Journal: Redox Biology

Article Title: A novel role of KEAP1/PGAM5 complex: ROS sensor for inducing mitophagy

doi: 10.1016/j.redox.2021.102186

Figure Lengend Snippet: KEAP1-dependent mitophagy does not involve NRF2 but another KEAP1 substrate PGAM5. A. Silencing of KEAP1 induces Parkin-EYFP translocation both in wt and NRF2-deficient mouse embryonic fibroblasts (MEFs). MEF-s were transfected with Parkin-EYFP and scrambled or KEAP1 shRNA encoding plasmid. **P < 0.01 and ***P < 0.001, n = 9 dishes, 20–35 fields per dish, one-way ANOVA followed by Sidak's multiple comparison test. B. NRF2 does not induce Parkin-EYFP translocation. PC6 cells were transfected with Parkin-EYFP, wt or constitutively active NRF2, or with KEAP1 shRNA. ****P < 0.0001 compared with the scrambled shRNA-expressing group, n = 6 dishes, 20 fields per dish, one-way ANOVA followed by Dunnett's multiple comparison test. C. NRF2 does not induce mitophagy. Primary cortical neurons were transfected with a mix of EGFP-LC3B and GFP-LC3C, mitochondrial Kate2 and wt or constitutively active NRF2, or with scrambled shRNA or KEAP1 shRNA. **P < 0.01 compared with the scrambled shRNA-expressing group, n = 5 dishes, 8 fields per dish, one-way ANOVA followed by Dunnett's multiple comparison test. D. Overexpression of PGAM5 induces Parkin-EYFP translocation. PC6 cells were transfected with Parkin-EYFP and plasmids of interest. ****P < 0.0001 compared with the control group, n = 3 dishes, 20 fields per dish, one-way ANOVA followed by Dunnett's multiple comparison test. E. Overexpression of PGAM5 induces mitophagy. Neurons were transfected with EGFP-LC3B, GFP-LC3C, mitochondrial Kate2 and plasmids of interest. *P < 0.05 and ****P < 0.0001 when compared with control group, n = 3 dishes, 7 fields per dish, one-way ANOVA followed by Dunnett's multiple comparison test. F. Representative superresolution Airyscan images of PC6 cells transfected with PGAM5, Parkin-EYFP and mitochondrial Kate 2 (upper panels) or PC6 cells (middle panels) or neurons (lower panels) transfected with PGAM5, EGFP-LC3B, GFP-LC3C and mitochondrial Kate2.

Article Snippet: PGAM5-Myc-DDK transcript variant 3 (isoform 2, UniProt #Q96HS1-2) , OriGene , Cat# RC201678.

Techniques: Translocation Assay, Transfection, shRNA, Plasmid Preparation, Comparison, Expressing, Over Expression, Control

Journal: Redox Biology

Article Title: A novel role of KEAP1/PGAM5 complex: ROS sensor for inducing mitophagy

doi: 10.1016/j.redox.2021.102186

Figure Lengend Snippet: KEAP1 controls the degradation of full-length PGAM5. A. KEAP1 interacts only with full-length PGAM5. The left panel shows that full-length PGAM5-FLAG, but not cleaved PGAM5, co-immunoprecipitates with KEAP1-HA in HEK cells. Note also that PGAM5 E79A/S80A co-immunoprecipitates with KEAP1 less efficiently than PGAM5-WT-FLAG. The right panel shows quantification of full length and cleaved PGAM5-WT from total cell lysate and KEAP1 interacting fraction. ****P < 0.0001 compared with respective total cell lysate group, n = 3 independent IP-s, one-way ANOVA followed by Sidak's multiple comparison test. B. KEAP1 overexpression induces degradation of PGAM5. The left panel shows the representative Western blot image of PGAM5 expression in the presence of KEAP1-FLAG or KEAP1 R380A/R415A-FLAG in HEK cells. Note also that KEAP1-FLAG but not KEAP1 R380A/R415A-FLAG is co-immunoprecipitating with PGAM5-HA. The right panel shows quantification of full length and cleaved PGAM5 from total cell lysate. **P < 0.01, ns: not significant, n = 4 independent experiments, one-way ANOVA followed by Sidak's multiple comparison test. C. KEAP1 silencing increases the level of endogenous PGAM5. Representative Western blot image (left) and analysis of PGAM5 expression (right) in PC6 cells expressing scrambled or KEAP1 shRNA. ***P < 0.001 and ****P < 0.0001, n = 4, t -test. D. PGAM5 is accumulating in the cytosol after proteasome inhibition. Representative superresolution Airyscan images showing PC6 cells expressing PGAM5-YPet and mitochondrially targeted Kate2 treated with DMSO or 25 μM MG132 for 5 h. E. Proteasome inhibition leads to cytosolic accumulation of full-length PGAM5. Representative Western blot image of PGAM5-Flag levels in cytosolic and mitochondrial fractions of PC6 cells treated DMSO or 25 μM MG132 for 5 h. ATP5A and GAPDH as markers of mitochondria and cytosol, respectively, demonstrate the purity of fractions.

Article Snippet: PGAM5-Myc-DDK transcript variant 3 (isoform 2, UniProt #Q96HS1-2) , OriGene , Cat# RC201678.

Techniques: Comparison, Over Expression, Western Blot, Expressing, shRNA, Inhibition

Journal: Redox Biology

Article Title: A novel role of KEAP1/PGAM5 complex: ROS sensor for inducing mitophagy

doi: 10.1016/j.redox.2021.102186

Figure Lengend Snippet: PGAM5 mediates the KEAP1-dependent mitophagy. A. KEAP1 but not KEAP1 R380A/R415A suppresses PGAM5-induced Parkin translocation. PC6 cells were transfected with Parkin-EYFP, wt KEAP1 or KEAP1 R380A/R415A and PGAM5. **P < 0.01 and ****P < 0.0001, n = 6 dishes, 25 fields per dish, Ordinary one-way ANOVA followed by Holm-Sidak's multiple comparison test. B. KEAP1 but not KEAP1 R380A/R415A suppresses PGAM5-induced mitophagy. Primary cortical neurons were transfected with a mix of EGFP-LC3B and GFP-LC3C, mitochondrially targeted Kate2, wt KEAP1 or KEAP1 R380A/R415A and PGAM5. ***P < 0.001 and ****P < 0.0001, n = 8 dishes, 5 fields per dish, Welch's ANOVA followed by Dunnett's T3 multiple comparison test. C. PGAM5 E79A/S80A-induced Parkin translocation is insensitive to KEAP1. PC6 cells were transfected with Parkin-EYFP, wt PGAM5 or PGAM5 E79A/S80A and wt KEAP1. **P < 0.01 and ****P < 0.0001, n = 6 dishes, 20 fields per dish, Welch's ANOVA followed by Dunnett's T3 multiple comparison test. D. PGAM5 E79A/S80A-induced mitophagy is insensitive to KEAP1. Primary cortical neurons were transfected with a mix of EGFP-LC3B and GFP-LC3C, mitochondrial Kate2, wt PGAM5 or PGAM5 E79A/S80A and wt KEAP1. ***P < 0.001 and ****P < 0.0001, n = 8 dishes, 7 fields per dish, Welch's ANOVA followed by Dunnett's T3 multiple comparison test. E. KEAP1 silencing does not induce Parkin translocation in the absence of PGAM5. PC6 cells were transfected with Parkin-EYFP and scrambled shRNA, KEAP1 shRNA, or/and PGAM5 shRNA expressing plasmids. ****P < 0.0001, n = 6 dishes, 20 fields per dish, one-way ANOVA followed Holms-Sidak's multiple comparison test. Interaction between the treatment P < 0.0001, Two-way ANOVA. F. KEAP1 silencing does not induce mitophagy in the absence of PGAM5. Primary cortical neurons were transfected with a mix of EGFP-LC3B and GFP-LC3C, mitochondrial Kate2 and scrambled shRNA, KEAP1 shRNA or/and PGAM5 shRNA expressing plasmids. ****P < 0.0001, n = 8 dishes, 7 cells per dish, one-way ANOVA followed by Sidak's multiple comparison test. Interaction between the treatment P = 0.0003, Two-way ANOVA. G. KEAP1 silencing does not lead to mitochondrial loss in the absence of PGAM5. The neurons were transfected with neuronal marker hSyn-EGFP, mitochondrial DsRed2 and KEAP1 shRNA or/and PGAM5 shRNA and mitochondrial density was quantified at the end of the axon. **P < 0.01, n = 76–85 axons from 8 dishes, Kruskal-Wallis test followed by Dunn's multiple comparisons test.

Article Snippet: PGAM5-Myc-DDK transcript variant 3 (isoform 2, UniProt #Q96HS1-2) , OriGene , Cat# RC201678.

Techniques: Translocation Assay, Transfection, Comparison, shRNA, Expressing, Marker

Journal: Redox Biology

Article Title: A novel role of KEAP1/PGAM5 complex: ROS sensor for inducing mitophagy

doi: 10.1016/j.redox.2021.102186

Figure Lengend Snippet: PGAM5 mediates the ROS-induced KEAP1-dependent mitophagy, A. KEAP1 interaction with PGAM5 is disrupted by menadione-induced ROS. Left panel - original blots of PGAM5-FLAG co-immunoprecipitates with KEAP1-HA in HEK cells treated with different concentrations of menadione for 4 h. Right panel - quantification of KEAP1 in immunoprecipitated fraction as well as the amount of full-length PGAM5-WT in total cell lysate. The grey curve depicts the amount of H 2 O 2 produced by the cells at these menadione concentrations (note that these data are derived from the standard curve depicted in ). n = 1. B. Menadione induces accumulation of full-length PGAM5. Representative Western blot image and analysis of endogenous full-length PGAM5 expression in PC6 cells treated with DMSO or 20 μM menadione for 4 h. **P < 0.01, n = 4, t -test. C. PGAM5 silencing inhibits menadione-induced Parkin translocation. PC6 cells expressing Parkin-EYFP and scrambled or PGAM5 shRNA were treated with DMSO or 20 μM menadione for 4 h. ****P < 0.0001, n = 5 dishes, 20 fields per dish, one-way ANOVA followed Holms-Sidak's multiple comparison test. Interaction between the treatments P = 0.0002, Two-way ANOVA. D. PGAM5 silencing inhibits laser irradiation-induced Parkin translocation. PC6 cells were transfected with mitochondrial ECFP, Parkin-EYFP, mitochondrial KillerRed and scrambled or PGAM5 shRNA encoding plasmid. Sub-population of mitochondria in the selected cell was irradiated using a 561 nm laser, and redistribution of Parkin-EYFP signal was visualized 2 h later. *P = 0.018, n = 42–46 cells from 8 individual dishes, Mann Whitney test. E. PGAM5 silencing inhibits menadione-induced mitophagy. Primary cortical neurons expressing EGFP-LC3B, GFP-LC3C, Kate2 targeted to the mitochondria and scrambled or PGAM5 shRNAs were treated with DMSO or 20 μM menadione for 4 h. ****P < 0.0001, n = 5 dishes, 5 cells per dish, one-way ANOVA followed by Sidak's multiple comparison test. Interaction between the treatments P < 0.0001, Two-way ANOVA.

Article Snippet: PGAM5-Myc-DDK transcript variant 3 (isoform 2, UniProt #Q96HS1-2) , OriGene , Cat# RC201678.

Techniques: Immunoprecipitation, Produced, Derivative Assay, Western Blot, Expressing, Translocation Assay, shRNA, Comparison, Irradiation, Transfection, Plasmid Preparation, MANN-WHITNEY

Journal: Redox Biology

Article Title: A novel role of KEAP1/PGAM5 complex: ROS sensor for inducing mitophagy

doi: 10.1016/j.redox.2021.102186

Figure Lengend Snippet: PGAM5 interferes with PINK1 processing independently of its phosphatase activity. A. PGAM5 mutant and isoform lacking phosphatase activity induce Parkin translocation. PC6 cells were transfected with Parkin-EYFP and PGAM5 wt, PGAM5 F244D or short PGAM5 isoform (isoform 2, UniProt #Q96HS1-2). ****P < 0.0001, n = 6 dishes, 20 fields per dish, Welch's ANOVA followed by Dunnett's T3 multiple comparisons test. B. PGAM5 mutant lacking PARL/OMA1 cleaving site does not induce Parkin translocation. PC6 cells were transfected with Parkin-EYFP and PGAM5 or PGAM5 S24F. ****P < 0.0001, n = 6 dishes, 20 fields per dish, Welch's ANOVA followed by Dunnett's T3 multiple comparisons test. C. TOMM7 overexpression increases the fraction of full-length PGAM5 in HEK293 cells expressing PGAM5-flag and TOMM7-myc. D. TOMM7 overexpression protects partially against PGAM5 induced Parkin translocation. PC6 cells were transfected with Parkin-EYFP, PGAM5 wt or/and TOMM7. ****P < 0.0001, n = 9 dishes, 20 fields per dish, Welch's ANOVA followed by Dunnett's T3 multiple comparisons test. E. OMA1 and PARL protect partially against PGAM5 induced Parkin translocation. PC6 cells were transfected with Parkin-EYFP, PGAM5 wt and PARL or OMA1. ****P < 0.0001, n = 6 dishes, 20 fields per dish, One-way ANOVA followed by Sidak's multiple comparisons test. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: PGAM5-Myc-DDK transcript variant 3 (isoform 2, UniProt #Q96HS1-2) , OriGene , Cat# RC201678.

Techniques: Activity Assay, Mutagenesis, Translocation Assay, Transfection, Over Expression, Expressing

Journal: Redox Biology

Article Title: A novel role of KEAP1/PGAM5 complex: ROS sensor for inducing mitophagy

doi: 10.1016/j.redox.2021.102186

Figure Lengend Snippet: Inhibitors of KEAP1–PGAM5 protein-protein interaction enhance mitophagy. A. Molecular docking simulation showing energetically most favorable interactions between a KEAP1 and non-electrophilic KEAP1 inhibitors. The table is showing hydrogen-bond pairings in KEAP1-inhibitor complexes. The length of the hydrogen bond is given in angstroms. B. Treatment with CPUY192018 disrupts KEAP1-PGAM5 interaction. PC6 cells were co-transfected with KEAP1-FLAG and PGAM5-HA and treated for 24 h with DMSO or 100 μM CPUY192018. KEAP1-FLAG was co-immunoprecipitating with PGAM5-HA in DMSO but not in CPUY192018 treated cells. C. Treatment with CPUY192018 increases the level of endogenous PGAM5. PC6 cells were treated with 100 μM CPUY192018 for 24 h. The left panel shows a representative Western blot and the right panel quantitative analysis. *P < 0.05, n = 4 samples, t -test. D. Effect of non-electrophilic KEAP1 inhibitors on mitochondrial membrane potential. PC6 cells were treated with KEAP1 inhibitors for 24 h, after which the cells were stained with ratiometric mitochondrial membrane potential sensor JC10. *P < 0.05 and ****P < 0.0001 when compared with DMSO treated group, n = 7–12 wells per data point, One-way ANOVA followed by Dunn's multiple comparisons test. E. Non-electrophilic KEAP1 inhibitors induce Parkin-EYFP translocation to mitochondria. PC6 cells expressing Parkin-EYFP were treated with DMSO or 100 μM of KEAP1 inhibitors for 24 h ***P < 0.001 and ****P < 0.0001 when compared with DMSO treated group, n = 6 dishes per group, 20 fields per dish, One-way ANOVA followed by Dunnett's multiple comparisons test. F and G. Non-electrophilic KEAP1 inhibitors enhance mitophagy but not general autophagy. Neurons expressing mitochondrial marker Kate2 and autophagosome markers EGFP-LC3B and GFP-LC3C were treated with DMSO or 100 μM of KEAP1 inhibitors for 24 h. The number of LC3 positive puncta colocalizing with mitochondria (F) as well as the total number of LC3 positive puncta (G) was counted by a blinded observer. **P < 0.01, ***P < 0.001 and ****P < 0.0001 when compared with DMSO treated group, n = 9–14 dishes (10 cells per dish), Welch's ANOVA followed by Dunnett's T3 multiple comparisons test. H. Treatment of primary cortical neurons with CPUY192018 (24 h) induces a concentration-dependent increase in mitochondrial colocalization with autophagosome markers EGFP-LC3B/GFP-LC3C. *P < 0.05 and ****P < 0.0001, when compared with DMSO, treated group, n = 16–18 dishes from 4 independent experiments (10 cells per dish), Kruskal-Wallis test followed by Dunn's multiple comparisons test, the dotted line shows the EC50.

Article Snippet: PGAM5-Myc-DDK transcript variant 3 (isoform 2, UniProt #Q96HS1-2) , OriGene , Cat# RC201678.

Techniques: Transfection, Western Blot, Membrane, Staining, Translocation Assay, Expressing, Marker, Concentration Assay

Journal: Redox Biology

Article Title: A novel role of KEAP1/PGAM5 complex: ROS sensor for inducing mitophagy

doi: 10.1016/j.redox.2021.102186

Figure Lengend Snippet:

Article Snippet: PGAM5-Myc-DDK transcript variant 3 (isoform 2, UniProt #Q96HS1-2) , OriGene , Cat# RC201678.

Techniques: Recombinant, Lysis, Extraction, Blocking Assay, Isolation, DC Protein Assay, Caspase-Glo Assay, shRNA, Generated, Plasmid Preparation, Synthesized, Variant Assay

Journal: iScience

Article Title: Development of chimeric antigen receptor (CAR)-T cells targeting A56 viral protein implanted by oncolytic virus

doi: 10.1016/j.isci.2024.109256

Figure Lengend Snippet: Essential domain of A56 for its localization on cell plasma membrane (A) Confocal immunofluorescence (IF) microscopy of OVV-infected A549 tumor cells showing A56 expression; nuclei (blue), OVV (green), A56 (red); scale bar, 10 μm. (B) Full A56 construct design compared with those of various A56 mutant constructs that do not have one or more domains; Sg, signaling domain; IgV, IgV-like domain; TDR, tandem repeat domain; S, stalk region; TM, transmembrane domain; CT, cytoplasmic tail. (C) Merged confocal images of A56 localization on the plasma membranes of U2-OS tumor cells upon full A56-expressing plasmid transduction compared to proteins enclosed within the Golgi or ER from transduction with mutant A56 vectors; nuclei (blue), A56 (green), Golgi apparatus or endoplasmic reticulum (red); scale bar, 10 μm.

Article Snippet: For A56 detection on the plasma membrane, the cells were labeled with 1μg of anti-A56 antibody (Mouse IgG1 w/o kappa light chain) (LakePharma, Variant 3) diluted in PBS supplemented with 2% FBS for 30 min at 4°C.

Techniques: Clinical Proteomics, Membrane, Immunofluorescence, Microscopy, Infection, Expressing, Construct, Mutagenesis, Plasmid Preparation, Transduction

Journal: iScience

Article Title: Development of chimeric antigen receptor (CAR)-T cells targeting A56 viral protein implanted by oncolytic virus

doi: 10.1016/j.isci.2024.109256

Figure Lengend Snippet: Localization of A56 on solid tumor plasma membrane and distribution in tumor tissue (A) OTS-412 were treated in A549, HCT-116, HeLa cancer cells at 0.05 and 0.1 MOI for 24 h followed by detection of A56 expression via flow cytometry. Dead cells were excluded by Zombie Aqua (ZA) staining. For intracellular detection of A56, fixation/permeabilization was performed before anti-A56 staining. (B) Analysis of A56 expression compared to A27L (a vaccinia virus cytosolic protein) expression on tumor tissue collected 4 days post-OV injection of HT-29 tumor-bearing mice. Scale bar, 100 px (=438 μm). (C) IF analysis of tumor-specific A56 expression and non-expression on other tissues from VX2 tumor-bearing New Zealand White rabbits collected on day (D) 5, 14, and 28 after intravenous injection of OTS-412/HU; The data shown are representative of six and two individual rabbits from the treatment and control groups, respectively. Scale bar, 200 μm. (D) Quantitation of A56 expression detected from each tissue of Figure 2 C presented in mean intensity of fluorescence. Data are represented as mean ± SEM. (E) Distribution of OTS-412 in the OTS-412/HU combination group presented in mean viral copies per 25 mg of each tissue and 1 mL of PBMC analyzed until D28 post virus injection. Data are represented as mean ± SEM. BM; bone marrow, AG; adrenal gland.

Article Snippet: For A56 detection on the plasma membrane, the cells were labeled with 1μg of anti-A56 antibody (Mouse IgG1 w/o kappa light chain) (LakePharma, Variant 3) diluted in PBS supplemented with 2% FBS for 30 min at 4°C.

Techniques: Clinical Proteomics, Membrane, Expressing, Flow Cytometry, Staining, Virus, Injection, Control, Quantitation Assay, Fluorescence

Journal: iScience

Article Title: Development of chimeric antigen receptor (CAR)-T cells targeting A56 viral protein implanted by oncolytic virus

doi: 10.1016/j.isci.2024.109256

Figure Lengend Snippet: In vitro and in vivo functionality of A56-specific A56 CAR-T cells (A) Real-time cytotoxicity of A56 CAR-T cells against HCT-116 cells infected with 0.05 MOI of OTS-412. (B) Quantification of A56 CAR-T cell cytotoxicity in various human cancer cell lines infected with OTS-412. (∗∗∗ p < 0.001, comparison of T cell groups; n = 2 for A549, HCT-116, HeLa, SK-MEL-5 and n = 3 for MCF7 and PC-3 by two-tailed unpaired t test). Data are represented as the mean value ± SEM. (C) Aggregation of A56 CAR-T cells targeting tumor cells observed every 12 h until day 4 in real-time. (D) Antigen-dependent anti-tumor efficacy of A56 CAR-T cells in HCT-116 tumor-bearing mice after intratumoral T cell injection following OTS-412 injection with or without HU (∗ p < 0.05, ∗∗ p < 0.01, comparison of T cell groups; saline, n = 5; treatment groups, n = 4, two-way ANOVA with multiple comparison). Data are represented as the mean value ± SD. (E) IHC analysis of A56 expression and infiltrated hCD3 + T cells in tumors isolated from HCT-116 tumor-bearing mice 21 days post-intravenous T cell treatment. Scale bar, 20 μm (left panel) and quantitation of tumor-infiltrating A56 CAR-T cells positive for hCD3 (right panel) (∗ p = 0.01, n = 3, two-tailed unpaired t test). Data are presented as the mean ± SD. MOI, multiplicity of infection; IHC, immunohistochemistry.

Article Snippet: For A56 detection on the plasma membrane, the cells were labeled with 1μg of anti-A56 antibody (Mouse IgG1 w/o kappa light chain) (LakePharma, Variant 3) diluted in PBS supplemented with 2% FBS for 30 min at 4°C.

Techniques: In Vitro, In Vivo, Infection, Comparison, Two Tailed Test, Injection, Saline, Expressing, Isolation, Quantitation Assay, Immunohistochemistry

Journal: iScience

Article Title: Development of chimeric antigen receptor (CAR)-T cells targeting A56 viral protein implanted by oncolytic virus

doi: 10.1016/j.isci.2024.109256

Figure Lengend Snippet:

Article Snippet: For A56 detection on the plasma membrane, the cells were labeled with 1μg of anti-A56 antibody (Mouse IgG1 w/o kappa light chain) (LakePharma, Variant 3) diluted in PBS supplemented with 2% FBS for 30 min at 4°C.

Techniques: Staining, Virus, Recombinant, Isolation, Saline, Plasmid Preparation, Cell Isolation, Expressing, Software

Journal: Cancer immunology research

Article Title: Efficacy of a cancer vaccine against ALK-rearranged lung tumors

doi: 10.1158/2326-6066.CIR-15-0089

Figure Lengend Snippet: A, EML4-ALK expression in ASB-XIV infected cells and in human EML4-ALK NSCLC cell lines (H3122 and H2228) was evaluated by immunoblotting with the indicated antibodies. B, Analysis of the Major Histocompatibility Complex (MHC) Class I (PE-H2Dd Ab) antigen expression on ASB-XIV cells by flow cytometry. C, Schematic representation of ALK vaccination protocol in BALB/c mice. Control mice were vaccinated with empty pDEST (Ctrl) and ALK vaccinated mice were vaccinated with pDEST-ALK (Vax). D, Cytotoxic activity in ALK vaccinated mice evaluated by an in vivo cytotoxicity assay. Horizontal bars represent means. E and F, Representative hematoxilin-eosin (H&E) sections of lungs injected with GFP-ASB-XIV cells (E) or EML4-ALK ASB-XIV cells (F). Histograms represent the number of tumors in control (Ctrl; n=3 mice) and ALK vaccinated mice (Vax; n=3 mice). Scale bars, 1mm (top) and 50μm (bottom). The total number of tumors was counted in the whole lung of each mouse. Data are represented from three independent experiments as mean (±SEM). ***, P<0.0001.

Article Snippet: Human ALK -rearranged NSCLC cell lines, H2228 (variant 3, E6;A20), DFCI032 and H3122 (variant 1, E13;A20) were obtained from the ATCC collection and were passaged for fewer than 6 months after receipt and resuscitation.

Techniques: Expressing, Infection, Western Blot, Immunopeptidomics, Flow Cytometry, Control, Activity Assay, In Vivo, Cytotoxicity Assay, Injection