Journal: Cancer Research

Article Title: Parkin Deficiency Suppresses Antigen Presentation to Promote Tumor Immune Evasion and Immunotherapy Resistance

doi: 10.1158/0008-5472.CAN-22-2499

Figure Lengend Snippet: Parkin deficiency in human ccRCC is associated with poor clinical outcome, limited antigen presentation capabilities, and impacts immunotherapy efficacy. Parkin ( PRKN ) downregulation in patients with KIRC is significantly associated with overall survival and tumor stage. A, PRKN gene expression is significantly downregulated in patient tumor samples vs. matched controls ( N = 72 matches samples, P value = 3E−07 via paired Student t test; FPKMs mapped reads data from GDC HTSeq-FPKM pipeline was used). Dashed lines represent cutoffs used to bin patients with KIRC into low, mid, high PRKN gene expression levels. B, PRKN gene expression levels are significantly associated with overall survival ( N = 538 tumor samples, P < 0.0001 via log-rank test for trend). C, PRKN expression is significantly associated with overall survival in patients with stage 4 tumors (log-rank P < 0.007). D, mRNA expression levels of PRKN on HEK293, 786-O, CAKI-I, and A498 cells cultured overnight in growth media. E, mRNA and protein expression levels of A498 Parkin KO or EV cells. F, A498 Parkin KO or EV cells were stimulated with IFNγ (10 ng/mL) for 18 hours. Then, immunoblotting analysis was performed on Parkin, HLA-A, TAP1, and PSMB8. Data represent two to three independent experiments. Immunoblot data represent two to three independent experiments. G, IFNγ ELISA analysis was performed on donor-derived NY-ESO-1-specific T cells (5 × 10 4 ) cocultured in a 1:1 ratio overnight with A498 Parkin KO or EV cells. Tumor cells were prestimulated with IFNγ (10 ng/mL) or mock-treated for 18 hours before the assay. Data are represented as mean ± SD. ***, P < 0.001; unpaired, two-tailed t test.

Article Snippet: The primary antibodies for TAP1, PSMB8, β2-microglobulin (D8P1H), HSP90 (C45G5), β-actin (E4D9Z), Vinculin (VCL; E1E9V), Parkin, PTEN, AMPKa, phospho-AMPKa (Thr172; 40H9), Akt, phospho-Akt (Ser473; D9E), phospho-GSK-3β (Ser9, D85E12), and GSK-3β (D5C5Z) were purchased from Cell Signaling Technology; HLA-A was purchased from Thermo Fisher Scientific; and MHC-I (ER-HR52) was obtained from Santa Cruz Biotechnology.

Techniques: Immunopeptidomics, Gene Expression, Expressing, Cell Culture, Western Blot, Enzyme-linked Immunosorbent Assay, Derivative Assay, Two Tailed Test

Journal: Cancer Research

Article Title: Parkin Deficiency Suppresses Antigen Presentation to Promote Tumor Immune Evasion and Immunotherapy Resistance

doi: 10.1158/0008-5472.CAN-22-2499

Figure Lengend Snippet: Parkin regulates the MHC-I-associated tumor APM and fosters tumor progression. A and B, mRNA expression levels of Prkn , H2-k1 , Tap1 , Psmb8 , and B2m in RENCA cells stably transfected with sh Prkn _1 (sh#1), sh Prkn _3 (sh#3), and empty vector control (pLKO.1) lentiviral vectors. Data are represented as mean ± SD. ***, P < 0.001; unpaired, two-tailed t test. C, Protein expression analyses of Parkin, Tap1, Psmb8, and B2m (immunoblotting) and Parkin vs. MHC-I (intracellular staining, flow cytometry) of pLKO.1 controls, sh#1, and sh#3 RENCA cells. Data represent two to three independent experiments. D, IFNγ ELISPOT analysis was performed on T cells isolated from the spleens of pLKO.1 RENCA tumor-bearing mice at day 25 after challenge. T cells were cocultured overnight with stimulator Parkin-positive (pLKO.1) or -negative (sh#1, sh#3) RENCA cells (5:1 ratio). E, Parkin-positive (pLKO.1) or -negative (sh#1, sh#3) RENCA cells (10 6 ) were injected in the back of BALB/c mice and tumor growth was monitored. Data are represented as mean ± SEM. *, P < 0.05; ***, P < 0.001; two-way ANOVA.

Article Snippet: The primary antibodies for TAP1, PSMB8, β2-microglobulin (D8P1H), HSP90 (C45G5), β-actin (E4D9Z), Vinculin (VCL; E1E9V), Parkin, PTEN, AMPKa, phospho-AMPKa (Thr172; 40H9), Akt, phospho-Akt (Ser473; D9E), phospho-GSK-3β (Ser9, D85E12), and GSK-3β (D5C5Z) were purchased from Cell Signaling Technology; HLA-A was purchased from Thermo Fisher Scientific; and MHC-I (ER-HR52) was obtained from Santa Cruz Biotechnology.

Techniques: Expressing, Stable Transfection, Transfection, Plasmid Preparation, Control, Two Tailed Test, Western Blot, Staining, Flow Cytometry, Enzyme-linked Immunospot, Isolation, Injection

Journal: Cancer Research

Article Title: Parkin Deficiency Suppresses Antigen Presentation to Promote Tumor Immune Evasion and Immunotherapy Resistance

doi: 10.1158/0008-5472.CAN-22-2499

Figure Lengend Snippet: Parkin regulates cytosolic tumor antigen processing and presentation and facilitates effector CD8 + T-cell cancer immunity. A–C, Mouse melanoma B16-OVA Prkn knockout (KO, CRISPr/Cas9) or EV control cells were stimulated for 18 hours with IFNγ (10 ng/mL) or mock-stimulated before the analyses. A, mRNA expression levels of Prkn , H2-k1 , Psmb8 , and Tap1 in B16-OVA Parkin KO or EV cells. Data are represented as mean ± SD. ** P < 0.01; *** P < 0.001; unpaired, two-tailed t test. B, Protein expression analyses of Parkin, Tap1, and Psmb8 (immunoblotting) and SIINFEKL-bound to H-2Kb (MHCi-OVA; intracellular staining, flow cytometry) of B16-OVA KO and EV cells. Data represent two to three independent experiments. C, IFNγ ELISPOT analysis was performed on OT.1 T cells (10 5 ) cocultured overnight with B16-OVA KO and EV cells in a 5:1 ratio. D, B16-OVA Parkin KO or EV cells (10 6 ) were injected in the back of C57BL/6 mice and tumor growth was monitored. Five mice per group were treated with anti-CD8 blocking antibody (aCD8) or isotype (ISO) control. E, Percentage of CD45 − /MHC-OVA + cells present in B16-OVA Parkin KO or EV isotype-treated tumors analyzed by flow cytometry. F, SIINFEKL-specific CD8 + T-cell tumor infiltration present in B16-OVA Parkin KO or EV isotype-treated tumors. G, mRNA expression levels of IFNγ in B16-OVA Parkin KO or EV isotype-treated tumors. E–G, Data are represented as mean ± SEM. ns, not significant; *, P < 0.05; **, P < 0.01; unpaired, two-tailed t test. H, B16-OVA Parkin KO or EV cells (10 6 ) were injected in the back of C57BL/6-Tg(TcraTcrb)1100Mjb/J (OT.1) mice (5 per group) and tumor growth was monitored. I, Survival rate analysis (Kaplan–Meier, log-rank test) was performed by day 25 posttumor challenge, including five mice per group. D and H, Data are represented as mean ± SEM. ***, P < 0.001; two-way ANOVA.

Article Snippet: The primary antibodies for TAP1, PSMB8, β2-microglobulin (D8P1H), HSP90 (C45G5), β-actin (E4D9Z), Vinculin (VCL; E1E9V), Parkin, PTEN, AMPKa, phospho-AMPKa (Thr172; 40H9), Akt, phospho-Akt (Ser473; D9E), phospho-GSK-3β (Ser9, D85E12), and GSK-3β (D5C5Z) were purchased from Cell Signaling Technology; HLA-A was purchased from Thermo Fisher Scientific; and MHC-I (ER-HR52) was obtained from Santa Cruz Biotechnology.

Techniques: Knock-Out, CRISPR, Control, Expressing, Two Tailed Test, Western Blot, Staining, Flow Cytometry, Enzyme-linked Immunospot, Injection, Blocking Assay

Journal: Frontiers in Molecular Biosciences

Article Title: Comprehensive Analysis of the Potential Immune-Related Biomarker Transporter Associated With Antigen Processing 1 That Inhibits Metastasis and Invasion of Ovarian Cancer Cells

doi: 10.3389/fmolb.2021.763958

Figure Lengend Snippet: The expression of transporter associated with antigen processing 1 (TAP1) in pan-cancer. (A) TAP1 expression of 31 kinds of normal tissues downloaded from genotype-tissue expression (GTEx) database. (B) TAP1 expression in 21 kinds of cancer cell lines derived from the cancer cell line encyclopedia (CCLE). (C) TAP1 expression in tumor samples and the adjacent normal samples of 20 cancers derived from the TCGA. (D) TAP1 expression in tumor samples and normal samples of 27 cancers derived from the cancer genome atlas (TCGA) and GTEx database. * p < 0.05. ** p < 0.01. *** p < 0.001.

Article Snippet: The membrane was incubated with the following primary antibodies TAP1 (1:3,000), MEF2A (1:2000, #12382-1-AP, Proteintech, United States), LEF1 (1:1000, #14972-1-AP, Proteintech), E-cadherin (1:10000, #20874-1-AP, Proteintech), vimentin (1:2000, #10366-1-AP, Proteintech), and beta actin (1:1000, # 20536-1-AP, Proteintech).

Techniques: Expressing, Derivative Assay

Journal: Frontiers in Molecular Biosciences

Article Title: Comprehensive Analysis of the Potential Immune-Related Biomarker Transporter Associated With Antigen Processing 1 That Inhibits Metastasis and Invasion of Ovarian Cancer Cells

doi: 10.3389/fmolb.2021.763958

Figure Lengend Snippet: The prediction ability of transporter associated with antigen processing 1 (TAP1) in pan-cancer prognosis. (A–C) The top three cancers correlated with TAP1 in disease-free survival (DFS). (D–F) The top three cancers correlated with TAP1 in disease-specific survival (DSS). (G–I) The top three cancers correlated with TAP1 in progression-free survival (PFS). (J–L) The top three cancers correlated with TAP1 in Overall survival (OS).

Article Snippet: The membrane was incubated with the following primary antibodies TAP1 (1:3,000), MEF2A (1:2000, #12382-1-AP, Proteintech, United States), LEF1 (1:1000, #14972-1-AP, Proteintech), E-cadherin (1:10000, #20874-1-AP, Proteintech), vimentin (1:2000, #10366-1-AP, Proteintech), and beta actin (1:1000, # 20536-1-AP, Proteintech).

Techniques:

Journal: Frontiers in Molecular Biosciences

Article Title: Comprehensive Analysis of the Potential Immune-Related Biomarker Transporter Associated With Antigen Processing 1 That Inhibits Metastasis and Invasion of Ovarian Cancer Cells

doi: 10.3389/fmolb.2021.763958

Figure Lengend Snippet: The correlation of transporter associated with antigen processing 1 (TAP1) with immune infiltration in multiple cancers. The top three cancers with the strongest correlation with 6 immune infiltration cells which included B cells, CD4+T cells, CD8+T cells, neutrophils, macrophages, and dendritic cells. The cancers were colon adenocarcinoma (COAD), kidney renal clear cell carcinoma (KIRC), and breast invasive carcinoma (BRCA), respectively.

Article Snippet: The membrane was incubated with the following primary antibodies TAP1 (1:3,000), MEF2A (1:2000, #12382-1-AP, Proteintech, United States), LEF1 (1:1000, #14972-1-AP, Proteintech), E-cadherin (1:10000, #20874-1-AP, Proteintech), vimentin (1:2000, #10366-1-AP, Proteintech), and beta actin (1:1000, # 20536-1-AP, Proteintech).

Techniques:

Journal: Frontiers in Molecular Biosciences

Article Title: Comprehensive Analysis of the Potential Immune-Related Biomarker Transporter Associated With Antigen Processing 1 That Inhibits Metastasis and Invasion of Ovarian Cancer Cells

doi: 10.3389/fmolb.2021.763958

Figure Lengend Snippet: The relationship between TAP1 and ESTIMATE score in multiple cancers. The ESTIMATE score was used to determine the correlation of TAP1 with immune cells and stromal cells in 33 tumors. TAP1 was positively correlated with ESTIMATE scores in cervical squamous cell carcinoma (CESC), ovarian cancer (OC), uveal melanoma (UVM), testicular germ cell tumors (TGCT), and thyroid carcinoma (THCA) ( p < 0.05).

Article Snippet: The membrane was incubated with the following primary antibodies TAP1 (1:3,000), MEF2A (1:2000, #12382-1-AP, Proteintech, United States), LEF1 (1:1000, #14972-1-AP, Proteintech), E-cadherin (1:10000, #20874-1-AP, Proteintech), vimentin (1:2000, #10366-1-AP, Proteintech), and beta actin (1:1000, # 20536-1-AP, Proteintech).

Techniques:

Journal: Frontiers in Molecular Biosciences

Article Title: Comprehensive Analysis of the Potential Immune-Related Biomarker Transporter Associated With Antigen Processing 1 That Inhibits Metastasis and Invasion of Ovarian Cancer Cells

doi: 10.3389/fmolb.2021.763958

Figure Lengend Snippet: The relationship between transporter associated with antigen processing 1 (TAP1) and immune checkpoint therapy in multiple cancers. (A) The relationship between TAP1 and 47 immune checkpoint genes. Color green represented negative correlation with TAP1 and color red represented positive correlation with TAP1. (B) The correlation of TAP1 with tumor mutation burden (TMB). (C) The correlation of TAP1 with microsatellite instability (MSI). * p < 0.05. ** p < 0.01. *** p < 0.001.

Article Snippet: The membrane was incubated with the following primary antibodies TAP1 (1:3,000), MEF2A (1:2000, #12382-1-AP, Proteintech, United States), LEF1 (1:1000, #14972-1-AP, Proteintech), E-cadherin (1:10000, #20874-1-AP, Proteintech), vimentin (1:2000, #10366-1-AP, Proteintech), and beta actin (1:1000, # 20536-1-AP, Proteintech).

Techniques: Mutagenesis

Journal: Frontiers in Molecular Biosciences

Article Title: Comprehensive Analysis of the Potential Immune-Related Biomarker Transporter Associated With Antigen Processing 1 That Inhibits Metastasis and Invasion of Ovarian Cancer Cells

doi: 10.3389/fmolb.2021.763958

Figure Lengend Snippet: The relationship between transporter associated with antigen processing 1 (TAP1) and neoantigen in various cancers. TAP1 was positively related to neoantigens in ovarian cancer (OC), cervical squamous cell carcinoma (CESC), lung adenocarcinoma (LUAD), breast invasive carcinoma (BRCA), skin cutaneous melanoma (SKCM), and brain lower grade glioma (LGG) ( p < 0.05), whereas it was negatively correlated in colon adenocarcinoma (COAD) and rectum adenocarcinoma (READ).

Article Snippet: The membrane was incubated with the following primary antibodies TAP1 (1:3,000), MEF2A (1:2000, #12382-1-AP, Proteintech, United States), LEF1 (1:1000, #14972-1-AP, Proteintech), E-cadherin (1:10000, #20874-1-AP, Proteintech), vimentin (1:2000, #10366-1-AP, Proteintech), and beta actin (1:1000, # 20536-1-AP, Proteintech).

Techniques: Immunopeptidomics

Journal: Frontiers in Molecular Biosciences

Article Title: Comprehensive Analysis of the Potential Immune-Related Biomarker Transporter Associated With Antigen Processing 1 That Inhibits Metastasis and Invasion of Ovarian Cancer Cells

doi: 10.3389/fmolb.2021.763958

Figure Lengend Snippet: Correlation of transporter associated with antigen processing 1 (TAP1) expression with the prognosis of ovarian cancer (OC) and cervical cancer (CC). (A) Immunohistochemical (IHC) analysis of transporter associated with antigen processing 1 (TAP1) protein expression in OC tissues. Original magnifications:×200 and ×400. i represents strong positive staining, ii represents moderately positive staining, and iii represents weakly positive staining. (B) IHC analysis of TAP1 protein expression in CC tissues. Original magnifications:×200 and ×400. i represents strong positive staining, ii represents moderately positive staining, and iii represents weakly positive staining. Scale bars 200 μm. (C) 5-Year Kaplan–Meier analysis of overall survival in OC patients on the basis of TAP1 protein expression. (D) 5-Year Kaplan–Meier analysis of overall survival in CC patients on the basis of the TAP1 protein expression. (E) The protein-protein interaction (PPI) network of TAP1 and top 26 correlated transcription factors (TFs) were identified using STRING ( https://string-db.org/cgi/input.pl ). Color blue represented TFs negatively correlated with TAP1, color red represented TFs positively correlated with TAP1, and color white represented TFs significantly correlated with the top 26 TFs in enrichment analysis. (F) Gene set enrichment analysis of transporter associated with antigen processing 1 (TAP1) and pivotal TFs in OC. (G) TAP1, MEF2A, LEF1, and β-actin protein expression in normal cell lines and cancer cell lines. Error bars represent the SD of triplicate measurements. * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: The membrane was incubated with the following primary antibodies TAP1 (1:3,000), MEF2A (1:2000, #12382-1-AP, Proteintech, United States), LEF1 (1:1000, #14972-1-AP, Proteintech), E-cadherin (1:10000, #20874-1-AP, Proteintech), vimentin (1:2000, #10366-1-AP, Proteintech), and beta actin (1:1000, # 20536-1-AP, Proteintech).

Techniques: Expressing, Immunohistochemical staining, Staining

Journal: Frontiers in Molecular Biosciences

Article Title: Comprehensive Analysis of the Potential Immune-Related Biomarker Transporter Associated With Antigen Processing 1 That Inhibits Metastasis and Invasion of Ovarian Cancer Cells

doi: 10.3389/fmolb.2021.763958

Figure Lengend Snippet: Downregulation of transporter associated with antigen processing 1 (TAP1) reduces the invasion and migration in ovarian cancer cells. (A) The volcano map of TAP1-related pathways by gene set variation analysis. (B) Enriching pathway analysis in ovarian cancer of TAP1. (C) The migration abilities of OVCAR3 and SKOV3 were photographed (i, ii) and measured (iii) by examining the wound closure after TAP1 knockdown using wound healing assays. Original magnifications, ×100. Scale bars 150 μm. (D) Transwell assays were photographed (i) and measured (ii) to detect the migration abilities after TAP1 knockdown in SKOV3 and OVCAR3 cells. Original magnifications, ×200. Scale bars 100 μm. (E) Colony formation assays were photographed (i) and measured (ii) to detect the proliferation abilities after TAP1 knockdown in SKOV3 and OVCAR3 cells. (F) Effects of TAP1 knockdown on migration-associated protein (E-cadherin and vimentin) and transcription factor (MEF2A and LEF1) were analyzed by western blotting in SKOV3 and OVCAR3 cells (i). Error bars of histogram (ii) represented the SD of triplicate measurements. * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: The membrane was incubated with the following primary antibodies TAP1 (1:3,000), MEF2A (1:2000, #12382-1-AP, Proteintech, United States), LEF1 (1:1000, #14972-1-AP, Proteintech), E-cadherin (1:10000, #20874-1-AP, Proteintech), vimentin (1:2000, #10366-1-AP, Proteintech), and beta actin (1:1000, # 20536-1-AP, Proteintech).

Techniques: Migration, Knockdown, Western Blot

Journal: Frontiers in Molecular Biosciences

Article Title: Comprehensive Analysis of the Potential Immune-Related Biomarker Transporter Associated With Antigen Processing 1 That Inhibits Metastasis and Invasion of Ovarian Cancer Cells

doi: 10.3389/fmolb.2021.763958

Figure Lengend Snippet: To establish the model of tumor metastasis, SKOV3 cells were injected into nude mice by the i.p. route and randomly treated with si-TAP1_01 or si-NC ( n = 4/group). (A) Representative images of metastatic tumors in the peritoneal cavity. Yellow arrows and rectangles highlighted visible tumor nodes and liver metastasis. (B,C) Number of metastasis and total weight of all metastases were shown (mean ± SEM). (D) Body weight growth of all mice were shown (mean ± SEM). (E) The protein expression of migration-associated protein (E-cadherin and vimentin) and transcription factor (MEF2A and LEF1) in the tumor of si-TAP1_01 group and si-NC group (i). Error bars of histogram (ii) represented the SD of quadruplicate measurements. * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: The membrane was incubated with the following primary antibodies TAP1 (1:3,000), MEF2A (1:2000, #12382-1-AP, Proteintech, United States), LEF1 (1:1000, #14972-1-AP, Proteintech), E-cadherin (1:10000, #20874-1-AP, Proteintech), vimentin (1:2000, #10366-1-AP, Proteintech), and beta actin (1:1000, # 20536-1-AP, Proteintech).

Techniques: Injection, Expressing, Migration

Journal: Alzheimer's & Dementia

Article Title: Differential impact of manganese on glutamate clearance, electroencephalography, and sleep in Alzheimer's disease

doi: 10.1002/alz.70821

Figure Lengend Snippet: RNA expression changes in primary astrocytes and cortical tissues following Mn exposure. (A) Primary astrocytes were obtained from the cortex of P4 pups from both WT and APP/PSEN1 mice. Astrocytes from each pup were then split into two T25 flasks so that astrocytes from a single animal were treated with 100 µM MnCl 2 • 4(H 2 O) and vehicle for within‐subject comparisons of mRNA measured by qPCR. Astrocytic expression of (B) Gfap , (C) Glast , (D) Glt‐1 , (E) Swell1 , and (F) Aqp4 was decreased following Mn exposure in vitro regardless of genotype. RNA expression of (G) Casp1 was increased in astrocytes from APP/PSEN1 pups. RNA obtained from cortical tissue obtained from 6‐month‐old mice treated with Mn or vehicle, as described in Figure , was run on a microarray plate probing for glutamatergic and GABAergic‐related genes. Comparisons between (H) vehicle or Mn‐exposed WT mice, (I) and Mn‐exposed WT and APP/PSEN1 mice, and (J) vehicle or Mn‐exposed APP/PSEN1 mice. Summary of genes with fold‐changes greater than 1.5 (dotted line on x‐axis) and with expression changes with p < 0.05 (dotted line on y‐axis) are summarized in Table S2. (B–G) All n's = 10–12. (H–J) Vehicle‐treated WT, n = 4; Mn‐treated WT, n = 5, saline‐treated APP/PSEN1, n = 4, and Mn‐treated APP/PSEN1, n = 4. Mn, manganese; qPCR, quantitative polymerase chain reaction; WT, wild‐type.

Article Snippet: Blots were incubated in one of the following antibodies in 5% milk in TBST [guinea pig anti‐GLT‐1 [1:4000] (glutamate transporter 1), EMD Millipore Corp cat # AB1783; rabbit anti‐GLAST [1:1000] (glutamate aspartate transporter 1), Novus cat # NB100‐1869; or mouse anti‐actin [1:1000], Santa Cruz Biotechnology cat# sc‐47778] at 4°C on a rotator overnight.

Techniques: RNA Expression, Expressing, In Vitro, Microarray, Saline, Real-time Polymerase Chain Reaction