Journal: Nature Communications

Article Title: Cathepsin D deficiency in mammary epithelium transiently stalls breast cancer by interference with mTORC1 signaling

doi: 10.1038/s41467-020-18935-2

Figure Lengend Snippet: a Flow cytometry-based monitoring of cre recombination reporter expression (Tomato: non-recombined, GFP: recombined) in rtTA-tTS;tetO-cre;Ctsd fl/fl ;mTmG PyMT cells treated for 0–4 days with doxycycline (Dox) ( n = 3 independent experiments). b Analysis of CTSD expression by Western blot (with TUBA as loading control) in lysates from cells used in a . Pro/SC, zymogen/single-chain form; HC, heavy chain; LC, light chain. In vitro competitive growth assay of non-recombined and recombined rtTA-tTS;tetO-cre;Ctsd fl/fl ;mTmG PyMT cells in 10% FCS ( c ) or 1% FCS ( d ) medium after a one-day pulse of Dox ( n = 3 independent experiments for d 0 and d 1 in c and d 28 in d , n = 4 independent experiments for the rest). e In vivo competitive growth assay of non-recombined and recombined 10% FCS rtTA-tTS;tetO-cre;Ctsd fl/fl ;mTmG PyMT cells. A one-day Dox-pulsed cell suspension of known ratio of non-recombined to recombined cells (left bar) was orthotopically transplanted into 6 recipient mice. The ratio was again determined by flow cytometry in the outgrown tumors (right bar) ( n = 6 animals; two-sided one-sample t-test). Line and bar charts show all data points with mean ± SD and p -value. Source data are provided as a Source Data file.

Article Snippet: Membranes were blocked with 3% BSA in TBS-Tween (0.1%) and incubated with primary antibodies for ACTB (MP, 691001; 1:2000), CDH2 (Cell Signaling, 4061; 1:500), CTSB (R&D, BAF965; 1:200), CTSD (R&D, AF1029; 1:200), CTSL (R&D, AF1515; 1:200), GFP (Cell Signaling, 2555; 1:1000), LAMP1 (Cell Signaling, 3243; 1:500), LAMTOR4 (Cell Signaling, 12284; 1:1000), LC3 (Cell Signaling, 2775; 1:500), mTOR (Cell Signaling, 2983; 1:1000), p-p38 MAPK (Cell Signaling, 9211; 1:1000), p38 MAPK (Cell Signaling, 9212; 1:1000), p62 (Cell Signaling, 5114; 1:500), p-P70S6K (Cell Signaling, 9205; 1:500), P70S6K (Enzo, ADI-KAP-CC035-E; 1:250), RHEB (Cell Signaling, 13879; 1:1000), RPTOR (Cell Signaling, 2280; 1:1000), RRAGC (Cell Signaling, 5466; 1:1000), p-S6 (Cell Signaling, 4857; 1:1000), S6 (Cell Signaling, 2317; 1:1000), TUBA (Sigma, T9026; 1:1000), and VIM (BD, 550513; 1:500) mostly overnight at RT.

Techniques: Flow Cytometry, Expressing, Western Blot, In Vitro, Growth Assay, In Vivo

Journal: Nature Communications

Article Title: Cathepsin D deficiency in mammary epithelium transiently stalls breast cancer by interference with mTORC1 signaling

doi: 10.1038/s41467-020-18935-2

Figure Lengend Snippet: a Flow cytometry analysis of LysoTracker TM Green intensity in Ctsd +/+ and Ctsd − /− PyMT cells cultured for 7–10 days in 10% FCS or 1% FCS medium or treated with Torin ( n = 3 independent experiments for Torin, n = 6 independent experiments for the rest; two-sided three-way ANOVA for the factors intensity of LysoTracker TM , Ctsd genotype and FCS). b Analysis of LAMP1 expression by Western blot (with TUBA as loading control) in 10% FCS and 1% FCS Ctsd +/+ and Ctsd − /− PyMT cells. Slight molecular weight changes due to different glycosylation pattern. Representative of 2 independent experiments. Quantification is below ( n = 1 experiment). c Relative Lamp1 expression determined by RT-PCR in 10% FCS and 1% FCS Ctsd +/+ and Ctsd − /− PyMT cells ( n = 3 independent experiments; two-sided two-sample t -test). Activity assay with substrates specific for aspartic ( d ) or cysteine ( e ) proteases in 10% FCS and 1% FCS Ctsd +/+ and Ctsd − /− PyMT cells ( n = 2 independent experiments). f Lysosomal activity assay using a quenched fluorogenic substrate in 10% FCS and 1% FCS Ctsd +/+ and Ctsd − /− PyMT cells ( n = 4 independent experiments for 10% FCS, n = 3 independent experiments for 1% FCS). Histogram including controls blocking the endocytic substrate uptake (Cytochalasin D) or lysosomal acidification (Bafilomycin A1) on the left, median fluorescence intensity (MFI) on the right. g Analysis of CTSL expression by Western blot (with TUBA as loading control) in 10% FCS and 1% FCS Ctsd +/+ and Ctsd -/- PyMT cells. Pro, zymogen; SC, single-chain form; HC, heavy chain. Representative of 2 independent experiments. Quantification is to the right ( n = 2 independent experiments). h Relative Ctsl expression determined by RT-PCR in 10% FCS and 1% FCS Ctsd +/+ and Ctsd − /− PyMT cells ( n = 3 independent experiments; two-sided two-sample t -test). i Analysis of CTSB expression by Western blot (with TUBA as loading control) in 10% FCS and 1% FCS Ctsd +/+ and Ctsd − /− PyMT cells. Pro, zymogen; SC, single-chain form. Representative of 2 independent experiments. Bar charts show all data points with mean + SD and p -value. Source data are provided as a Source Data file.

Article Snippet: Membranes were blocked with 3% BSA in TBS-Tween (0.1%) and incubated with primary antibodies for ACTB (MP, 691001; 1:2000), CDH2 (Cell Signaling, 4061; 1:500), CTSB (R&D, BAF965; 1:200), CTSD (R&D, AF1029; 1:200), CTSL (R&D, AF1515; 1:200), GFP (Cell Signaling, 2555; 1:1000), LAMP1 (Cell Signaling, 3243; 1:500), LAMTOR4 (Cell Signaling, 12284; 1:1000), LC3 (Cell Signaling, 2775; 1:500), mTOR (Cell Signaling, 2983; 1:1000), p-p38 MAPK (Cell Signaling, 9211; 1:1000), p38 MAPK (Cell Signaling, 9212; 1:1000), p62 (Cell Signaling, 5114; 1:500), p-P70S6K (Cell Signaling, 9205; 1:500), P70S6K (Enzo, ADI-KAP-CC035-E; 1:250), RHEB (Cell Signaling, 13879; 1:1000), RPTOR (Cell Signaling, 2280; 1:1000), RRAGC (Cell Signaling, 5466; 1:1000), p-S6 (Cell Signaling, 4857; 1:1000), S6 (Cell Signaling, 2317; 1:1000), TUBA (Sigma, T9026; 1:1000), and VIM (BD, 550513; 1:500) mostly overnight at RT.

Techniques: Flow Cytometry, Cell Culture, Expressing, Western Blot, Molecular Weight, Reverse Transcription Polymerase Chain Reaction, Activity Assay, Blocking Assay, Fluorescence

Journal: International Journal of Nephrology

Article Title: The Role of Cathepsin B in Peritoneal Fibrosis due to Peritoneal Dialysis

doi: 10.1155/2019/4150656

Figure Lengend Snippet: Procathepsin B (a) and cathepsin B (b) level in the supernatant of cultured human peritoneal mesothelial cells following various stimuli. Procathepsin B and cathepsin B level in the supernatant after the addition of various concentrations of mannitol and glucose and LPS are shown as fold increases compared with the control level in untreated cells. Procathepsin B and cathepsin B level was increased by 4.25% glucose and LPS. OC, G , and LPS means osmotic control, glucose, and lipopolysaccharide, respectively. ∗ p < 0.05 compared with the control; # p < 0.05 compared with its osmotic control.

Article Snippet: The other 30 mice were treated with daily intraperitoneal injections of 0.3 ml chlorhexidine solution for 3 days and were then divided into three groups and treated with normal saline 0.3 ml (group 2), 0.5 μ g/ml mouse cathepsin B (R&D Systems) 0.3 ml (group 3), or 0.5 μ g/ml cathepsin B 0.15 ml plus 5 μ g/ml mouse cystatin C (Sino Biological, Beijing, China) 0.15 ml (group 4) for a further 3 days.

Techniques: Cell Culture

Journal: International Journal of Nephrology

Article Title: The Role of Cathepsin B in Peritoneal Fibrosis due to Peritoneal Dialysis

doi: 10.1155/2019/4150656

Figure Lengend Snippet: Protein expression of MMPs, TIMPs, and uPA in the supernatant of cultured human peritoneal mesothelial cells after treatment with cathepsin B. Cathepsin B increased MMP-1, MMP-2, MMP-3, and TIMP-1 protein expression, but had no effect on uPA. TIMP-2 was increased by 1 ng/ml cathepsin B but decreased by 5 ng/ml. MMP, TIMP, and uPA means matrix metalloproteinase, tissue inhibitor of metalloproteinase, and urokinase-type plasminogen activator, respectively. ∗ p < 0.05 compared with protein expression without cathepsin B treatment, # p < 0.05 compared with protein expression after1 ng/ml cathepsin B treatment.

Article Snippet: The other 30 mice were treated with daily intraperitoneal injections of 0.3 ml chlorhexidine solution for 3 days and were then divided into three groups and treated with normal saline 0.3 ml (group 2), 0.5 μ g/ml mouse cathepsin B (R&D Systems) 0.3 ml (group 3), or 0.5 μ g/ml cathepsin B 0.15 ml plus 5 μ g/ml mouse cystatin C (Sino Biological, Beijing, China) 0.15 ml (group 4) for a further 3 days.

Techniques: Expressing, Cell Culture

Journal: International Journal of Nephrology

Article Title: The Role of Cathepsin B in Peritoneal Fibrosis due to Peritoneal Dialysis

doi: 10.1155/2019/4150656

Figure Lengend Snippet: Microscopic examination of anterior abdominal wall in the mouse fibrosis model. Submesothelial thickness was increased from 31 ± 9 μ m in the control group (a) to 333 ± 59 μ m in the chlorhexidine-treated group (b). The addition of cathepsin B to the chlorhexidine-treated mice reduced the thickness of the submesothelial layer by 192 ± 25 μ m (c), while the simultaneous addition of cathepsin B and cystatin C had no significant effect (280 ± 37 μ m) (d).

Article Snippet: The other 30 mice were treated with daily intraperitoneal injections of 0.3 ml chlorhexidine solution for 3 days and were then divided into three groups and treated with normal saline 0.3 ml (group 2), 0.5 μ g/ml mouse cathepsin B (R&D Systems) 0.3 ml (group 3), or 0.5 μ g/ml cathepsin B 0.15 ml plus 5 μ g/ml mouse cystatin C (Sino Biological, Beijing, China) 0.15 ml (group 4) for a further 3 days.

Techniques: