klk4 human  (Sino Biological)

Journal: Cancers

Article Title: The RSL3 Induction of KLK Lung Adenocarcinoma Cell Ferroptosis by Inhibition of USP11 Activity and the NRF2-GSH Axis

doi: 10.3390/cancers14215233

Figure Lengend Snippet: The inhibition of NRF2-GSH axis sensitizes RSL3-induced KLK LUAD cell ferroptosis. ( A ) The KEGG analysis. The data showed that the ferroptosis pathway is the top hit in the KEGG pathway enrichment analysis of KLK LUAD A549 cells after knockdown of NRF2 expression. ( B ) qRT-PCR. The gene expression involved in ferroptosis was validated using qRT-PCR in indicated cells. Slc7a11, solute carrier family 7 member 11; Fth , ferritin heavy chain 1; Ftl , ferritin light chain. ( C ) Western blot. Protein expression was confirmed using Western blot analysis of NRF2, SLC7A11, GCLC, GCLM and GPX4 in indicated cells. GCLC, Glutamate-Cysteine Ligase Catalytic Subunit, glutamate-cysteine ligase, modifier subunit; GCLM, Glutamate-Cysteine Ligase, Modifier Subunit; GPX4, glutathione peroxidase 4. ( D ) GSH and GSSG assay. The level of changes in GSH+GSSG, GSH and GSSG in indicated cells was assayed. ( E , F ) Cell viability assays. A549 cells were treated with dimethyl sulfoxide (DMSO) or RSL3 (4 μM) for 12 h and H2122 cells were treated with DMSO or RSL3 (8 μM) for 12 h and then subjected to CCK-8 assay. Indicated cells were pretreated with DFO (100 μM) or NAC (5 mM) for 4 h ( F ). ( G ) Lipid peroxidation and flow cytometric FACS assays. A549 cells were treated with dimethyl sulfoxide (DMSO) or RSL3 (4 μM) for 12 h and H2122 cells were treated with DMSO or RSL3 (8 μM) for 12 h and then subjected to the FACS assay. * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001 versus the control group.

Article Snippet: Human KLK LUAD cell lines A549 and H2122 were originally obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA).

Techniques: Inhibition, Knockdown, Expressing, Quantitative RT-PCR, Gene Expression, Western Blot, GSSG Assay, CCK-8 Assay, Control

Journal: Cancers

Article Title: The RSL3 Induction of KLK Lung Adenocarcinoma Cell Ferroptosis by Inhibition of USP11 Activity and the NRF2-GSH Axis

doi: 10.3390/cancers14215233

Figure Lengend Snippet: RSL3 suppression of NRF2 protein expression in KLK LUAD cells during ferroptosis. Indicated cells were treated with dimethyl sulfoxide (DMSO), RSL3 (4 or 8 μM), or Erastin (20 or 30 μM) individually for 24 h and then subjected to Western blotting analysis of NRF2, SLC7A11, GCLC, GCLM, and GPX4 proteins. Expression change of each protein was quantified by using the Image-Quant LAS 4000.

Article Snippet: Human KLK LUAD cell lines A549 and H2122 were originally obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA).

Techniques: Expressing, Western Blot

Journal: Cancers

Article Title: The RSL3 Induction of KLK Lung Adenocarcinoma Cell Ferroptosis by Inhibition of USP11 Activity and the NRF2-GSH Axis

doi: 10.3390/cancers14215233

Figure Lengend Snippet: RSL3 inhibition of the NRF2-GSH axis activity during ferroptosis of KLK LUAD cells in vitro. ( A ) Western blot. Indicated cells were treated with dimethyl sulfoxide (DMSO)or RSL3 (1, 4 or 8 μM) individually for 12 h and then subjected to Western blotting analysis of NRF2 protein. ( B – D ) Indicated cells were treated with dimethyl sulfoxide (DMSO) or RSL3 (8 μM) for 12 h. After treatment, cells were subjected to qRT-PCR analysis of Nrf2 . ( B ), Hmox1 ( heme oxygenase 1 ), Fth1 , Ftl , Slc7a11 , Gclc , Gclm and Gpx4 . ( C ) or GSH and GSSG analysis of changes in cellular GSH+GSSG, GSH and GSSG levels ( D ). ( E ) NRF2 overexpression was assayed by using Western blot. ( F ) GSH and GSSG assays. The relative fold reduction in GSH+GSSG, GSH and GSSG in indicated cells was assayed. The data are expressed as mean ± SD. * p < 0.05, ** p < 0.01, and *** p < 0.001 versus the control group.

Article Snippet: Human KLK LUAD cell lines A549 and H2122 were originally obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA).

Techniques: Inhibition, Activity Assay, In Vitro, Western Blot, Quantitative RT-PCR, Over Expression, Control

Journal: Cancers

Article Title: The RSL3 Induction of KLK Lung Adenocarcinoma Cell Ferroptosis by Inhibition of USP11 Activity and the NRF2-GSH Axis

doi: 10.3390/cancers14215233

Figure Lengend Snippet: RSL3 inhibition of the NRF2-GSH axis during ferroptosis of KLK LUAD cells in vivo. ( A ) Nude mouse tumor cell xenograft assay. Eight-week-old immunodeficient nude mice (n = 6 per group) were subcutaneously injected with cells 5 × 10 6 A549 cells and treated with RSL3 (100 mg/kg intratumorally, twice a day for one week) after the tumor cell xenograft volume reached 100 mm 3 . After that, mice were sacrificed and tumor weight was measured. ( B ) The malondialdehyde (MDA) analysis. The relative MDA level in tumor cell xenografts was analyzed in indicated group. ( C ) Western blot. Level of NRF2 protein was assayed using Western blot in tumor cell xenografts. ( D ) Immunohistochemistry. Level of NRF2 in tumor cell xenografts was assayed using immunohistochemistry. Magnification factor, 40. ( E ) qRT-PCR. Level of Slc7a11 and Gclc mRNA in tumor cell xenografts was assayed using qRT-PCR. The data were expressed as mean ± SD. * p < 0.05, ** p < 0.01, and *** p < 0.001 versus the control group.

Article Snippet: Human KLK LUAD cell lines A549 and H2122 were originally obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA).

Techniques: Inhibition, In Vivo, Xenograft Assay, Injection, Western Blot, Immunohistochemistry, Quantitative RT-PCR, Control

Journal: Cancers

Article Title: The RSL3 Induction of KLK Lung Adenocarcinoma Cell Ferroptosis by Inhibition of USP11 Activity and the NRF2-GSH Axis

doi: 10.3390/cancers14215233

Figure Lengend Snippet: RSL3 reduction of NRF2 expression by promoting of NRF2 protein ubiquination in KLK LUAD cells. ( A ) Western blot. Indicated cells were treated with dimethyl sulfoxide (DMSO) or RSL3 (8 μM) for 8 h and then with DMSO or MG132 (25 mM) for additional 4 h and then subjected to Western blotting analysis of NRF2 protein. Expression change of each protein was quantified by using the Image−Quant LAS 4000. ( B ) Immunoprecipitation−Western blot. Interaction of endogenous NRF2 with ubiquitin was assayed, i.e., indicated cells were treated with DMSO or RSL3 (8 μM) for 8 h and then with DMSO or MG132 (25 mM) for additional 4 h and subjected to whole−cell lysis, immunoprecipitation (IP) with an anti-NRF2 antibody, and then Western blotting (WB) with an anti−Ubiquitin antibody.

Article Snippet: Human KLK LUAD cell lines A549 and H2122 were originally obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA).

Techniques: Expressing, Western Blot, Immunoprecipitation, Ubiquitin Proteomics, Lysis

Journal: Cancers

Article Title: The RSL3 Induction of KLK Lung Adenocarcinoma Cell Ferroptosis by Inhibition of USP11 Activity and the NRF2-GSH Axis

doi: 10.3390/cancers14215233

Figure Lengend Snippet: RSL3 directly targeting of NRF2 de-ubiquitinating enzyme USP11 in promotion of NRF2 ubiquination in KLK LUAD cells. ( A ) Molecular docking analysis using Schrodinger software. The data showed the interaction of USP11 with RSL3 and the residue of Tyr833, Phe831 and Gln402 was predicted as the essential amino acid residues for RSL3 binding. ( B ) Eukaryotic purification of wild type and mutant USP11-1 (Y833E, Q402E) proteins. ( C ) Affinity assay. We constructed the coding sequences of the wild type and mutant USP11 (Y833E, Q402E) using PCR and subcloned into the expression vector P35. The resulted vectors were transformed into human embryonic kidney 293 (HEK293) cells for expression of USP11 and USP11-1 proteins, which were then purified by the affinity chromatography. The Octet platform was used to detect and analyze molecule interactions of wild type and mutant USP11-1 with RSL3, based on bio-layer interferometry (BLI). ( D ) Interaction between endogenous NRF2 and ubiquitin under a basal condition and RSL3 treatment in negative control (NC) and USP11 overexpressed KLK LAUD cells after treated with dimethyl sulfoxide (DMSO) or RSL3 (8 μM) for 12 h.

Article Snippet: Human KLK LUAD cell lines A549 and H2122 were originally obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA).

Techniques: Software, Residue, Binding Assay, Purification, Mutagenesis, Construct, Expressing, Plasmid Preparation, Transformation Assay, Affinity Chromatography, Ubiquitin Proteomics, Negative Control

Journal: Cancers

Article Title: The RSL3 Induction of KLK Lung Adenocarcinoma Cell Ferroptosis by Inhibition of USP11 Activity and the NRF2-GSH Axis

doi: 10.3390/cancers14215233

Figure Lengend Snippet: The working model. In KLK LUAD cells, RSL3 is able to inhibit multiple gene activities, i.e., in addition to inhibit GPX4 activity, RSL3 can also inhibit USP11 activity, thus induce NRF2 protein ubiquitination and degradation to reduce the GSH production but induce KLK LUAD cell ferroptosis.

Article Snippet: Human KLK LUAD cell lines A549 and H2122 were originally obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA).

Techniques: Activity Assay, Ubiquitin Proteomics

Journal: The Journal of Clinical Investigation

Article Title: Methylation in pericytes after acute injury promotes chronic kidney disease

doi: 10.1172/JCI135773

Figure Lengend Snippet: (A) The scheme showing the model of 3D capillary formation by mixing pericytes (green) and HUVECs (red) in collagen gels. (B) A representative bright-field image demonstrating the cross sections of capillary tubes 24 hours after mixing pericytes and HUVECs in collagen gels. Scale bars: 250 μm. Original magnification, ×4. (C) A representative immunofluorescent image showing capillary formation (red) 24 hours after mixing the pericytes and CellTracker Red–stained HUVECs in collagen gels. Scale bars: 250 μm. Original magnification, ×4. (D) Dot chart showing the vascular density in the collagen gels in the presence of coculture with qPericytes (qPC), aPericytes (aPC, myofibroblasts), aPericytes after Aza treatment for 3 days (aPC + Aza), or in the absence of pericyte coculture (HUVEC alone) for 24 hours. n = 10. (E) Images showing the collapse of collagen gels induced by kallikrein (KLK) in gels with HUVECs in the presence or absence of pericyte coculture. Scale bars: 250 μm. Original magnification, ×4. (F) Line chart showing the percentage of collapsed area in gels with HUVECs in the presence or absence of pericyte coculture at the indicated time points after KLK administration. n = 10. (G) Dot charts showing the expression of Timp3, Angpt1, and Angpt2 assessed by quantitative PCR. The expression levels were normalized to Gapdh. Horizontal bars represent the mean, error bars represent the SEM. n = 4. *P < 0.05, **P < 0.01, ***P < 0.001 by 1-way ANOVA with post hoc Tukey’s correction.

Article Snippet: Twenty-four hours later, human plasma kallikrein (KLK) (Enzyme Research Laboratories) was added to the cultures.

Techniques: Staining, Expressing, Real-time Polymerase Chain Reaction

Journal: Frontiers in Medicine

Article Title: Measurement of Bradykinin Formation and Degradation in Blood Plasma: Relevance for Acquired Angioedema Associated With Angiotensin Converting Enzyme Inhibition and for Hereditary Angioedema Due to Factor XII or Plasminogen Gene Variants

doi: 10.3389/fmed.2020.00358

Figure Lengend Snippet: Biotechnological inhibitors used in the present study.

Article Snippet: The samples were incubated for up to 120 min under rotary agitation (300 rpm) without any added stimulant or with recombinant human tissue kallikrein (KLK-1, 10 nM; gift from DiaMedica, Inc.), with the particulate contact system activator Pacific Hemostasis Kontact-APTTTM (2% v/v; used without the calcium supplement; composed mainly of magnesium aluminum silica and rabbit brain phospholipid; ThermoFisher Scientific) or with recombinant tissue plasminogen activator (tPA, 169 nM; Cathflo, Roche).

Techniques: Clinical Proteomics