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

Journal: Cell

Article Title: A blood atlas of COVID-19 defines hallmarks of disease severity and specificity

doi: 10.1016/j.cell.2022.01.012

Figure Lengend Snippet:

Article Snippet: CD22 (HIB22)-159Tb , Fluidigm , Cat# 3159005B, RRID: AB_2892688.

Techniques: Mass Cytometry, Flow Cytometry, Recombinant, Staining, Selection, Antibody Labeling, Labeling, Isolation, Sample Prep, Luminex, Quantitative Proteomics, Generated, Gene Expression, Clone Assay, Marker, Expressing, Mass Spectrometry, Derivative Assay, RNA Sequencing, Sequencing, Illumina Sequencing, Software, Variant Assay