Journal: Aging (Albany NY)

Article Title: Cathepsin V drives lung cancer progression by shaping the immunosuppressive environment and adhesion molecules cleavage

doi: 10.18632/aging.205278

Figure Lengend Snippet: CTSV contributes to the metastasis of lung cancer cells both in vitro and in vivo . ( A – C ) A549, NCI-H292, and NCI-H1975, lung cancer cells transfected with the indicated negative control plasmids, shCTSV#1 or shCTSV#3, for 48 h were analyzed by Western blotting. The results were repeated for three biologically independent experiments. ( D ) The indicated stable cells transfected with shCTSV#1 and HA-CTSV for 48 h were subjected to Western blotting. The grouping of blots cropped from different parts of the same gel. ( E – H ) The indicated stable cells were analyzed by cell migration and invasion assays. The results are the mean ± SD of three biologically independent experiments. * P < 0.05, ** P < 0.01, *** P < 0.001 were analyzed by using the student’s t -test. NS represents no significance. ( I ) Bioluminescent imaging analysis of mice. Representative images are shown ( n = 6 mice per group). The tumors were isolated at the end of the experiments. ( J – M ) CTSV-HA-medium or CTSV-HA-high NCI-H1975 and A549 cells were transiently transfected with sgRNAs as indicated and then analyzed by Western blotting, qRT-PCR, cell migration, and invasion assays. The results showed that with the increase in CTSV expression (2–5 times the normal value), the promoting effect of CTSV on migration and invasion also improved. The grouping of blots cropped from different parts of the same gel. ( N – P ) H&E staining of lungs from representative tumor-bearing nude mice. Scale bars, 5 mm. Kaplan-Meier survival curves of the nude mice in the vector and CTSV-HA groups. Quantification of lung nodules. Data are shown as the mean ± SD and P -values; two-tailed Student’s t -tests were used to analyze the data. **** P -value < 0.0001. Full-length blots are shown in .

Article Snippet: We used 200 μl of serum-free DMEM to seed (1 × 10 5 ) A549, NCI-H1975, and NCI-H292 cells in the 24-well Boyden chamber (BD Biosciences, USA) with an 8 m pore size.

Techniques: In Vitro, In Vivo, Transfection, Negative Control, Western Blot, Migration, Imaging, Isolation, Quantitative RT-PCR, Expressing, Staining, Plasmid Preparation, Two Tailed Test

Journal: Aging (Albany NY)

Article Title: Cathepsin V drives lung cancer progression by shaping the immunosuppressive environment and adhesion molecules cleavage

doi: 10.18632/aging.205278

Figure Lengend Snippet: CTSV promotes lung cancer metastasis by cleaving off adhesion proteins. ( A – C ) Immunofluorescence analysis was performed using an anti-actin antibody in the indicated stable cells. Scale bar, 50 μm. ( D ) The indicated A549/NCI-H1975 cells were analyzed by adhesion assay. ( E ) Protein mass spectrometry analysis of CTSV binding partner candidates in the indicated stable cells. ( F , G ) A549 and NCI-H1975 cells expressing CTSV-HA-medium and CTSV-HA-high were analyzed by Western blotting using anti-fibronectin, anti-E-cadherin and anti-N-cadherin antibodies. ( H – J ) A549, NCI-H1975, and NCI-H292 cells transfected with negative control plasmids, shCTSV#1 or shCTSV#3 for 48 h were analyzed by Western blotting using anti-fibronectin, anti-E-cadherin and anti-N-cadherin antibodies. The grouping of blots cropped from different parts of the different gels. Data are shown as the mean ± SD and repeated for three biologically independent experiments. P -values are shown; two-tailed Student’s t -test. Abbreviation: NC: negative control shRNA. Vector, vector-only control. The representative data are repeated for three biologically independent experiments. Student’s t -tests were used for data analysis; * P < 0.05, ** P -value < 0.01, *** P -value < 0.001 and **** P -value < 0.0001.

Article Snippet: We used 200 μl of serum-free DMEM to seed (1 × 10 5 ) A549, NCI-H1975, and NCI-H292 cells in the 24-well Boyden chamber (BD Biosciences, USA) with an 8 m pore size.

Techniques: Immunofluorescence, Cell Adhesion Assay, Mass Spectrometry, Binding Assay, Expressing, Western Blot, Transfection, Negative Control, Two Tailed Test, shRNA, Plasmid Preparation

Journal: Aging (Albany NY)

Article Title: Cathepsin V drives lung cancer progression by shaping the immunosuppressive environment and adhesion molecules cleavage

doi: 10.18632/aging.205278

Figure Lengend Snippet: Fibronectin, E-cadherin, and N-cadherin regulate cell adhesion, migration, and invasion. ( A – C ) NCI-H1975 and A549 cells were transfected with shRNAs targeting fibronectin and then analyzed by Western blotting, migration and invasion assay. ( D – F ) NCI-H1975 and A549 cells were transfected with shRNAs targeting E-cadherin and then analyzed by Western blotting, migration and invasion assay. ( G – I ) NCI-H1975 and A549 cells were transfected with shRNAs targeting N-cadherin and then analyzed by Western blotting, migration and invasion assay. The grouping of blots cropped from different parts of the different gels. Data are shown as the mean ± SD and repeated for three biologically independent experiments. P -values are shown; two-tailed Student’s t -test. Abbreviation: NC: negative control shRNA. Vector, vector-only control. The representative data are repeated for three biologically independent experiments. Student’s t -tests were used for data analysis; * P < 0.05, ** P -value < 0.01, *** P -value < 0.001 and **** P -value < 0.0001.

Article Snippet: We used 200 μl of serum-free DMEM to seed (1 × 10 5 ) A549, NCI-H1975, and NCI-H292 cells in the 24-well Boyden chamber (BD Biosciences, USA) with an 8 m pore size.

Techniques: Migration, Transfection, Western Blot, Invasion Assay, Two Tailed Test, Negative Control, shRNA, Plasmid Preparation

Journal: Aging (Albany NY)

Article Title: Cathepsin V drives lung cancer progression by shaping the immunosuppressive environment and adhesion molecules cleavage

doi: 10.18632/aging.205278

Figure Lengend Snippet: CTSV cleaves off fibronectin, E-cadherin, and N-cadherin. ( A , B ) Stable CTSV-HA A549 cells were subjected to coimmunoprecipitation assays using anti-fibronectin, anti-E-cadherin, and anti-N-cadherin antibodies, followed by Western blotting thrice. The grouping of blots cropped from different parts of the different gels. ( C ) Lysates of A549 cells were subjected to IP using anti-fibronectin, anti-E-cadherin, and anti-N-cadherin antibodies and control IgG, then detected by immunoblotting. ( D – F ) 1 μg recombinant human fibronectin, E-cadherin, and N-cadherin were incubated with 1 μg recombinant human CTSV at 37°C for the indicated periods. Fibronectin, E-cadherin, and N-cadherin fragments were detected using anti-fibronectin, anti-E-cadherin, and anti-N-cadherin antibodies. HA is shown as a control. The grouping of blots cropped from different parts of the same gels.

Article Snippet: We used 200 μl of serum-free DMEM to seed (1 × 10 5 ) A549, NCI-H1975, and NCI-H292 cells in the 24-well Boyden chamber (BD Biosciences, USA) with an 8 m pore size.

Techniques: Western Blot, Recombinant, Incubation

Journal: Aging (Albany NY)

Article Title: Cathepsin V drives lung cancer progression by shaping the immunosuppressive environment and adhesion molecules cleavage

doi: 10.18632/aging.205278

Figure Lengend Snippet: CTSV antibody blockade inhibits adhesion, migration, and invasion in lung cancer cells. ( A ) The schematic diagram for the experimental procedures of generating CTSV antibodies. ( B ) A549 cells treated with CTSV antibody were then subjected to immunoblotting assay using anti-fibronectin, anti-E-cadherin, and anti-N-cadherin antibodies. The grouping of blots cropped from different parts of the same gels. Full-length blots are shown in . ( C – E ) Adhesion assays were performed for the indicated stable A549, NCI-H1975, and NCI-292 cells pre-treated with CTSV antibody. ( F – K ) The indicated stable A549, NCI-H1975, and NCI-H292 cells were treated with CTSV antibody and examined by migration and invasion assay. The representative data are repeated for three biologically independent experiments. Student’s t -tests were used for data analysis; ** P -value < 0.01, *** P -value < 0.001, and **** P -value < 0.0001.

Article Snippet: We used 200 μl of serum-free DMEM to seed (1 × 10 5 ) A549, NCI-H1975, and NCI-H292 cells in the 24-well Boyden chamber (BD Biosciences, USA) with an 8 m pore size.

Techniques: Migration, Western Blot, Invasion Assay

Journal: Aging (Albany NY)

Article Title: Cathepsin V drives lung cancer progression by shaping the immunosuppressive environment and adhesion molecules cleavage

doi: 10.18632/aging.205278

Figure Lengend Snippet: CTSV inhibits T cell activity in vitro . ( A – K ) Co-cultured the indicated stable A549 and NCI-H1975 cells with PBMCs for 48 h, then the remaining cells were fixed and dyed with 1% crystal violet. Western Blotting analysis of the remaining A549 and NCI-H1975 cells using anti-cleaved casepase3/−7 antibodies ( B , E , I , J ). Caspase 3/7 activity was measured using the Caspase-Glo 3/7 assay. Each bar represents the mean ± SEM ( C , F , K ). ( L , M ) The indicated stable A549 and NCI-H1975 cells were pre-treated with CTSV antibodies and then co-cultured with PBMCs for 48 h. Next, the remaining cells were fixed and dyed with 1% crystal violet. ( N , O ) Western Blotting analysis of the remaining A549 and NCI-H1975 cells from ( L , M ) using anti-cleaved casepase3/−7 antibody. ( P ) Caspase 3/−7 activity was measured using the Caspase-Glo 3/7 assay. ** P -value < 0.01 and *** P -value < 0.001.

Article Snippet: We used 200 μl of serum-free DMEM to seed (1 × 10 5 ) A549, NCI-H1975, and NCI-H292 cells in the 24-well Boyden chamber (BD Biosciences, USA) with an 8 m pore size.

Techniques: Activity Assay, In Vitro, Cell Culture, Western Blot, Caspase-Glo Assay

Journal: Aging (Albany NY)

Article Title: Cathepsin V drives lung cancer progression by shaping the immunosuppressive environment and adhesion molecules cleavage

doi: 10.18632/aging.205278

Figure Lengend Snippet: Blockade CTSV suppresses tumor metastases in lung cancer. ( A – C ) Lung metastasis nodules after tail vein injection of A549 cells ( n = 3 mice per group). ( D ) Kaplan-Meier analysis of the indicated mice. ( E ) Intravenous injection of A549 cells for metastasis analysis. Representative images of antibody-treated mice and bioluminescent imaging (BLI) are shown ( n = 3 mice per group). The representative data are repeated for three independent biological experiments. Student’s t -tests were used for data analysis; **** P -value < 0.0001.

Article Snippet: We used 200 μl of serum-free DMEM to seed (1 × 10 5 ) A549, NCI-H1975, and NCI-H292 cells in the 24-well Boyden chamber (BD Biosciences, USA) with an 8 m pore size.

Techniques: Injection, Imaging