Journal: Journal of molecular medicine (Berlin, Germany)

Article Title: The KISS1 metastasis suppressor appears to reverse the Warburg effect by shifting from glycolysis to mitochondrial beta-oxidation

doi: 10.1007/s00109-017-1552-2

Figure Lengend Snippet: KISS1-mediated beta-oxidation induction is a result of AMPK phosphorylation of ACC at serine 79. (a) mRNA expression of early lipogenic genes, ACLY, ACC1, ACC2, and FASN in C1861.9Vector (V), C8161.9KFM59 (KFM), C8161.9KFM59/shKISS1 (K/shK), and C8161.9KFMΔSS6 (ΔSS) cells were quantified by qRT-PCR. (b & c) ACC, FASN, phos-ACC at serine 79, phos-AMPKβ at serine 108, AMPKβ, phos-AMPKα at threonine 172, and AMPKα proteins from C1861.9Vector, C8161.9KFM59, C8161.9KFM59/shKISS1, and C8161.9KFMΔSS6 whole cell lysate were quantified using immunoblot analysis with beta-actin as loading control. Densitometry of proteins was first normalized to beta-actin and then pACC:ACC ratios were determined. (a-c) N ≥ 3; error bars, SEM; * P ,< 0.05 (b & c) N≥ 3; mean ± SEM

Article Snippet: Primary antibodies were purchased from following manufacturers and were used at the titers listed: Acc (Cell Signaling, Danvers, MA cat# 3676, 1:500), phos-Acc Ser79 (Cell Signaling cat#3661, 1:500), FASN (Cell Signaling cat#3180, 1:500), AMPK-α (Cell Signaling cat# 2603, 1:500), phos-AMPK-α Thr172 (Cell Signaling cat#2535, 1:500), AMPK-β1/2 (Cell Signaling cat#4150, 1:500), phos- AMPK-β1/2 Ser108 (Cell Signaling cat#4181, 1:500), hexokinase II (Cell Signaling cat#2867, 1:500), VDAC (Millipore cat# AB10527) , PGC1α (Millipore cat# ST1202, 1:1,000), β-actin (Sigma cat#A5441, 1:5,000), GAPDH (Santa Cruz Biotechnology, Santa Cruz, CA cat#sc-365062, 1:3000), and KISS1 (ref. [ 26 ], 1:500).

Techniques: Phospho-proteomics, Expressing, Quantitative RT-PCR, Western Blot, Control

Journal: Journal of molecular medicine (Berlin, Germany)

Article Title: The KISS1 metastasis suppressor appears to reverse the Warburg effect by shifting from glycolysis to mitochondrial beta-oxidation

doi: 10.1007/s00109-017-1552-2

Figure Lengend Snippet: PGC-1α appears to be required for KISS1-mediated induction of beta-oxidation-related gene expression and AMPK-mediated phosphorylation of ACC. A PCR Array was used to probe expressions of various genes in C1861.9Vector (V), C8161.9KFM59 (KFM), and C8161.9KFM59/shPGC-1α (K/shP) cells. Selected upregulated genes are shown in (a & b). (c) mRNA expression of beta-oxidation-related genes, ACC1, ACC2, ACLY, and PPARα in C1861.9Vector, C8161.9KFM59, and C8161.9KFM59/shPGC-1α#1(K/shP1), and C8161.9KFM59/shPGC-1α#2 (K/shP2)cells were quantified by qRT-PCR. (d) PGC-1α, phos-ACC at serine 79, ACC, phos-AMPKβ at serine 108, AMPKβ, phos-AMPKα at threonine 172, AMPKα, and KISS1 proteins from C1861.9Vector, C8161.9KFM59, C8161.9KFMΔSS6(ΔSS), C8161.9KFM59/shPGC-1α#1 and C8161.9KFM59/shPGC-1α#2 whole cell lysates were quantified using immunoblot analysis with beta-actin as loading control. (a-c) N ≥ 3; error bars, SEM; * P , <0.05

Article Snippet: Primary antibodies were purchased from following manufacturers and were used at the titers listed: Acc (Cell Signaling, Danvers, MA cat# 3676, 1:500), phos-Acc Ser79 (Cell Signaling cat#3661, 1:500), FASN (Cell Signaling cat#3180, 1:500), AMPK-α (Cell Signaling cat# 2603, 1:500), phos-AMPK-α Thr172 (Cell Signaling cat#2535, 1:500), AMPK-β1/2 (Cell Signaling cat#4150, 1:500), phos- AMPK-β1/2 Ser108 (Cell Signaling cat#4181, 1:500), hexokinase II (Cell Signaling cat#2867, 1:500), VDAC (Millipore cat# AB10527) , PGC1α (Millipore cat# ST1202, 1:1,000), β-actin (Sigma cat#A5441, 1:5,000), GAPDH (Santa Cruz Biotechnology, Santa Cruz, CA cat#sc-365062, 1:3000), and KISS1 (ref. [ 26 ], 1:500).

Techniques: Gene Expression, Phospho-proteomics, Expressing, Quantitative RT-PCR, Western Blot, Control

Journal: Journal of translational medicine

Article Title: Unraveling resistance mechanisms in anti-CD19 chimeric antigen receptor-T therapy for B-ALL: a novel in vitro model and insights into target antigen dynamics.

doi: 10.1186/s12967-024-05254-z

Figure Lengend Snippet: Fig. 5 Observation of CD19-BBζ-CAR expression in relapsed Nalm-6 cells and salvage treatment. A Detection of FMC63 and CD247 transcripts and 4-1BB gene of CAR in CD19+ Nalm-6 (red) and relapsed CD19− Nalm-6 cells (blue) by qRT-PCR. Data of left bar graph represent the relative quantification using ACTB as the internal reference. Error bars represent s.d. The data are the representative of three independent experiments. B Expression of CD19 and CAR on CD19+ Nalm-6 cells and relapsed CD19− Nalm-6 cells analyzed by flow cytometry (representative of 3 experiments). Merge Graphs, the blue dots represent CD19− Nalm-6 cells and the red dots represent Nalm-6 cells. C Confocal imaging of Nalm-6 cells and relapsed CD19− Nalm-6 cells using Alexa Flour 488-conjugated anti-CD19 antibody (green), Alexa Flour 647-conjugated anti-CAR19 antibody (red), and DAPI (blue). D Lentiviral integration sites of CAR transduced Nalm-6 cells were analyzed by linear-amplification mediated PCR (LAM-PCR) and visualized with Circos plots. The integration sites across the genome and genomic features were shown from outer to inner circle: (1) cytogenetic bands; (2) genes that harbor these integration sites along with a bar chart showing the reads of integration sites; (3) the distribution of integration sites, with colored circles representing different gene functional regions of the host sequence: purple for promoter region, green for intron region, and red for distal intergenic region. E Phenotype changes of Nalm-6 cells transduced with small amount of CD19 CAR lentiviruses detected by flow cytometry over time. Gating was based on the same cells stained with isotype-matched antibody. F Dynamics of CD19− B phenotype in relapsed cells after co-culture with different ratios (5×, 20×) of Nalm-6 cells. Gating was based on the same cells stained with isotype-matched antibody. G Relapsed CD19− Nalm-6 cells were tested by qPCR specific for VSV-G sequence. H Comparison of in vitro efficacy of CD19-, CD22-, CD19/CD22- and CD22×CD19- CAR T cells. Cocultures with the relapsed cells were performed at 1:5, 1:1, and 5:1 E: T ratios, and lysis efficacies were detected by the LDH release assay Declarations

Article Snippet: The cells were then washed twice and stained with phycoerythrin (PE) streptavidin (BD bioscience, USA) for 15 min. CART-22 cells and CART-22/19 cells were washed once and incubated with CD22 Fc Alexa Fluor® 647 Protein (R&D Systems, USA) for 15 min. To detect in vitro cytotoxicity of CART-19 cells, transduced and untransduced T cells were co-cultured with Nalm-6 cells (total 1 × 106 cells) at E: T ratios (0.2:1, 0.5:1, 1:1, 5:1) for 6, 24 and 72 h. Cells were pipetted to incubate with antibodies for 30 min at room temperature in the dark and washed twice with PBS.

Techniques: Expressing, Quantitative RT-PCR, Quantitative Proteomics, Flow Cytometry, Imaging, Amplification, Functional Assay, Sequencing, Transduction, Staining, Co-Culture Assay, Comparison, In Vitro, Lysis, Lactate Dehydrogenase Assay

Journal: Nature Communications

Article Title: Antigen glycosylation regulates efficacy of CAR T cells targeting CD19

doi: 10.1038/s41467-022-31035-7

Figure Lengend Snippet: a Western blot of lysates from WT or SPPL3 KO Nalm6 and OCI-Ly10 cells probed for CD19. Protein electrophoresis was performed on a 6% polyacrylamide gel. Representative of n = 4 individual experiments. b Western blot of lysates from WT or SPPL3 KO Nalm6 cells that were either untreated (UT), treated with PNGase F (PNG), or treated with Endoglycosidase H (Endo H) and then probed for CD19. Representative data of n = 3 individual experiments. Electrophoresis performed on a 6% polyacrylamide gel. c Median Fluorescence Intensity of CD19 as detected by FMC63-APC on the surface of WT or SPPL3 KO Nalm6 cells. d Western blot analysis of lysates from cells treated with kifunensine (16 ng/mL) for 10 days and then probed for CD19. Representative of n = 2 individual experiments. e Survival of WT, untreated SPPL3 KO , and kifunensine-treated SPPL3 KO Nalm6 over time in co-culture with CART19 cells (E:T ratio 0.25:1) statistical annotations reflect differences between SPPL3 KO + kifu and WT and SPPL3 KO + kfiu and SPPL3 KO (lower). f Western blot of lysates from WT or SPPL3 KO Nalm6 cells probed for CD22. Representative of n = 4 individual experiments. g Survival of WT or SPPL3 KO Nalm6 cells over time after combination with CART22 (E:T ratio 0.25:1). e , g Representative data from n = 3 individual experiments with distinct donor T cells. Error bars reflect mean ± standard error of the mean (s.e.m.). * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 by two-way ANOVA with Bonferroni correction for multiple comparisons. Source Data are provided as a Source Data file.

Article Snippet: Proteins were labeled with SPPL3 (EMD Millipore #MABS1910), CD19 (#3574), CD22 (#67434), Actin (#4970) or GAPDH (#2118, all from Cell Signaling, all diluted 1:1000), followed by secondary antibody staining using either anti-rabbit (Cell Signaling #7074, diluted 1:5000), or anti-mouse (Cell Signaling #7076, diluted 1:5000) HRP-linked antibodies followed by visualization.

Techniques: Western Blot, Protein Electrophoresis, Electrophoresis, Fluorescence, Co-Culture Assay