhibition of human LGMN regulates the processing of (pro)-cathepsins; however mouse LGMN is not inhibited by mouse CST6.

Article Snippet: Protease inhibitor activity of recombinant mouse Cst6 (R&D Systems) against recombinant mouse CtsB (R&D Systems) was determined by measuring the hydrolysis of the fluorescent Z-Leu-Arg-AMC substrate (R&D Systems) (4).

Techniques: Activity Assay, Control, Inhibition, Functional Assay

Journal: Nature medicine

Article Title: MST1 is a novel regulator of apoptosis in pancreatic beta-cells

doi: 10.1038/nm.3482

Figure Lengend Snippet: (a,b) Adenovirus-mediated GFP or MST1 overexpression in human islets for 96h. (a) Insulin secretion during 1h-incubation with 2.8 mM (basal) and 16.7 mM glucose (stimulated), normalized to insulin content and basal secretion at GFP control. The insulin stimulatory index denotes the ratio of secreted insulin during 1h-incubation with 16.7 mM and 2.8 mM glucose, respectively and insulin content analyzed after GSIS and normalized to whole islet protein. (b) MST1 and PDX1 immunoreactivity were analyzed by Western blotting. Lower panel shows densitometry analysis from at least 3 independent experiments normalized to actin. Right panel shows PDX1 target genes including SLC2A2, GCK and Insulin analyzed by RT-PCR. (c-d) HEK293 cells were transfected with plasmids encoding Myc-MST1 and GFP-PDX1. (c) A kinase-dead MST1 (dn-MST1: K59R) was co-transfected with GFP-PDX1 (left panel). At 48 h after transfection, HEK293 cells were treated with cycloheximide (CHX) for 8h (middle panel). At 36h after transfection, HEK293 cells were treated with the proteasome inhibitor MG-132 for 6h (right panel). PDX1 and MST1 were analyzed by western blotting. (d) In vivo ubiquitination assay in HEK293 cells transfected with GFP-PDX1 and HA-ubiquitin, alone or together with Myc-MST1 or MST1-K59 expression plasmids for 48h (left) and human islets transfected with HA-ubiquitin and infected with Ad-GFP or Ad-MST1 for 48h (right; 2 different donors). MG-132 was added during the last 6h of the experiment. Cell lysates were immunoprecipitated with an anti-PDX1 antibody followed by immunoblotting with ubiquitin antibody to detect ubiquitinated PDX1. (e) HEK293 cells were transfected with GFP-PDX1 alone or together with Myc-MST1 for 48h. Reciprocal co-immunoprecipitations performed using anti-GFP and anti-Myc antibodies and western blot analysis performed with precipitates and input fraction using anti-Myc and anti-GFP antibodies, respectively. (f) In vitro kinase assay was performed by incubating recombinant MST1 and PDX1 proteins and analyzed by NuPAGE followed by western blotting using pan-phospho-threonine specific, PDX1 and MST1 antibodies. (g) Lysates of HEK293 cells transfected with PDX1-WT or PDX1-T11A expression-plasmids were immunoprecipitated with PDX1 antibody and subjected to an in vitro kinase assay using recombinant MST1. Phosphorylation reactions were analyzed by Western blotting using p-T11-PDX1 specific and pan-phospho threonine antibodies (left panel). HEK293 cells were transfected with PDX1-WT or PDX1-T11A alone or together with MST1 expression-plasmids for 48h. MST1 and PDX1 were analyzed by western blotting (middle panel). PDX-1-WT or PDX1-T11A co-transfected with MST1 in HEK293 cells for 36h and treated with CHX, western blot analysis for PDX1 and densitometry analysis of bands (right panel). (h) Human islets transfected with GFP, PDX1-WT or PDX1-T11A expression-plasmids and western blot analysis for PDX1. (i,j) human islets were infected with Ad-GFP or Ad-MST1 for 72h. (i) Insulin secretion during 1h-incubation with 2.8 mM (basal) and 16.7 mM glucose (stimulated), normalized to insulin content and basal secretion at control. The insulin stimulatory index denotes the ratio of secreted insulin during 1h-incubation with 16.7 mM and 2.8 mM glucose, respectively. (j) PDX1 target genes in human islets analyzed by RT-PCR and levels normalized to tubulin and shown as change from PDX1-WT transfected islets. All western blots show representative results from at least 3 independent experiments from 3 different donors (human islets). Tubulin/Actin was used as loading control. RT-PCR (b,j) and GSIS (a,i) show pooled results from 3 independent experiments from 3 different donors. Results shown are means ±SE. *p<0.05 MST-OE compared to control, **p<0.05 PDX-1T11A-MST1 compared to PDX-1WT-MST1.

Article Snippet: TaqMan(R) Gene Expression Assays were used for pdx1 (Hs00426216_m1), SLC2A2 (Hs01096905_m1), GCK (Hs01564555_m1), insulin (Hs02741908_m1), PPIA (Hs99999904_m1), BCL2L11 (Hs01083836_m1) and tubulin (Hs00362387_m1) for human and PDX1 (Rn00755591_m1), SLC2A2 (Rn00563565_m1), GCK (Rn00688285_m1), INS1 (Rn02121433_g1), INS2 (Rn01774648_g1), PPIA (Rn00690933_m1) and tuba1a (Rn01532518_g1) for rat.

Techniques: Over Expression, Incubation, Control, Western Blot, Reverse Transcription Polymerase Chain Reaction, Transfection, In Vivo, Ubiquitin Proteomics, Expressing, Infection, Immunoprecipitation, In Vitro, Kinase Assay, Recombinant, Phospho-proteomics

Journal: Nature medicine

Article Title: MST1 is a novel regulator of apoptosis in pancreatic beta-cells

doi: 10.1038/nm.3482

Figure Lengend Snippet: (a-d) Human islets transfected with MST1 siRNA or control siScr and treated with the cytokines mixture IL/IF, 33.3 mM glucose or the mixture of 33.3 mM glucose and 0.5 mM palmitate (33.3Pal) for 72h. (a) Beta-cell apoptosis analyzed by double staining of TUNEL and insulin. An average number of 11390 insulin-positive beta-cells were counted for each treatment condition in 3 independent experiments from 3 different donors. (b) Western blotting confirmed successful (~80%) MST1 depletion in human islets. MST1, pMST1, BIM, pH2B, caspase-9 and caspase-3 cleavage analyzes by western blotting. Right panel shows densitometry analysis from at least 3 independent experiments normalized to actin. (c) RT-PCR for BCL2L11 performed in human islets and levels normalized to tubulin shown as change from siScr control transfected islets. (d) Insulin stimulatory index denotes the ratio of secreted insulin during 1h-incubation with 16.7 mM and 1h-incubation with 2.8 mM glucose. (e,f) Islets were isolated from Mst1 −/− mice and their WT littermates and exposed to the cytokines mixture IL/IF or the mixture of 33.3 mM glucose and 0.5 mM palmitate (33.3Pal) for 72 hours. (e) beta-cell apoptosis analyzed by double staining for TUNEL and insulin. An average number of 24180 insulin-positive beta-cells were counted for each treatment condition in 3 independent experiments. (f) Insulin stimulatory index denotes the ratio of secreted insulin during 1h-incubation with 16.7 mM and 1h-incubation with 2.8 mM glucose. (g-i) Stable INS-1E clones were generated by transfection of vectors for shMst1 and shScr control and treated with the cytokines mixture IL/IF or 22.2 or 33.3 mM glucose for 72h. (g) Mst1, Bim, Pdx1, caspase-3 and PARP cleavage were analyzed by western blotting. Right panel shows densitometry analysis from at least 3 independent experiments normalized to actin. (h) Insulin stimulatory index. (i) PDX1 target genes in shMst1 and shScr control INS-1E cells normalized to tubulin and shown as change from shScr control INS1-E clones. Western blots (b,g) show representative results from 3 independent experiments from 3 different donors (human islets). Actin was used as loading control. TUNEL data (a,e), GSIS (d,f,h) or RT-PCR (c,i) show pooled results from 3 independent experiments. Results shown are means ±SE. *p<0.05 compared to siScr (a,b,c,d), WT (e,f) or shScr untreated controls (g,h,i) , **p<0.05 compared to siScr (a,b,c,d), WT (e,f) or shScr (g,h,i) at the same treatment conditions.

Article Snippet: TaqMan(R) Gene Expression Assays were used for pdx1 (Hs00426216_m1), SLC2A2 (Hs01096905_m1), GCK (Hs01564555_m1), insulin (Hs02741908_m1), PPIA (Hs99999904_m1), BCL2L11 (Hs01083836_m1) and tubulin (Hs00362387_m1) for human and PDX1 (Rn00755591_m1), SLC2A2 (Rn00563565_m1), GCK (Rn00688285_m1), INS1 (Rn02121433_g1), INS2 (Rn01774648_g1), PPIA (Rn00690933_m1) and tuba1a (Rn01532518_g1) for rat.

Techniques: Transfection, Control, Double Staining, TUNEL Assay, Western Blot, Reverse Transcription Polymerase Chain Reaction, Incubation, Isolation, Clone Assay, Generated

Journal: Nature medicine

Article Title: MST1 is a novel regulator of apoptosis in pancreatic beta-cells

doi: 10.1038/nm.3482

Figure Lengend Snippet: (a-g) Mst1 −/− mice (n=15) and their WT littermates (n=14) were injected with 40 mg/kg streptozotocin or citrate buffer for 5 consecutive days. (a) Random fed blood glucose measurements after last STZ injection (day 0) over 21 days and intraperitoneal glucose tolerance test (ipGTT) performed at day 17. (b) Insulin secretion during an ipGTT measured before (0 min) and 30 min after glucose injection and data are expressed as ratio of secreted insulin at 30 min/0 min (stimulatory index). (c) The ratio of secreted insulin and glucose is calculated at fed state. (d-g) Mice were sacrificed at day 22. (d) Beta-cell mass and quantitative analyses from triple stainings for TUNEL or Ki67, insulin and DAPI expressed as percentage of TUNEL- or Ki67-positive beta-cells ±SE. The mean number of beta-cells scored was 23121 for each treatment condition. (e) The pancreatic area of alpha- (stained in red) and beta-cells (stained in green) are given as percentage of the whole pancreatic section from 10 sections spanning the width of the pancreas . (f,g) Representative double-staining for Bim (red, f ) or Pdx1 (red, g ) and insulin (green) is shown from STZ-treated Mst1 −/− mice and controls. White arrows indicate areas of cytosolic Pdx1 localization and its total absence in WT-STZ mice. (h-j) b Mst1 −/− mice with specific deletion in the beta-cells using the Cre-Lox system (n=5) and their Rip-Cre (n=3) and fl/fl controls (n=3) were injected with 40 mg/kg STZ for 5 consecutive days. (h) Random fed blood glucose measurements after last STZ injection (day 0) over 32 days and ipGTT at day 32. (i) Insulin secretion during an ipGTT measured before (0 min) and 30 min after glucose injection and data are expressed as ratio of secreted insulin at 30 min/0 min (stimulatory index). The ratio of secreted insulin and glucose is calculated at fed state (right panel). (j) Mice were sacrificed at day 32. Beta-cell mass analysis and results from triple stainings for TUNEL or Ki67, insulin and DAPI expressed as percentage of TUNEL- or Ki67-positive beta-cells ±SE. Data show means ± SE. *p<0.05 WT-STZ compared to WT saline injected mice, **p<0.05 MST1 −/− -STZ compared to WT-STZ mice. #p<0.05 b MST -STZ compared to fl/fl-STZ or Cre-STZ mice.

Article Snippet: TaqMan(R) Gene Expression Assays were used for pdx1 (Hs00426216_m1), SLC2A2 (Hs01096905_m1), GCK (Hs01564555_m1), insulin (Hs02741908_m1), PPIA (Hs99999904_m1), BCL2L11 (Hs01083836_m1) and tubulin (Hs00362387_m1) for human and PDX1 (Rn00755591_m1), SLC2A2 (Rn00563565_m1), GCK (Rn00688285_m1), INS1 (Rn02121433_g1), INS2 (Rn01774648_g1), PPIA (Rn00690933_m1) and tuba1a (Rn01532518_g1) for rat.

Techniques: Injection, TUNEL Assay, Staining, Double Staining, Saline

Journal: Nature medicine

Article Title: MST1 is a novel regulator of apoptosis in pancreatic beta-cells

doi: 10.1038/nm.3482

Figure Lengend Snippet: (a-e) bMST1 −/− mice (fl/fl-Cre; n=12) and the Cre control mice (n=12) were fed a normal (ND) or high fat/ high sucrose diet (“Surwit”; HFD) for 20 weeks. (a) Random fed blood glucose measurements, (b) intraperitoneal glucose tolerance test (ipGTT) and (c) insulin secretion during an ipGTT measured before (0 min), 15 and 30 min after glucose injection. (d,e) Mice were sacrificed at week 21. (d) Islets were isolated from all 4 treatment groups, cultured overnight and subjected to an in vitro GSIS assay. Insulin secretion during 1h-incubation with 2.8 mM (basal) and 16.7 mM glucose (stimulated), normalized to insulin content. The insulin stimulatory index denotes the ratio of secreted insulin during 1h-incubation with 16.7 mM and 2.8 mM glucose, respectively. (e) Beta-cell mass analysis and results from triple stainings for TUNEL or Ki67, insulin and DAPI expressed as percentage of TUNEL- or Ki67-positive beta-cells ±SE. The mean number of beta-cells scored was 23121 for each treatment condition. *p<0.05 Cre HFD compared Cre ND mice. **p<0.05 b Mst1 −/− -HFD compared to Cre HFD mice. (f) Our view on how diabetic stimuli lead to activation of MST1. Active MST1 triggers cytochrome c release and mitochondrial-dependent apoptosis by modulating Bim/Bax/Bcl2/Bcl-xL through JNK/AKT signaling. Active caspase-9 then triggers cleavage of caspase-3, which triggers the caspase-3-dependent cleavage of MST1 to its constitutively active fragment, which leads to further MST1 activation and processing of caspase-3 by a positive feedback mechanism, and acceleration of beta-cell death occurs. Cleaved MST1 translocates to the nucleus and directly phosphorylates PDX1 (we do not exclude the possibility that MST1 targets PDX1 also in cytoplasm) and histone H2B. PDX1 then shuttles to cytosol, where it marks for ubiquitination and subsequent degradation by proteasome machinery and beta-cell function is impaired. Histone H2B phosphorylation by MST1 also induces chromatin condensation, one of the characteristic features of apoptosis.

Article Snippet: TaqMan(R) Gene Expression Assays were used for pdx1 (Hs00426216_m1), SLC2A2 (Hs01096905_m1), GCK (Hs01564555_m1), insulin (Hs02741908_m1), PPIA (Hs99999904_m1), BCL2L11 (Hs01083836_m1) and tubulin (Hs00362387_m1) for human and PDX1 (Rn00755591_m1), SLC2A2 (Rn00563565_m1), GCK (Rn00688285_m1), INS1 (Rn02121433_g1), INS2 (Rn01774648_g1), PPIA (Rn00690933_m1) and tuba1a (Rn01532518_g1) for rat.

Techniques: Control, Injection, Isolation, Cell Culture, In Vitro, Incubation, TUNEL Assay, Activation Assay, Ubiquitin Proteomics, Cell Function Assay, Phospho-proteomics

Journal: The FASEB Journal

Article Title: Cathepsin B as a potential cystatin M/E target in the mouse hair follicle

doi: 10.1096/fj.201700267r

Figure Lengend Snippet: Figure 1. Phenotype of rescued Tg(INV-Cst6)Cst6ichq/ichq mice. A) Tg(INV-Cst6)Cst6ichq/ichq mice survived and showed periodic hair loss. After 4 mo the progenies became completely bald. Keratitis and thickening of the cornea were observed in Tg(INV-Cst6)Cst6ichq/ichq mice from 4 to 5 mo. The mice shown are 9 and 32 wk old. Inset: magnified view of an affected eye. B) Keratitis and metaplasia of the corneal epithelium in Tg(INV- Cst6)Cst6ichq/ichq mice. H&E staining of the eye and the cornea in WT and Tg(INV-Cst6)Cst6ichq/ichq mice. C) Immunofluores- cence staining for the expression of loricrin (LOR) and filaggrin (FLG) in the cornea. Scale bars, 100 mm.

Article Snippet: Subsequently, standards, controls, and samples (undiluted up to 323 diluted) were incubated for 1 h, followed by incubation with monoclonal rat anti-mouse Cst6 (R&D Systems) in PBS/1% normal rabbit serum/0.1% bovine serum albumin/0.05% Tween-20 for 30 min. Next, wells were incubated with goat anti-rat biotinylated antibody (Vector Laboratories) for 30min, followed by a final incubation with avidin-biotinylated horseradish peroxidase complex (Vector Laboratories) for 30 min.

Techniques: Staining, Expressing

Journal: The FASEB Journal

Article Title: Cathepsin B as a potential cystatin M/E target in the mouse hair follicle

doi: 10.1096/fj.201700267r

Figure Lengend Snippet: Figure 2. Destruction of the HFs in Tg(INV-Cst6)Cst6ichq/ichq mice. A) Immunofluorescence double labeling of INV and Cst6 in the epidermis and the HF of WT mice. Cst6 was expressed in the stratum granulosum and halfway up the HF, including the area around the bulge, whereas INV expression was also seen in the stratum granulosum, but remained only in the proximal part of the HF. B) Immunohistological labeling in WT mice to detect the location of the bulge area (arrows) using the stem cell markers CD34 and keratin 15 and the proliferation marker Ki67. C) H&E staining of Tg(INV-Cst6)Cst6ichq/ichq mice from 11, 13, and 16 wk showed disappearance of the HFs. Scale bar, 100 mm.

Article Snippet: Subsequently, standards, controls, and samples (undiluted up to 323 diluted) were incubated for 1 h, followed by incubation with monoclonal rat anti-mouse Cst6 (R&D Systems) in PBS/1% normal rabbit serum/0.1% bovine serum albumin/0.05% Tween-20 for 30 min. Next, wells were incubated with goat anti-rat biotinylated antibody (Vector Laboratories) for 30min, followed by a final incubation with avidin-biotinylated horseradish peroxidase complex (Vector Laboratories) for 30 min.

Techniques: Labeling, Expressing, Marker, Staining

Journal: The FASEB Journal

Article Title: Cathepsin B as a potential cystatin M/E target in the mouse hair follicle

doi: 10.1096/fj.201700267r

Figure Lengend Snippet: Figure 3. Inhibition of mouse CtsB by mouse Cst6. A) BMV109 labels active cysteine cathepsins, such as CTSB and CTSX (14) in RAW cell lysates (murine Mf cell line). Lane 1, no inhibitor; lane 2, JPM-OEt (pan-cathepsin inhibitor, 50 mM); lane 3, CA-074 (CtsB inhibitor, 10 mM); lane 4, Z-FY (t-BU)DMK (CtsL inhibitor, 10 mM); lane 5, mouse Cst6 (18 mM); and lane 6, heat inactivation. Note that CA-074 and Cst6 inhibited CtsB (lane 3 and 5). M = precision plus protein dual-color marker. B) The Ki for the inhibition of CtsB by Cst6 was determined by measuring the residual enzymatic activity of a fixed concentration of enzyme, incubated with increasing concentrations of the inhibitor. An Easson-Stedman plot (inset) was used to calculate the Ki, according to the following equation: [I]/1 2 a = (Ki/a) + E0, were I is the inhibitor concentration, E0 is the enzyme concentration at time 0, and a is the fractional activity. The plot yielded a straight line with a Ki slope of 0.98 nM.

Article Snippet: Subsequently, standards, controls, and samples (undiluted up to 323 diluted) were incubated for 1 h, followed by incubation with monoclonal rat anti-mouse Cst6 (R&D Systems) in PBS/1% normal rabbit serum/0.1% bovine serum albumin/0.05% Tween-20 for 30 min. Next, wells were incubated with goat anti-rat biotinylated antibody (Vector Laboratories) for 30min, followed by a final incubation with avidin-biotinylated horseradish peroxidase complex (Vector Laboratories) for 30 min.

Techniques: Inhibition, Chromosome Transmission Fidelity Colony Color Assay, Activity Assay, Concentration Assay, Incubation

Journal: The FASEB Journal

Article Title: Cathepsin B as a potential cystatin M/E target in the mouse hair follicle

doi: 10.1096/fj.201700267r

Figure Lengend Snippet: Figure 4. CtsB and Cst6 colocalize in the mouse HF. A) Immunofluorescence double staining for Cst6 (green) and CtsB (red) in WT mice revealed that colocalization of both proteins was found in the proximal part of the HF as well as the lower region where the bulge area resides. Cst6 is also expressed in the epidermis, whereas no CtsB is observed. Scale bar, 100 mm. B) Cst6 and CtsB expression in mouse epidermis and HFs of Tg(INV-Cst6)Cst6ichq/ichq mice. No immunofluorescence staining for Cst6 was observed in the lower region of the HF (close to the bulge, arrowheads). Scale bar, 100 mm. C) Schematic presentation of INV, Cst6, and CtsB localization in the HF and epidermis of WT mice and the situation in rescued transgenic Tg(INV-Cst6)Cst6ichq/ichq mice.

Article Snippet: Subsequently, standards, controls, and samples (undiluted up to 323 diluted) were incubated for 1 h, followed by incubation with monoclonal rat anti-mouse Cst6 (R&D Systems) in PBS/1% normal rabbit serum/0.1% bovine serum albumin/0.05% Tween-20 for 30 min. Next, wells were incubated with goat anti-rat biotinylated antibody (Vector Laboratories) for 30min, followed by a final incubation with avidin-biotinylated horseradish peroxidase complex (Vector Laboratories) for 30 min.

Techniques: Double Staining, Expressing, Staining, Transgenic Assay

Journal: The FASEB Journal

Article Title: Cathepsin B as a potential cystatin M/E target in the mouse hair follicle

doi: 10.1096/fj.201700267r

Figure Lengend Snippet: Figure 5. Regulation of epidermal protease activity by CST6. Model of the regulatory role of CST6 in processes that control epidermal cornification, desquamation and HF maintenance. 1) Inhibition of CTSL activity by CST6 is important in the cornification process, as CTSL is the elusive processing and activating enzyme for (TGM)-3. CTSL is also able to process CTSD, which in turn can activate TGM-1. 2) Inhibition of CTSV regulates desquamation, as CTSV is able to degrade (corneo)-desmosomal proteins, such as desmoglein-1, desmocollin-1, and corneodesmosin. As CTSV is expressed only in humans, murine CTSL probably controls the specific functional enzymatic activities of both human CTSL and CTSV. 3) The findings in the present study suggest that inhibition of CtsB by CST6 protects HF maintenance in mice. 4) Inhibition of human LGMN regulates the processing of (pro)-cathepsins; however mouse LGMN is not inhibited by mouse CST6.

Article Snippet: Subsequently, standards, controls, and samples (undiluted up to 323 diluted) were incubated for 1 h, followed by incubation with monoclonal rat anti-mouse Cst6 (R&D Systems) in PBS/1% normal rabbit serum/0.1% bovine serum albumin/0.05% Tween-20 for 30 min. Next, wells were incubated with goat anti-rat biotinylated antibody (Vector Laboratories) for 30min, followed by a final incubation with avidin-biotinylated horseradish peroxidase complex (Vector Laboratories) for 30 min.

Techniques: Activity Assay, Control, Inhibition, Functional Assay

Journal: Scientific Reports

Article Title: The key regulation of LncRNA MALAT during reprogramming of primary mouse hepatocytes into insulin producing cells

doi: 10.1038/s41598-025-08106-y

Figure Lengend Snippet: Primary mouse hepatocytes differentiation into IPC. (A) Schematic presentation of the step-wise differentiation protocol used to generate IPCs from primary mouse hepatocytes. (B) Photographs of primary mouse hepatocytes differentiated into IPCs. Scale bar = 400 μm and 200 μm. (C) Identifying PDX1 cells using immunofluorescence staining with PDX1 antibody and expression of β-cell development-related genes. (D) Detection of insulin-producing cells using immunofluorescence staining with insulin and C-peptide antibodies and expression of the insulin gene. ( n = 3, ** P < 0.01, *** P < 0.001).

Article Snippet: Equal amounts of protein were loaded and resolved on 10% SDS polyacrylamide gels followed by transfer onto PVDF membranes, which were then blocked with 5% non-fat milk in Tris-buffered saline and 0.1% tween-20 (TBST) for 1 h at room temperature and then incubated with their corresponding primary antibodies, FoxA2(1:1000, 8186 T, CST), PI3K(1:1000, 4292 S, CST), PDX1(1:1000, TA321081, OriGene), and β-actin(1:2000, TA811000, OriGene), overnight at 4 °C.

Techniques: Immunofluorescence, Staining, Expressing

Journal: Scientific Reports

Article Title: The key regulation of LncRNA MALAT during reprogramming of primary mouse hepatocytes into insulin producing cells

doi: 10.1038/s41598-025-08106-y

Figure Lengend Snippet: Expression of lncRNA MALAT1/miR-124-3p/PI3K and genes associated with pancreatic development during primary mouse hepatocytes differentiation into IPCs. (A-D) RT-qPCR showing expression of lncRNA MALAT1/miR-124-3p/PI3K and FoxA2 ( n = 3, ** p < 0.01, *** p < 0.001). (C) Western blot results show significantly higher protein expression levels of PDX1 in primary mouse hepatocytes after induction ( n = 3, * p < 0.05, ** p < 0.01).

Article Snippet: Equal amounts of protein were loaded and resolved on 10% SDS polyacrylamide gels followed by transfer onto PVDF membranes, which were then blocked with 5% non-fat milk in Tris-buffered saline and 0.1% tween-20 (TBST) for 1 h at room temperature and then incubated with their corresponding primary antibodies, FoxA2(1:1000, 8186 T, CST), PI3K(1:1000, 4292 S, CST), PDX1(1:1000, TA321081, OriGene), and β-actin(1:2000, TA811000, OriGene), overnight at 4 °C.

Techniques: Expressing, Quantitative RT-PCR, Western Blot

Journal: Scientific Reports

Article Title: The key regulation of LncRNA MALAT during reprogramming of primary mouse hepatocytes into insulin producing cells

doi: 10.1038/s41598-025-08106-y

Figure Lengend Snippet: Functional analysis after IPCs transplantation in T1DM mice. (A) Fasting blood glucose levels of mice, glucose levels were monitored using tail-vein blood samples. (B, C) Serum TC, TG, HDL-C, LDL-C, and insulin levels at the end of the experiment. ( n = 5, * p < 0.05 versus control group, # p < 0.05 versus T1DM group, respectively). (D) Histological section of pancreas tissue. (E) Immunofluorescence staining of β-cell markers (Insulin) and α-cell markers (Glucagon) in pancreas tissue. (F)Immunofluorescence staining of β-cell markers (Insulin and PDX1) in transplanted kidney grafts, and statistical results of the overlapping fluorescence intensity. All fluorescently stained cells were observed under ×20 objective, and representative images are shown. ( n = 5, * p < 0.05 T1DM + IPCs group, respectively).

Article Snippet: Equal amounts of protein were loaded and resolved on 10% SDS polyacrylamide gels followed by transfer onto PVDF membranes, which were then blocked with 5% non-fat milk in Tris-buffered saline and 0.1% tween-20 (TBST) for 1 h at room temperature and then incubated with their corresponding primary antibodies, FoxA2(1:1000, 8186 T, CST), PI3K(1:1000, 4292 S, CST), PDX1(1:1000, TA321081, OriGene), and β-actin(1:2000, TA811000, OriGene), overnight at 4 °C.

Techniques: Functional Assay, Transplantation Assay, Control, Immunofluorescence, Staining, Fluorescence

Journal: PLOS ONE

Article Title: Iron metabolism disorders of patients with chronic paracoccidioidomycosis

doi: 10.1371/journal.pone.0282218

Figure Lengend Snippet: Comparison of functional iron parameter values before treatment and at the time of clinical cure of patients with chronic paracoccidioidomycosis.

Article Snippet: The soluble transferrin receptor (sTfR) level was determined using an enzyme-linked immunosorbent assay (human sTfR ELISA Kit; Elabscience).

Techniques: Comparison, Functional Assay

Journal: Cancers

Article Title: Pancreatic Lineage Specifier PDX1 Increases Adhesion and Decreases Motility of Cancer Cells

doi: 10.3390/cancers13174390

Figure Lengend Snippet: Effect of PDX1 on cell migration and invasion. Colo357, PANC-1, MiaPaCa-2, BxPC-3, and SW620 cell migration ( A , B ) analysis and Colo357, PANC-1 cell invasion analysis ( C , D ) were performed, as described in “Materials and Methods.” Representative pictures of three independent studies are shown. Magnification ×175. The white stripe corresponds to a scale of 100 µm. Data are presented as the mean ± SD. * p < 0.05, ** p < 0.01 compared with control group, Manna–Whitney test.

Article Snippet: The membranes were then incubated with indicated primary antibodies rabbit anti-PDX1 (1:1000; Cat. no. 5679; Cell Signaling Technology, Danvers, MA, USA) and mouse anti-GAPDH (1:60,000; Cat. no. 10494–1-AP; Santa Cruz Biotechnology Inc., Dallas, TX, USA) at 4 °C overnight, followed by incubation with horseradish peroxidase (HRP)-conjugated secondary antibodies (1:5000; Cat. no. sc-2054; Santa Cruz Biotechnology Inc.) at room temperature for 1 h. Mouse monoclonal anti-GAPDH antibody was used as a loading control.

Techniques: Migration, Control

Journal: Cancers

Article Title: Pancreatic Lineage Specifier PDX1 Increases Adhesion and Decreases Motility of Cancer Cells

doi: 10.3390/cancers13174390

Figure Lengend Snippet: ( A ) Schematic of the lentiviral construct used to generate cultures of pancreatic cancer cells ectopically expressing PDX1. ( B ) Western blot analysis of PDX1 protein content in transduced pancreatic cancer cell lines BxPC-3, Colo357, MiaPaCa-2, PANC-1, and colon cancer line SW620. Red numbers indicate the relative amount of PDX1 protein in transduced Colo357-PDX1, MiaPaCa-2-PDX1, PANC-1-PDX1, BxPC-3-PDX1, and SW620-PDX1 cells. The values are given relative to the level of PDX1 in Colo357-PDX1 cells. GAPDH protein detection in cell extracts was used as a normalizing control. ( C ) Growth kinetics of PDX1-expressing cultures of pancreatic cancer cell lines and corresponding control cells by MTS test performing. Data are normalized relative to values on the first day of the experiment. Three independent experiments involving five–six time-point were conducted. Error bars refer to SD. Cancer cells transduced with lentivirus without the PDX1 gene were used as control. * p ≤ 0.05, ** p ≤ 0.01.

Article Snippet: The membranes were then incubated with indicated primary antibodies rabbit anti-PDX1 (1:1000; Cat. no. 5679; Cell Signaling Technology, Danvers, MA, USA) and mouse anti-GAPDH (1:60,000; Cat. no. 10494–1-AP; Santa Cruz Biotechnology Inc., Dallas, TX, USA) at 4 °C overnight, followed by incubation with horseradish peroxidase (HRP)-conjugated secondary antibodies (1:5000; Cat. no. sc-2054; Santa Cruz Biotechnology Inc.) at room temperature for 1 h. Mouse monoclonal anti-GAPDH antibody was used as a loading control.

Techniques: Construct, Expressing, Western Blot, Control, Transduction

Journal: Cancers

Article Title: Pancreatic Lineage Specifier PDX1 Increases Adhesion and Decreases Motility of Cancer Cells

doi: 10.3390/cancers13174390

Figure Lengend Snippet: Influence of the PDX1 gene on the motility genes expression. ( A ) Motility genes expression. The data are shown as the mean ± SD ( n = 3). * p < 0.05, ** p < 0.01. ( B ) Functional protein association network of genes with changed expression based on STRING database ( https://string-db.org/Version11.0b , Date: 17 October 2020 to 12 August 2021).

Article Snippet: The membranes were then incubated with indicated primary antibodies rabbit anti-PDX1 (1:1000; Cat. no. 5679; Cell Signaling Technology, Danvers, MA, USA) and mouse anti-GAPDH (1:60,000; Cat. no. 10494–1-AP; Santa Cruz Biotechnology Inc., Dallas, TX, USA) at 4 °C overnight, followed by incubation with horseradish peroxidase (HRP)-conjugated secondary antibodies (1:5000; Cat. no. sc-2054; Santa Cruz Biotechnology Inc.) at room temperature for 1 h. Mouse monoclonal anti-GAPDH antibody was used as a loading control.

Techniques: Expressing, Functional Assay

Journal: Cancers

Article Title: Pancreatic Lineage Specifier PDX1 Increases Adhesion and Decreases Motility of Cancer Cells

doi: 10.3390/cancers13174390

Figure Lengend Snippet: The sensitivity of Control and PDX1-expressing Colo357 and PANC-1 cells to TGFβ1-induced epithelial-mesenchymal transition (EMT). ( A , B ) Morphological changes in PANC-1-Control/PDX1 ( A ) and Colo357-Control/PDX1 ( B ) cells after treatment with TGFβ1 factor for 24 h. Magnification ×175. The white and black stripes correspond to a scale of 100 µm. Red arrows indicate examples of fibroblast-like changes. ( C ) Changes in cell parameters cells after treatment with TGFβ1 factor for 24 h: length, width, length-to-width ratio. Data are presented as the mean ± SD. * p < 0.05, ** p < 0.01 compared with control group, Mann–Whitney test.

Article Snippet: The membranes were then incubated with indicated primary antibodies rabbit anti-PDX1 (1:1000; Cat. no. 5679; Cell Signaling Technology, Danvers, MA, USA) and mouse anti-GAPDH (1:60,000; Cat. no. 10494–1-AP; Santa Cruz Biotechnology Inc., Dallas, TX, USA) at 4 °C overnight, followed by incubation with horseradish peroxidase (HRP)-conjugated secondary antibodies (1:5000; Cat. no. sc-2054; Santa Cruz Biotechnology Inc.) at room temperature for 1 h. Mouse monoclonal anti-GAPDH antibody was used as a loading control.

Techniques: Control, Expressing, MANN-WHITNEY

Journal: Cancers

Article Title: Pancreatic Lineage Specifier PDX1 Increases Adhesion and Decreases Motility of Cancer Cells

doi: 10.3390/cancers13174390

Figure Lengend Snippet: Changes in the expression of EMT marker genes upon treatment of Colo357 with TGFβ1 factor for 48 h. ( A ) Changes in EMT marker gene expression when PANC 1 cells were treated with TGFβ1 factor for 48 h. The data are shown as the mean of relative expression ± SD ( n = 3). *, # , $ p < 0.05 (*- PANC-1-PDX1 over PANC-1-Control, # -PANC-1-Control + TGFb1 over PANC-1-Control, $ -PANC-1-PDX1 + TGFb1 over PANC-1-PDX1). ( B ) Changes in the expression of EMT marker genes upon treatment of Colo357 with TGFβ1 factor for 48 h. The data are shown as the mean of relative expression ± SEM ( n = 3). *, # , $ p < 0.05 (*-Colo357-PDX1 over Colo357-Control, # -Colo357-Control + TGFb1 over Colo357-Control, $ -Colo357-PDX1 + TGFb1 over Colo357-PDX1). Values of relative expression are highlighted as heatmaps (green-increased expression level, blue-decreased level).

Article Snippet: The membranes were then incubated with indicated primary antibodies rabbit anti-PDX1 (1:1000; Cat. no. 5679; Cell Signaling Technology, Danvers, MA, USA) and mouse anti-GAPDH (1:60,000; Cat. no. 10494–1-AP; Santa Cruz Biotechnology Inc., Dallas, TX, USA) at 4 °C overnight, followed by incubation with horseradish peroxidase (HRP)-conjugated secondary antibodies (1:5000; Cat. no. sc-2054; Santa Cruz Biotechnology Inc.) at room temperature for 1 h. Mouse monoclonal anti-GAPDH antibody was used as a loading control.

Techniques: Expressing, Marker, Gene Expression, Control

Journal: Cancers

Article Title: Pancreatic Lineage Specifier PDX1 Increases Adhesion and Decreases Motility of Cancer Cells

doi: 10.3390/cancers13174390

Figure Lengend Snippet: PDX1 gene expression increases cell adhesion. A quantitation of adhesion and cell attachment by cell binding to collagen type I, fibronectin, poly-L-lysine, and BSA (as a control). Data are given as the mean cell attachment ± SEM ( n = 3). * p < 0.05.

Article Snippet: The membranes were then incubated with indicated primary antibodies rabbit anti-PDX1 (1:1000; Cat. no. 5679; Cell Signaling Technology, Danvers, MA, USA) and mouse anti-GAPDH (1:60,000; Cat. no. 10494–1-AP; Santa Cruz Biotechnology Inc., Dallas, TX, USA) at 4 °C overnight, followed by incubation with horseradish peroxidase (HRP)-conjugated secondary antibodies (1:5000; Cat. no. sc-2054; Santa Cruz Biotechnology Inc.) at room temperature for 1 h. Mouse monoclonal anti-GAPDH antibody was used as a loading control.

Techniques: Gene Expression, Quantitation Assay, Cell Attachment Assay, Binding Assay, Control