Journal: bioRxiv

Article Title: Inhibition of PIKfyve kinase induces senescent cell death by suppressing lysosomal exocytosis and leads to improved outcomes in a mouse model of idiopathic pulmonary fibrosis

doi: 10.1101/2025.03.19.644224

Figure Lengend Snippet: A. List of the most differentially up- and down-regulated (in blue) surface proteins that were common between S-dox and S-IR cells compared to NS cells. B, B-i. Validation of differential protein expression of CATC, LYAG, TPP1, CA2D1 and CAVN2 in the whole cell lysate of NS and S-dox IMR-90 cells. p16 was used for validation of senescence induction, β-Actin was used as a loading control. C. Validation of the differential CATC, LYAG and TPP1 protein expression in the whole lysate of NS and S-dox ECs. D. Western blot comparing total CATC, LYAG, TPP1, CA2D1 and CAVN2 protein expression in NS cells, and IMR-90 cells kept in low serum medium for one (NS1) or four (NS2) days to induce quiescence. E - E-i. Western blot analysis of the CO8A1, IBP7, TPP1, CATC, LYAG, CA2D1 and CAVN2 protein isolated from the membrane and cytosolic fractions of NS and S-dox cells. Na⁺/K⁺ ATPase was used to confirm successful fraction separation. F. Representative immunofluorescence images of NS, S-dox and RS IMR-90 cells labelled with cell surface markers identified in the Surfaceome-TriCEPS dataset (TPP1, CATC and CA2D1) in red and Hoechst nuclear staining in blue. Scale bar = 75 µm. F-i . Quantification of median fluorescence intensity per cell. Multiple images were collected per sample and all clearly visible cells per sample were analyzed. Unpaired t-test, **p < 0.01, ****p < 0.0001; n≥3.

Article Snippet: The following primary antibodies were used: anti-γ-H2AX (pSer139) (Novus Biologicals; NB100-74435), anti-HMGB1 (abcam; ab18256), anti-cathepsin C (CATC) (Santa Cruz; sc-74590), anti-TPP1 (Millipore; MABW1806), anti-CA2D1 (CACNA2D) (Thermo Fisher; MA3-921), anti-LAMP1 (Santa Cruz, clone H4A3).

Techniques: Expressing, Control, Western Blot, Isolation, Membrane, Immunofluorescence, Staining, Fluorescence

Journal: Biochemical pharmacology

Article Title: Pharmacological inhibition of cathepsin S and of NSPs-AAP-1 (a novel, alternative protease driving the activation of neutrophil serine proteases).

doi: 10.1016/j.bcp.2024.116114

Figure Lengend Snippet: Fig. 5. Consequences of pharmacological CatS inhibition on the maturation of proCatC and proNSPs in HL-60 cells. HL-60 cells were cultured for 7 days in the presence of 10 µM of IcatSRO-11 or IcatS#54 or after adding DMSO containing medium alone. A) Western blot analysis of cell lysates using antiCatC antibody directed against the 23-kDa heavy chain of CatC demonstrating the accumulation of ~ 35-kDa fragment composed of truncated propeptide, the heavy chain and the light chain in presence of IcatSRO-11 or IcatS#54. Western blot analysis of cell lysates using antiCatS antibody as protein control showing not impact of inhibitors on proCatS processing. The percentage of proteolytic activities of CatC, PR3 and CatG was determined using fluorogenic substrates GF-AMC, Abz-VAD(nor)VADYQ- EDDnp, and Abz-TPFSGQ-EDDnp, respectively. Similar results were obtained in three independent experiments. B) Western blot analysis of cell lysates incubated with or without human α1PI using antiPR3 or antiCatG antibody. The de novo formation of irreversible α1PI-NSP complexes of about 75-kDa revealing activation of PR3 and CatG in presence of IcatS#54.

Article Snippet: Antibodies: murine anti-human CatC antibody directed against the heavy chain of CatC [15] (sc-74590), rabbit anti-human MPO antibody directed against the heavy chain (C-16-R, sc-16128-R) and rabbit anti-mouse CatG (sc-33207) were purchased from Santa Cruz Biotechnology (Heidelberg, Germany).

Techniques: Inhibition, Cell Culture, Western Blot, Control, Incubation, Activation Assay

Journal: Biochemical pharmacology

Article Title: Pharmacological inhibition of cathepsin S and of NSPs-AAP-1 (a novel, alternative protease driving the activation of neutrophil serine proteases).

doi: 10.1016/j.bcp.2024.116114

Figure Lengend Snippet: Fig. 6. Processing of proCatC, proCatS and proNSPs in HL-60 cells cultured in the presence of compound 1 supplemented with IcatCXPZ-01. HL-60 cells were cultured for 14 days in the presence of compound 1 or after adding DMSO containing buffer alone. IcatCXPZ-01 was added at day 7. Processing of proCatC, proCatS and proNSPs in cell lysates were analyzed by western blotting. A) Western blot analysis of cell lysates using anti-CatC antibody directed against the 23-kDa heavy chain of CatC demonstrating the accumulation of ~ 35-kDa fragment in presence of compound 1. Proteolytic activities of NSPs are inhibited in presence of compound 1 supplemented with IcatCXPZ-01. The percentage of proteolytic activities of CatC, PR3 and CatG was determined as in Fig. 5. Results obtained in three independent experiments were shown. B) Western blot analysis of cell lysates using anti-CatS antibody showing alteration of proCatS processing with accumulation of inter mediate precursor in presence of compound 1 supplemented with IcatCXPZ-01. Similar results were obtained in three independent experiments. Incubation of cells only with compound 1 (data not shown) or IcatCXPZ-01 for 14 days did not alter the processing of proCatS (see Fig. 7C). C) Western blot analysis of cell lysates incubated with or without α1PI using anti-PR3 antibody. (Left) PR3 stability in cell lysates incubated without α1PI. (Right) PR3 in same cell lysates incubated with or without α1PI. The decrease of ~ 75-kDa irreversible α1PI-PR3 complexes formation and the detection of free PR3 in presence of compound 1 supplemented with IcatCXPZ-01 revealing unprocessing of PR3 zymogens. Similar results were obtained in three independent experiments. D) Diagram summary of proNSPs activation pathways in HL-60 cells. ProNSPs are activated by CatC (in blue) and NSPs-AAP-1 (in dark yellow) dependent pathways in HL-60 cells. CatC dependent activation pathway is initiated by proteolytic processing and maturation of proCatS. Combination of compound 1 with IcatCXPZ-01 partially inhibits the maturation of proCatS. The nitrile dipeptidyl CatC inhibitor IcatCXPZ-01 inhibits both CatC and NSPs-AAP-1 dependent maturation of proNSPs in HL-60 cells.

Article Snippet: Antibodies: murine anti-human CatC antibody directed against the heavy chain of CatC [15] (sc-74590), rabbit anti-human MPO antibody directed against the heavy chain (C-16-R, sc-16128-R) and rabbit anti-mouse CatG (sc-33207) were purchased from Santa Cruz Biotechnology (Heidelberg, Germany).

Techniques: Cell Culture, Western Blot, Incubation, Activation Assay

Journal: Biochemical pharmacology

Article Title: Pharmacological inhibition of cathepsin S and of NSPs-AAP-1 (a novel, alternative protease driving the activation of neutrophil serine proteases).

doi: 10.1016/j.bcp.2024.116114

Figure Lengend Snippet: Fig. 10. Activation of CatC and NSPs in Ctss−/−C57BL/6 mice. A) Western blot analysis of WT and Ctss−/−bone marrow (BM) cell lysates using anti-CatS antibodies. B) Western blot analysis of bone marrow cells and blood neutrophils lysates using anti-CatC antibodies. Presence of mature immunoreactive CatC as revealed by the ~ 20-kDa heavy chain in Ctss−/−mice indicates that CatS is not required in proCatC maturation. C) Western blot analysis of bone marrow cells and blood neutrophils lysates incubated with or without human α1PI using anti-NE antibodies. The de novo formation of irreversible α1PI-scNE complexes of about 80-kDa reveals that NE is proteolytically active despite the absence of CatS. NE produced by murine neutrophils may be present in a cleaved (two-chains (tc)) and uncleaved (single chain (sc)) [64]. Similar results were obtained in three independent experiments.

Article Snippet: Antibodies: murine anti-human CatC antibody directed against the heavy chain of CatC [15] (sc-74590), rabbit anti-human MPO antibody directed against the heavy chain (C-16-R, sc-16128-R) and rabbit anti-mouse CatG (sc-33207) were purchased from Santa Cruz Biotechnology (Heidelberg, Germany).

Techniques: Activation Assay, Western Blot, Incubation, Produced

Journal: Biochemical pharmacology

Article Title: Pharmacological inhibition of cathepsin S and of NSPs-AAP-1 (a novel, alternative protease driving the activation of neutrophil serine proteases).

doi: 10.1016/j.bcp.2024.116114

Figure Lengend Snippet: Fig. 11. Activation of NSPs in Ctsc−/− C57BL/6 mice. A) Western blot analysis of bone marrow cell lysates using antiCatC, antiNE and antiCatG antibodies. Significantly reduced level of immunoreactive NE and CatG in Ctsc−/−mice bone marrow lysates compared with WT shows the elimination of NSP zymogens in absence of CatC (n = 4 animals). sc: single chain/tc: two chains (upper and lower bands). Myeloperoxidase (MPO) served as control for protein content. B) Residual activity of CatC, NE, PR3 and CatG in Ctsc−/−mice bone marrow lysates. Proteolytic activities were measured using fluorogenic substrates. Monitored selective NE, PR3 and CatG activities indicates the activation of NSPs in absence of CatC. The histograms show the mean ± SD of CatC (n = 5 animals) and NSP activities (n = 5 animals), each done in duplicates. Statistical analysis of the data was performed using a nonparametric test (Mann Whitney test), **p < 0.01. C) Consequences of CatC depletion in Ctsc−/−mice on the fate of proNSPs. Activation and degradation pathways of proNSPs identified in PLS patients are conserved in CatC defi cient mice.

Article Snippet: Antibodies: murine anti-human CatC antibody directed against the heavy chain of CatC [15] (sc-74590), rabbit anti-human MPO antibody directed against the heavy chain (C-16-R, sc-16128-R) and rabbit anti-mouse CatG (sc-33207) were purchased from Santa Cruz Biotechnology (Heidelberg, Germany).

Techniques: Activation Assay, Western Blot, Control, Activity Assay, MANN-WHITNEY