Journal: Biosensors

Article Title: Duplex Vertical-Flow Rapid Tests for Point-of-Care Detection of Anti-dsDNA and Anti-Nuclear Autoantibodies

doi: 10.3390/bios14020098

Figure Lengend Snippet: The general principle of the VFA setup. dsDNA and Hep-2 cell nuclear antigens are spotted on the left as test zones, and biotinylated BSA at low and high concentrations are spotted on the right of the nitrocellulose membrane (NCM) as part of control zones. After wetting the NCM, the sample containing anti-dsDNA and/or ANAs is loaded for them to bind to their respective capture antigen substrate on the NCM, thus forming an antigen–antibody immuno-complex. Biotinylated anti-human IgG is then added for it to bind with the anti-dsDNA or ANAs. Finally, streptavidin-conjugated GNPs are added for them to bind with anti-IgG in test zones and with biotinylated BSA in control zones, displaying a green colorimetric signal on the NCM. Between each step, the membrane is washed to remove unbound materials and to prevent nonspecific binding.

Article Snippet: Bovine serum albumin (BSA), Tween-20, sodium chloride (NaCl), Tris-HCl, phosphate-buffered saline (PBS, pH 7.4), polyethylene glycol with an average mol wt of 6000 (PEG 6000), and polyvinylpyrrolidone with an average mol wt of 40,000 (PVP40) were purchased from Sigma (St. Louis, MO, USA), diluted in MilliQ water and then filtered using a 0.2 μm filter (Pall Science, New York, NY, USA). dsDNA (Sigma), Hep-2 cell lysate (source of nuclear antigens for ANA testing; Novus Biologicals), Bovine AGE-BSA Biotinylated Protein (R&D system, Minneapolis, MN, USA), human anti-dsDNA standard plasma (708 IU/mL, PSG Lot ID: 20400), biotinylated anti-IgG (Abcam, Eugene, OR, USA), and 150 nm streptavidin-conjugated GNP (Nanocomposix, San Diego, CA, USA) were purchased for VFA development.

Techniques: Membrane, Control, Binding Assay

Journal: Experimental & Molecular Medicine

Article Title: PRKCSH contributes to TNFSF resistance by extending IGF1R half-life and activation in lung cancer

doi: 10.1038/s12276-023-01147-1

Figure Lengend Snippet: a Quantitative analysis of PRKCSH mRNA expression levels between normal ( n = 396) and cancerous ( n = 1135) lung tissues, including lung adenocarcinoma (LUAD) ( n = 585) and lung squamous cell carcinoma (LUSC) ( n = 550). The gene expression profiles were obtained from The Cancer Genome Atlas and Genotype-Tissue Expression databases. Data are presented as the mean ± the two-tailed Student determined SD. Statistical significance’s t test. b Representative images of immunohistochemical staining of PRKCSH in LUAD ( n = 74 LUAD tissues; n = 74 adjacent tissues) (scale bar = 50 μm). Quantitative analysis of PRKCSH expression levels in paired clinical samples. c Representative images of immunohistochemical staining of PRKCSH in LUSC ( n = 75 of LUSC tissues; n = 75 of adjacent tissues) (scale bar = 50 μm). Quantitative analysis of PRKCSH expression levels in paired clinical samples. d Kaplan‒Meier plot of the first progression rate or overall survival rate of patients with lung cancer stratified by PRKCSH mRNA expression level. Patients were divided into two groups: high PRKCSH mRNA expression vs. low PRKCSH mRNA expression. The significance of differences was determined by the two-sided log-rank test. e Two-dimensional hierarchical clustering shows top-ranked pathways in transcriptome analysis showing negatively coexpressed genes relative to PRKCSH mRNA expression (blue) and positively coexpressed genes (red) in LUAD and LUSC tissues. Gene-set enrichment analysis of negatively coexpressed genes with PRKCSH mRNA in LUSC tissues. NES, normalized enrichment score; FDR q , false discovery rate q value. f NK cell-A549 cell killing assay in tumor 3D spheroid culture (scale bar = 200 μm). The tumor organoids of A549-shCon and A549-shPRK cells were treated with NK-92 cells at the indicated E:T ratio for 5 days. The NK cell-mediated cytotoxicity against tumor organoids was analyzed by measuring spheroid volume and GFP signal intensity. Data are shown as the means ± SDs of three independent assays. The statistical significance of differences between two groups was determined with the two-tailed Student’s t test. ** p < 0.01. g NK cell-A549 cell killing assay in tumor cell 2D culture. A549-shCon and A549-shPRK cells were seeded onto culture dishes following treatment of NK-92 cells at the indicated E:T ratio for 4 h. The NK cell-mediated cytotoxicity against tumor cells was measured using a lactate dehydrogenase release assay. h The inhibition assay of NK cell cytotoxicity with neutralizing antibodies against TNF-α or TRAIL in tumor 3D spheroid culture (scale bar = 200 μm). The tumor organoids of A549-shCon and A549-shPRK cells were treated with a complex of the neutralizing antibody and NK-92 cells for 5 days. The NK cell cytotoxicity against tumor organoids was analyzed by measuring spheroid volume and GFP signal intensity. i The inhib i tion assay of NK cell cytotoxicity with neutralizing antibodies against TNF-α or TRAIL in tumor cell 2D culture. A549-shCon and A549-shPRK cells were treated with the complex of the neutralizing antibody and NK-92 cells for 4 h. The NK cell-mediated cytotoxicity against tumor cells was measured by a lactate dehydrogenase release assay. j Immunoblot analysis of TNF-R1 and TRAIL-R2 expression in A549-shCon and A549-shPRK cells. Representative immunoblots and quantitative analysis of gene expression levels in A549-shCon and A549-shPRK cells are shown. ACTB was used as a loading control.

Article Snippet: The following antibodies were used for IP: PRKCSH (Proteintech, 12148-1-AP), Cas8 (Cell Signaling Technology, 9746), and IGF1R (Cell Signaling Technology, 9750).

Techniques: Expressing, Gene Expression, Two Tailed Test, Immunohistochemical staining, Staining, Release Assay, Inhibition, Western Blot, Control

Journal: Experimental & Molecular Medicine

Article Title: PRKCSH contributes to TNFSF resistance by extending IGF1R half-life and activation in lung cancer

doi: 10.1038/s12276-023-01147-1

Figure Lengend Snippet: a Analysis of IGF1R and EGFR pathway activation in PRKCSH-deficient cells. A549-shCon and A549-shPRK cells or other lung cancer cells, including H292R and H1299 (p53 null) cells transfected with siPRK#2 or siCon, were treated with or without 50 ng/ml TRAIL for the indicated times. The phosphorylation of IGF1R, EGFR, AKT, and ERK1/2 in cell lysates was analyzed by immunoblotting. Representative immunoblots and quantitative analysis of phosphorylated protein levels in each cell line are shown. The quantitative data are shown as the means ± SDs of three independent assays. The statistical significance of differences between two groups was determined with the two-tailed Student’s t test. b The effect of IGF1R overexpression on TNFSF-mediated tumor cell killing in lung cancer cells. A549-shPRK cells were infected with lentivirus containing IGF1R cDNA plasmid, followed by treatment with TRAIL at the indicated concentrations for 24 h. TNFSF-mediated cytotoxicity was analyzed by MTT assay, and activation of caspases was analyzed by immunoblotting. H292R and H1299 (p53 null) cells were transfected with siRNA to IGF1R, followed by treatment with TRAIL at the indicated concentrations for 24 h. TNFSF-mediated cytotoxicity was analyzed by MTT assay, and activation of caspases was analyzed by immunoblotting. c The effect of IGF1R overexpression or knockdown on TNFSF-mediated phosphorylation of AKT and ERK1/2 in lung cancer cells. A549-shPRK cells were infected with lentivirus containing IGF1R cDNA plasmid, or H292R and H1299 (p53 null) cells were transfected with siRNA to IGF1R. Cells were treated with TRAIL for the indicated times, followed by determination of AKT and ERK1/2 phosphorylation by immunoblotting. Representative immunoblots and quantitative analysis of phosphorylated protein levels in each cell line are shown. d The effect of IGF1R overexpression or knockdown on TNFSF-mediated Mcl-1 expression in lung cancer cells. A549-shPRK cells were infected with lentivirus containing IGF1R cDNA plasmid, or H292R and H1299 (p53 null) cells were transfected with siRNA to IGF1R. Cells were treated with TRAIL for the indicated times, followed by determining the expression of Mcl-1L and Mcl-1S by immunoblotting. Representative immunoblots and quantitative analysis of Mcl-1L and Mcl-1S protein levels in each cell line are shown. ** p < 0.01, * p < 0.05.

Article Snippet: The following antibodies were used for IP: PRKCSH (Proteintech, 12148-1-AP), Cas8 (Cell Signaling Technology, 9746), and IGF1R (Cell Signaling Technology, 9750).

Techniques: Activation Assay, Transfection, Phospho-proteomics, Western Blot, Two Tailed Test, Over Expression, Infection, Plasmid Preparation, MTT Assay, Knockdown, Expressing

Journal: Experimental & Molecular Medicine

Article Title: PRKCSH contributes to TNFSF resistance by extending IGF1R half-life and activation in lung cancer

doi: 10.1038/s12276-023-01147-1

Figure Lengend Snippet: a Analysis of IGF1R and EGFR protein levels in PRKCSH-deficient cells. A549-shCon and A549-shPRK cells or other lung cancer cells, including H292R and H1299 (p53 null) transfected with siPRK#2 or siCon, were analyzed by immunoblotting using anti-IGF1R and EGFR antibodies. Representative immunoblots and quantitative analysis of IGF1R and EGFR protein levels in each cell line are shown. The quantitative data are shown as the means ± SDs of three independent assays. The statistical significance of differences between two groups was determined with the two-tailed Student’s t test. b Analysis of IGF1R mRNA expression levels in PRKCSH-deficient cells. The expression levels of IGF1R mRNA in A549-shCon and A549-shPRK cells or other lung cancer cells, including H292R and H1299 (p53 null) cells transfected with siPRK#2 or siCon, were analyzed by qPCR. Data are quantitative analysis of IGF1R mRNA expression in each cell line. c Analysis of IGF1R protein expression levels on the cell surface of PRKCSH-deficient cells. The expression of IGF1R protein on the cell surface of A549-shCon and A549-shPRK cells or other lung cancer cells, including H292R and H1299 (p53 null) cells transfected with siPRK#2 or siCon, was analyzed by flow cytometry. Representative flow cytometry data and quantitative analysis of IGF1R expression levels on the cell surface of each cell line are shown. d The effect of PRKCSH depletion on the IGF1R protein half-life in lung cancer cells. A549-shCon and A549-shPRK cells were treated with cycloheximide for the indicated times. The half-life of IGF1R protein in cell lysates was analyzed using immunoblotting. Representative immunoblots and quantitative analysis of IGF1R protein levels are shown. e The effect of proteasome or lysosome inhibitors on the regulation of IGF1R protein half-life in lung cancer cells. A549-shCon and A549-shPRK cells were treated with MG132 (proteasome inhibitor) or chloroquine (lysosome inhibitor) for 24 h. SQSTM1 was used as a lysosome inhibition control, and ACTB was used as a loading control. f Analysis of protein‒protein interactions among endogenous IGF1R, EGFR, and PRKCSH in lung cancer cells. The protein extracts from A549 cells were pulled down with anti-PRKCSH or anti-IGF1R antibodies, and the interacting proteins were identified by immunoblotting. g Scheme of the protein domains expressed in wild-type and mutant PRKCSH plasmids. WT, carboxyl-terminal Flag-tagged full-length PRKCSH; ΔG2B, deletion mutant of the G2B domain in WT; ΔS/G2B, deletion mutant of the signal peptide and G2B domain in WT; ΔMRH, deletion mutant of MRH in ΔG2B; MRH, deletion of the EP domain in ΔG2B. h Mapping domain of PRKCSH related to the interaction with IGF1R. A549 cells were transfected with WT PRKCSH and each PRKCSH deletion mutant plasmid. The protein extracts were pulled down with an anti-IGF1R antibody, and the interacting proteins were determined by immunoblotting with an anti-Flag antibody. i Mapping domain of PRKCSH associated with regulation of IGF1R protein half-life. A549-shPRK cells were transfected with WT and each deletion mutant of PRKCSH plasmids. IGF1R protein expression levels were determined by immunoblotting. j Analysis of the interaction between IGF1R and HERPUD1 in lung cancer cells. A549-shCon and A549-shPRK cells were treated with MG132 for 24 h. The protein extracts from each cell were pulled down with an anti-IGF1R antibody, and the interaction between IGF1R and HERPUDP1 protein was determined using immunoblotting. k The role of WT and each deletion mutant of PRKCSH in the inhibition of the interaction between IGF1R and HERPUD1. A549-shPRK cells were transfected with each plasmid, followed by treatment with MG132 for 24 h. The protein extracts from each cell were pulled down with an anti-IGF1R antibody, and the interaction between IGF1R and HERPUDP1 protein was determined using immunoblotting. l The effect of PRKCSH overexpression on IGF1R protein half-life. H460 lung cancer cells were transfected with WT or ΔG2B mutant plasmid. IGF1R protein expression levels were determined using immunoblotting. m-n The effect of PRKCSH overexpression on TNFSF-mediated tumor cell killing in H460 lung cancer cells. H460 cells were transfected with PRKCSH WT or ΔG2B mutant plasmid, followed by treatment with TRAIL for 24 h. TNFSF-mediated cytotoxicity was analyzed using MTT assay ( m ), and activation of caspases was analyzed using immunoblotting ( n ). ** p < 0.01, * p < 0.05. NS, nonsignificant.

Article Snippet: The following antibodies were used for IP: PRKCSH (Proteintech, 12148-1-AP), Cas8 (Cell Signaling Technology, 9746), and IGF1R (Cell Signaling Technology, 9750).

Techniques: Transfection, Western Blot, Two Tailed Test, Expressing, Flow Cytometry, Inhibition, Control, Mutagenesis, Plasmid Preparation, Over Expression, MTT Assay, Activation Assay

Journal: Experimental & Molecular Medicine

Article Title: PRKCSH contributes to TNFSF resistance by extending IGF1R half-life and activation in lung cancer

doi: 10.1038/s12276-023-01147-1

Figure Lengend Snippet: a Quantitative analysis of IGF1R mRNA expression levels between normal ( n = 396) and cancerous ( n = 1135) lung tissues, including lung adenocarcinoma (LUAD) ( n = 585) and lung squamous cell carcinoma (LUSC) ( n = 550). The gene expression profiles were obtained from The Cancer Genome Atlas and Genotype-Tissue Expression databases. Data are presented as the mean ± SD. Statistical significance was determined by the two-tailed Student’s t test. b Representative immunohistochemical staining of IGF1R protein in LUAD ( n = 74 of LUAD tissues; n = 74 of adjacent tissues) (scale bar = 50 μm). Quantitative analysis of IGF1R expression levels in paired clinical samples. c Representative immunohistochemical staining of IGF1R protein in LUSC ( n = 75 of LUSC tissues; n = 75 of adjacent tissues) (scale bar = 50 μm). Quantitative analysis of IGF1R expression levels in paired clinical samples. d Analysis of the correlation between PRKCSH and IGF1R mRNA levels in lung cancer tissues using the gene expression profiles obtained from The Cancer Genome Atlas database. Correlation analysis was performed using Pearson’s rank correlation coefficient. e Analysis of the correlation between PRKCSH and IGF1R protein levels in lung cancer tissues using the immunohistochemical data determined in LUAD and LUSC tissues. f Kaplan‒Meier plot of the first progression rate, overall survival rate, or post progression survival rate of patients with lung cancer stratified by IGF1R mRNA expression level. Patients were divided into two groups: high IGF1R mRNA expression vs. low IGF1R mRNA expression. Statistical differences were determined by the two-sided log-rank test. g Two-dimensional hierarchical clustering shows top-ranked pathways in transcriptome analysis showing negatively coexpressed genes relative to IGF1R mRNA expression (blue) and positively coexpressed genes (red) in LUAD and LUSC tissues. Gene-set enrichment analysis of coexpressed genes with IGF1R mRNA in LUSC tissues. NES, normalized enrichment score; FDR q , false discovery rate q value. h ‒ j The effect of PRKCSH knockdown on the NK cell-mediated antitumor effect using the tumor-bearing IL-2Rg-deficient NOD/SCID (NIG) mouse model (each group, n = 10). A549-shCon and A549-shPRK cells were inoculated subcutaneously into the mice, and human IL2-NK-92 cells were injected intravenously into the mice at 3 weeks after inoculation ( h ). Tumor volume and weight were measured on Day 40 after inoculation. Representative tumor-bearing mice and enucleated tumors ( i ) and quantitative analysis of tumor volume and weight ( j ) are shown. k Representative immunohistochemical staining of cell death (cleaved PARP1) and NK cell infiltration (NKG2D) in tumor tissues of xenograft mice (each group, n = 5) (scale bar = 50 μm). Quantitative analysis of cell death and NK cell infiltration levels.

Article Snippet: The following antibodies were used for IP: PRKCSH (Proteintech, 12148-1-AP), Cas8 (Cell Signaling Technology, 9746), and IGF1R (Cell Signaling Technology, 9750).

Techniques: Expressing, Gene Expression, Two Tailed Test, Immunohistochemical staining, Staining, Knockdown, Injection