ctsd  (Proteintech)

Journal: Clinical and Translational Medicine

Article Title: Hypoxia‐induced secretory autophagy in cancer‐associated fibroblasts promotes ECM remodelling through serglycin secretion in oral squamous cell carcinoma

doi: 10.1002/ctm2.70556

Figure Lengend Snippet: Under hypoxia, most SRGN is secreted extracellularly via the secretory autophagy pathway in CAFs. (A) Prediction of SRGN signal peptide structure using SignalP 6.0. (B) WB analysis of SRGN expression in normoxic and hypoxic CAFs with or without BFA (.2 µM, 24 h). (C) Detection of SRGN secretion levels in the supernatant of normoxic and hypoxic CAFs with or without BFA by ELISA. (D) IF showing co‐localization of SRGN and GM130 in normoxic and hypoxic CAFs with or without BFA. Scale bar = 10 µm. (E) IF observation of LAMP1 protein expression in normoxic and hypoxic CAFs and changes in LysoSensor intensity. Scale bar = 50 µm. (F, G) WB analysis of LAMP1 and CTSD protein expression levels in normoxic and hypoxic CAFs, along with quantitative analysis. (H) IF showing co‐localization of SRGN and LC3B in normoxic and hypoxic CAFs with or without 3‐MA. Scale bar = 10 µm. (I, J) WB analysis of LAMP1, P62, CTSD, SRGN, and LC3B protein expression levels in normoxic and hypoxic CAFs treated with BFA, CQ (60 µM), and Baf A1 (20 nM) for 24 h, along with quantitative analysis of SRGN expression. (K) Detection of SRGN secretion levels in the supernatant of normoxic and hypoxic CAFs treated with CQ and Baf A1 by ELISA. ns, not significant; * p < .05; ** p < .01; *** p < .001.

Article Snippet: The primary antibodies used are listed as follows: SRGN (1:1000, A25426, ABclonal), ATG5 (1:1000, 10181‐2‐AP, Proteintech), LC3B (1:2000, ab192890, Abcam, USA), Beclin 1 (1:1000, 11306‐1‐AP, Proteintech), β‐actin (1:4000, 20536‐1‐AP, Proteintech), LAMP1 (1:4000, 21997‐1‐AP, Proteintech), P62 (1:4000, 18420‐1‐AP, Proteintech), CTSD (1:4000, 21327‐1‐AP, Proteintech), MMP9 (1:1000, bs‐4593R, Bioss, China), MMP2 (1:1000, CY5189, Abways, China), and MMP11 (1:1000, CY5778, Abways).

Techniques: Expressing, Enzyme-linked Immunosorbent Assay

Journal: Molecular Cancer

Article Title: Lung endothelial PEAR1 induces tumor cell dormancy

doi: 10.1186/s12943-025-02488-3

Figure Lengend Snippet: PEAR1 sequesters CTSD and LOXL2 to promote tumor cell dormancy. A , MDA-MB-231 cells were treated with increasing concentrations of the extracellular domain of recombinant human PEAR1 containing a His-tag and immobilized on Ni-NTA beads. Cells incubated with Ni-NTA beads only served as a control group. Cell count was determined by flow cytometry analysis ( n = 3 independent experiments). B , MDA-MB-231 cells were treated daily for 4 days with PEAR1 immobilized on Ni-NTA beads, and cells were analyzed by immunoblotting using anti-GAPDH and anti-p27 antibodies. Shown is a representative immunoblot and the statistical analysis ( n = 3 independently performed experiments). C , D , MDA-MB-231 cells expressing mVenus-p27K − were transfected with control siRNA or siRNA directed against SVEP1 or LOXL2 and were incubated in the absence or presence of PEAR1 (100 nM). After 36 h, the cell count (C) and the percentage of p27-positive cells (D) was determined ( n = 3 independent experiments). E , Procedure to search for interaction partners of PEAR1 within the conditioned medium of tumor cells using a pull-down assay with PEAR1 immobilized on Ni-NTA beads. Control Ni-NTA beads and the recombinant extracellular part of human epidermal growth factor receptor immobilized on Ni-NTA beads served as controls. F , The plot shows the proteins that were specifically co-precipitated with PEAR1 compared to control samples. Proteins only found with PEAR1 are indicated by green dots, and proteins found with both PEAR1 and EGFR are represented by grey dots. G , Recombinant CTSD, SEMA6D or LOXL2 were incubated alone or together with purified His-tagged PEAR1. PEAR1 was precipitated with Ni-NTA beads, and precipitates were analyzed by immunoblotting. Shown are representative of 3 independently performed experiments. H , I , MDA-MB-231 cells co-expressing mCherry-luciferase and mVenus-p27K − were co-cultured with HUVECs for the indicated time periods, and cells were treated daily without or with CTSD (100 nM), LOXL2 (100 nM) or SEMA6D (1000 nM). The cell count (H) was assessed using flow cytometry. At the end of the experiment, the percentage of p27-positive cells (l) was determined by flow cytometry ( n = 3 independent experiments). J , K , Co-cultures of HUVECs and MDA-MD-231 cells were incubated for the indicated time periods with 100 nM PEAR1 alone or in combination with 100 nM CTSD or LOXL2. Assessment of cell count (J) was conducted utilizing flow cytometry, and, after 4 days, the percentage of p27-positive cells (K) was analyzed by flow cytometry ( n = 3 independent experiments). L , M , MDA-MB-231 cells co-expressing mCherry and mVenus-p27K − were transfected with control siRNA or siRNA directed against CTSD, LOXL2 or both, and were cultured in the absence or presence of the recombinant extracellular part of human PEAR1 (rhPEAR1; 100 nM). After 96 h, cells were counted (L), and p27 expression was determined (M) ( n = 3 independently performed experiments). Shown are mean values ± S.E.M.; * , p ≤ 0.05; ** , p ≤ 0.01; *** , p ≤ 0.001; n.s., non-significant (2-way ANOVA and Bonferroni’s post-hoc test (A-D, H, J, L, M); multiple two-tailed t-test, Bayesian moderated, Benjamin, Krieger, and Yekutieli-corrected, false discovery rate = 1% (F); one-way ANOVA with Tukey’s multiple comparison test (I, K))

Article Snippet: 6x-His tagged mouse recombinant CTSD protein (catalog #50127-M08H) was purchased from Sinobiological.

Techniques: Recombinant, Incubation, Control, Cell Counting, Flow Cytometry, Western Blot, Expressing, Transfection, Pull Down Assay, Purification, Luciferase, Cell Culture, Two Tailed Test, Comparison

Journal: Molecular Cancer

Article Title: Lung endothelial PEAR1 induces tumor cell dormancy

doi: 10.1186/s12943-025-02488-3

Figure Lengend Snippet: Suppression of CTSD in tumor cells increased tumor cell dormancy both in vitro and in vivo . A , B , The indicated tumor cells co-expressing mCherry-luciferase and mVenusp27K − were subjected to stable transduction with either a scrambled control shRNA (shScr) or an shRNA targeting Ctsd (shCtsd). Subsequently, tumor cells were co-cultured with MLECs and treated daily with either a vehicle control or recombinant mouse CTSD (100 nM) over a period of 96 h, and the percentages of p27-positive E0771 (A) and B16F10 (B) cells were quantified ( n = 3 independent experiments). C - J , The indicated tumor cells without or with stable knock-down of CTSD (shScr or shCtsd, respectively) co-expressing mCherry-luciferase and mVenus-p27K − were i.v. injected into wild-type animals. Lung metastasis was assessed by determining the number of visible metastatic nodules 12 days after injection of E0771 (C) or B16F10 (D) as well as by analyzing isolated lungs by bioluminescence imaging (E and F) ( n = 4 mice per group). The proportion of p27-positive tumor cells present in lung digests collected 1, 5, and 12 days after i.v. injection of E0771 (G) or B16F10 (H) was determined by flow cytrometry ( n = 4 mice per group). Cryosections of whole lungs were stained with Hoechst 33342, and antibodies against mVenus, mCherry and Ki67. Thereafter, entire lung sections were imaged, enabling the categorization of tumor cells into three distinct groups: solitary tumor cells (TC) (1 cell) and those present in micrometastasis (2–8 cells) and macrometastasis (> 8 cells). The bar graphs show the total numbers of proliferating (Ki67-positive) and dormant (Ki67-negative and p27-positive) tumor cells per area for E0771 (I) and B16F10 cells (J) without (shScr) or with CTSD knock-down (shCtsd) ( n = 4 mice per group; 3 sections per mice). K , Schematic representation of the proposed role of endothelial PEAR1 in promoting tumor cell dormancy. EC, endothelial cell; TC, tumor cell. Shown are mean values ± S.E.M.; * , P ≤ 0.05; ** , P ≤ 0.01; *** , P ≤ 0.001; n.s., non-significant (2-way ANOVA and Bonferroni’s post-hoc test (A, B, G, H, I (solitary TCs and micrometastasis and J); two-tailed, unpaired t-test with Welch’s correction (C); two-tailed, unpaired t-test (D, E); Mann-Whitney U test, unpaired, two-tailed (F); Kruskal-Wallis test with Dunn’s multiple comparison test (I (macrometastasis))

Article Snippet: 6x-His tagged mouse recombinant CTSD protein (catalog #50127-M08H) was purchased from Sinobiological.

Techniques: In Vitro, In Vivo, Expressing, Luciferase, Transduction, Control, shRNA, Cell Culture, Recombinant, Knockdown, Injection, Isolation, Imaging, Staining, Two Tailed Test, MANN-WHITNEY, Comparison