Journal: International Journal of Molecular Sciences

Article Title: A Metal-Free, Disulfide Oxidized Form of Superoxide Dismutase 1 as a Primary Misfolded Species with Prion-Like Properties in the Extracellular Environments Surrounding Motor Neuron-Like Cells

doi: 10.3390/ijms22084155

Figure Lengend Snippet: Generation of motor neuron models of ALS in which misfolded and aggregated hSOD1 are intracellularly deposited. NSC-34 cells were transiently transfected with expression vectors encoding human wild-type superoxide dismutase 1 (hSOD1 WT ) or the ALS-causing hSOD1 mutants (G93A, G37R, D90A, and G85R) tagged with green fluorescent protein (GFP), treated with all trans -retinoic acid, and cultured for 72 h. ( a ) Western blots of hSOD1 in the Nonidet P-40 (NP-40) soluble and insoluble factions from NSC-34 cells, β-tubulin was used as a loading control. No Vec., untransfected cells; ( b ) florescence microscopy of cells transfected with hSOD1 WT GFP or the ALS-linked hSOD1 mutant GFP. Arrows indicate protein aggregations containing hSOD1 GFP. Scale bar = 10 µm; ( c ) enzyme-linked immunosorbent assay signals of (black) EDI-positive and (gray) C4F6-positive misfolded SOD1 in the NP-40 soluble fraction. All data are shown as the mean ± SD, n = 3 in each transfection.

Article Snippet: For experiments on intracellular propagation of hSOD1 WT misfolding by the conditioned medium, wild-type SOD1 with untagged GFP was also cloned into pCMV3 from human cDNA, (HG11727-UT, Sino Biological, Beijing, China).

Techniques: Transfection, Expressing, Cell Culture, Western Blot, Microscopy, Mutagenesis, Enzyme-linked Immunosorbent Assay

Journal: International Journal of Molecular Sciences

Article Title: A Metal-Free, Disulfide Oxidized Form of Superoxide Dismutase 1 as a Primary Misfolded Species with Prion-Like Properties in the Extracellular Environments Surrounding Motor Neuron-Like Cells

doi: 10.3390/ijms22084155

Figure Lengend Snippet: Decrease in the total level of extracellular hSOD1 mutants, but not murine SOD1, in the conditioned medium. The NSC-34 cells were transiently transfected with human wild-type superoxide dismutase 1 (hSOD1 WT ) and the ALS-causing hSOD1 mutants (G93A, G37R, D90A, and G85R) fused with green fluorescent protein (GFP). After 72 h of transfection, the conditioned medium from the cells was collected; ( a ) Western blots of hSOD1-GFP and endogenous murine SOD1 in the medium, β-tubulin was used as a marker for intracellular non-secreted protein to confirm whether cell lysis or debris could be contaminated in the conditioned medium. The amount of protein loaded for sodium dodecyl sulfate polyacrylamide gel electrophoresis was validated by Ponceau S staining; ( b , c ) quantification of the absolute total protein level of extracellular ( b ) hSOD1 and ( c ) murine SOD1. Data are shown as the mean ± SD, n = 3 in each transfection. Statistical analysis was performed using one-way ANOVA followed by the Tukey–Kramer post hoc test. ** p < 0.01 vs. hSOD1 WT .

Article Snippet: For experiments on intracellular propagation of hSOD1 WT misfolding by the conditioned medium, wild-type SOD1 with untagged GFP was also cloned into pCMV3 from human cDNA, (HG11727-UT, Sino Biological, Beijing, China).

Techniques: Transfection, Western Blot, Marker, Lysis, Polyacrylamide Gel Electrophoresis, Staining

Journal: International Journal of Molecular Sciences

Article Title: A Metal-Free, Disulfide Oxidized Form of Superoxide Dismutase 1 as a Primary Misfolded Species with Prion-Like Properties in the Extracellular Environments Surrounding Motor Neuron-Like Cells

doi: 10.3390/ijms22084155

Figure Lengend Snippet: The thiol/disulfide redox balance of extracellular hSOD1 shifts toward oxidation because of increased oxidative stress in the conditioned medium. The conditioned medium of the NSC-34 cells expressing human wild-type superoxide dismutase 1 (hSOD1 WT )-green fluorescence protein (GFP), the ALS-linked hSOD1-GFP, or GFP alone was treated with 100 mM iodoacetamide to block artificial oxidation of the thiol group of proteins. ( a ) Non-reducing Western blots of disulfide oxidized (S-S) and reduced (-SH) forms of hSOD1-GFP in the medium. β-ME = beta-mercaptoethanol; ( b ) quantification of absolute levels of (black) hSOD1 S-S and (gray) hSOD1 SH in the medium; ( c ) Western blots of 4-hydroxynonenal (4-HNE)-conjugated proteins in the conditioned medium; ( d ) densitometrical calculation of relative levels of 4-HNE-conjugated proteins in the medium. The amount of protein loaded for sodium dodecyl sulfate polyacrylamide gel electrophoresis was validated by Ponceau S staining. Data are expressed as the mean ± SD. Statistical analysis was performed using one-way ANOVA followed by the Tukey–Kramer post hoc test, n = 3 for each transfection. ** p < 0.01 vs. no vector.

Article Snippet: For experiments on intracellular propagation of hSOD1 WT misfolding by the conditioned medium, wild-type SOD1 with untagged GFP was also cloned into pCMV3 from human cDNA, (HG11727-UT, Sino Biological, Beijing, China).

Techniques: Expressing, Fluorescence, Blocking Assay, Western Blot, Polyacrylamide Gel Electrophoresis, Staining, Transfection, Plasmid Preparation

Journal: International Journal of Molecular Sciences

Article Title: A Metal-Free, Disulfide Oxidized Form of Superoxide Dismutase 1 as a Primary Misfolded Species with Prion-Like Properties in the Extracellular Environments Surrounding Motor Neuron-Like Cells

doi: 10.3390/ijms22084155

Figure Lengend Snippet: The population of extracellular Cu/Zn-deficient hSOD1 was increased in the conditioned medium. The conditioned medium from the NSC-34 cells transfected with human wild-type superoxide dismutase 1 (hSOD1 WT )-green fluorescence protein (GFP), the ALS-linked hSOD1-GFP, or GFP alone, was treated with 100 mM iodoacetamide to block artificial oxidation of the thiol group of proteins. Then, extracellular human and murine SOD1 enzymatic activity in the medium was measured by a SOD Assay Kit-WST. The level of active SOD1 in the medium was calculated based on a calibration curve that was generated from a Cu/Zn SOD1 standard. The extracellular level of active ( a ) hSOD1 and ( b ) endogenous murine SOD1 in the medium; ( c ) the medium was treated with 1 mM CuSO 4 , 1 mM ZnSO 4 , or both, for 24 h, and active hSOD1 levels in the medium were quantified using a SOD Assay Kit-WST. All data are expressed as the mean ± SD. Statistical analysis was performed using one-way ANOVA followed by the Tukey–Kramer post hoc test, n = 3 for each treatment or for each transfection. ** p < 0.01 vs. H 2 O-added medium that corresponds to the same transfection.

Article Snippet: For experiments on intracellular propagation of hSOD1 WT misfolding by the conditioned medium, wild-type SOD1 with untagged GFP was also cloned into pCMV3 from human cDNA, (HG11727-UT, Sino Biological, Beijing, China).

Techniques: Transfection, Fluorescence, Blocking Assay, Activity Assay, Generated

Journal: International Journal of Molecular Sciences

Article Title: A Metal-Free, Disulfide Oxidized Form of Superoxide Dismutase 1 as a Primary Misfolded Species with Prion-Like Properties in the Extracellular Environments Surrounding Motor Neuron-Like Cells

doi: 10.3390/ijms22084155

Figure Lengend Snippet: Extracellular misfolded human superoxide dismutase 1 (hSOD1) presents as a metal-free, disulfide oxidized form (apo-SOD1 S-S ). Enzyme-linked immunosorbent assay signals of ( a ) EDI-positive misfolded SOD1; ( b ) C4F6-positive misfolded SOD1; ( c ) total hSOD1, in the conditioned medium treated with or without both 1 mM CuSO 4 and 1 mM ZnSO 4 for 24 h. Data are expressed as the mean ± SD, n = 3 for each treatment per each transfection. Statistical analysis was performed using a two-tailed unpaired Student’s t test. ** p < 0.01 vs. Cu/Zn-untreated medium that corresponds to the same transfection. N.S., not significant; ( d ) the extracellular levels of different folding states of hSOD1 in the mediums; ( e ) extracellular misfolded SOD1 in the medium was detected by immunoprecipitation with C4F6 followed by non-reducing Western blotting to determine the thiol/disulfide redox balance of the misfolded species. β-ME = beta-mercaptoethanol.

Article Snippet: For experiments on intracellular propagation of hSOD1 WT misfolding by the conditioned medium, wild-type SOD1 with untagged GFP was also cloned into pCMV3 from human cDNA, (HG11727-UT, Sino Biological, Beijing, China).

Techniques: Enzyme-linked Immunosorbent Assay, Transfection, Two Tailed Test, Immunoprecipitation, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: A Metal-Free, Disulfide Oxidized Form of Superoxide Dismutase 1 as a Primary Misfolded Species with Prion-Like Properties in the Extracellular Environments Surrounding Motor Neuron-Like Cells

doi: 10.3390/ijms22084155

Figure Lengend Snippet: Extracellular misfolded apo-SOD1 S-S induces intracellular propagation of hSOD1 WT misfolding in recipient cells. A procedure to investigate intracellular prion-like propagation of human wild-type superoxide dismutase 1 (hSOD1 WT ) misfolding was performed using immunoprecipitation of the Nonidet P (NP-40) soluble fraction with C4F6 followed by Western blotting with antibodies to ( a , c ) SOD1 or (f) green fluorescence protein (GFP). The conditioned media from NSC-34 cells transfected with hSOD1-GFP containing misfolded apo-SOD1 S-S were used as a donor medium, whereas NSC-34 cells transfected with GFP-untagged hSOD1 WT were used as a recipient cell. ( a ) The recipient cells were exposed to conditioned medium pretreated with or without C4F6 to remove misfolded apo-SOD1 S-S ; ( b ) relative level of intracellular misfolded hSOD1 WT in the recipient cells exposed to medium pretreated with or without C4F6, ** p < 0.01 vs. C4F6-untreated cells that correspond to the same transfection, n = 3 for each treatment; ( c ) the recipient cells were pretreated with endocytosis inhibitors, 50 nM wortmannin, or 100 µM 5-( N -ethyl- N -isopropyl) amiloride (EIPA), for 1 h, and the cells were exposed to the medium for 24 h; ( d ) relative level of intracellular misfolded hSOD1 WT in endocytosis-impaired cells, ** p < 0.01 vs. DMSO-treaded cells that were exposed to the corresponding medium, n = 3 for each treatment; ( e ) the recipient cells expressing GFP-tagged hSOD1 WT were exposed to 0.5 µg/mL purified hSOD1 WT proteins with a disulfide bond (Cu/Zn SOD1 S-S , Zn SOD1 S-S , and apo-SOD1 S-S ) for 24 h, n = 3 for each treatment; ( f ) relative level of intracellular misfolded hSOD1 WT cells treated with the purified hSOD1 WT protein, ** p < 0.01 vs. HEPES-treated cells, N.S. = not significant. All data are expressed as the mean ± SD. Statistical analysis was performed using one-way ANOVA followed by the Tukey–Kramer post hoc test.

Article Snippet: For experiments on intracellular propagation of hSOD1 WT misfolding by the conditioned medium, wild-type SOD1 with untagged GFP was also cloned into pCMV3 from human cDNA, (HG11727-UT, Sino Biological, Beijing, China).

Techniques: Immunoprecipitation, Western Blot, Fluorescence, Transfection, Expressing, Purification

Journal: International Journal of Molecular Sciences

Article Title: A Metal-Free, Disulfide Oxidized Form of Superoxide Dismutase 1 as a Primary Misfolded Species with Prion-Like Properties in the Extracellular Environments Surrounding Motor Neuron-Like Cells

doi: 10.3390/ijms22084155

Figure Lengend Snippet: The conditioned medium containing extracellular misfolded metal free, disulfide oxidized superoxide dismutase 1 (apo-SOD1 S-S ) exerted cytotoxicity to motor neuron-like cells. (white) NSC-34 cells expressing green fluorescence protein (GFP) untagged human wild-type SOD1 (hSOD1 WT ) were exposed to conditioned medium from NSC-34 harboring hSOD1-GFP containing misfolded apo-SOD1 S-S for 24 h. Cell viability and cytotoxicity were assessed using ( a ) a Cell Counting Kit 8 assay and ( b ) a lactate dehydrogenase (LDH) release assay, respectively. (black) Extracellular misfolded apo-SOD1 S-S was removed from the conditioned medium by immunoprecipitation with C4F6. (gray) As a control, the medium was immunoprecipitated with normal mouse IgG. In the data set of cell viability, the results are expressed as the cell viability of recipient cells relative to that of untransfected cells, which were incubated with normal culture medium, Dulbecco’s modified Eagle’s medium and F-12 with GlutaMAX™ containing 1% ( v / v ) fetal bovine serum and 0.1 mM non-essential amino acids, instead of the conditioned medium. In the data set of cytotoxicity, the results are given as the amounts of released LDH to the medium relative to that of cells exposed to 100 μM H 2 O 2 for 24 h. All data are expressed as the mean ± SD, n = 3 for each treatment. Statistical analysis was performed using one-way ANOVA followed by the Tukey–Kramer post hoc test. ** p < 0.01.

Article Snippet: For experiments on intracellular propagation of hSOD1 WT misfolding by the conditioned medium, wild-type SOD1 with untagged GFP was also cloned into pCMV3 from human cDNA, (HG11727-UT, Sino Biological, Beijing, China).

Techniques: Expressing, Fluorescence, Cell Counting, Lactate Dehydrogenase Assay, Immunoprecipitation, Incubation, Modification

Journal: Molecular Systems Biology

Article Title: Molecular profiling reveals features of clinical immunity and immunosuppression in asymptomatic P. falciparum malaria

doi: 10.15252/msb.202110824

Figure Lengend Snippet:

Article Snippet: After surface staining, cells were fixed and permeabilized with a Maxpar nuclear antigen staining buffer set (Fluidigm) and then stained with a 161Dy‐conjugated anti‐T‐bet (clone 4B10; Fluidigm) antibody for 45 min at room temperature.

Techniques: Control, Enzyme-linked Immunosorbent Assay, Marker, Infection, Purification, Flow Cytometry, Blocking Assay, Generated, Recombinant, Saline, Staining, Software, Cytometry

Journal: International Journal of Molecular Sciences

Article Title: Discovery of NFκB2-Coordinated Dual Regulation of Mitochondrial and Nuclear Genomes Leads to an Effective Therapy for Acute Myeloid Leukemia

doi: 10.3390/ijms25158532

Figure Lengend Snippet: Mitochondria (ATP synthase, Complex V)-targeted therapy sensitized blasts to TKI treatments but nuclear genomes of blasts underwent reprogramming to survive. (N = 3). ( A ) Representative FC plots of different experimental groups with NO TX, 80 nM gilteritinib (GILT), 100 nM oligomycin (OA), and GILT + OA on viability dye and CD44 expression; Red circle indicates CD44+/viability dye- (negative) viable blasts; Right table: Cumulative percentage data of viable CD44+ cells in different treatment groups; ( B ) Gene expression of GDF15 , a biomarker for mitochondrial diseases or oxidative stress, was analyzed by qPCR. Data of mRNA expressions show the fold change (normalized to β-actin ) of GDF15 in different treatment groups; ( C ) Gene expression of NRF1 (nuclear respiratory factor 1), a key transcription regulator of mitochondrial biogenesis, was analyzed by qPCR. Data of mRNA expressions show the fold change (normalized to β-actin ) of NRF1 in different treatment groups; ( D ) Representative FC histograms showing expression of NRF1 in viable MV4-11 cells that had IgG-staining control (orange plot line); NO TX (gray plot line); 100 nM oligomycin (OA)-treated experimental groups (red plot line); Right table: Cumulative MFI (mean fluorescence intensity) data of NRF1 + viable cells of different treatment groups; ( E ) Gene expressions of NFκB1 and NFκB2 , transcription factors that rapidly respond to cellular stimuli, were analyzed by qPCR. Data of mRNA expressions show the fold change (normalized to β-actin ) of NFκB1 and NFκB2 in different treatment groups; Where applicable, data are means ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.005.

Article Snippet: MV4-11 cells were grown in 6-well plates and transduced with human shRNA- NFκB2 lentiviral particles, which contain 4 sets of unique 29mer target-specific shRNA targeting NFκB2 ( P100/P52 ) (Catalog#: TL311187V, OriGene, Rockville, MD, USA).

Techniques: Expressing, Biomarker Assay, Staining, Control, Fluorescence

Journal: International Journal of Molecular Sciences

Article Title: Discovery of NFκB2-Coordinated Dual Regulation of Mitochondrial and Nuclear Genomes Leads to an Effective Therapy for Acute Myeloid Leukemia

doi: 10.3390/ijms25158532

Figure Lengend Snippet: New transgenic cell lines of NFκB -family genes reveal that NFκB2 overexpression (OE) promotes gene expressions of TFAM, TFB2M and NRF1 in vitro. (N = 3). ( A ) Schematic diagram of lentiviral expression constructs containing open-reading frames (ORF) of human NFκB2, RELB , or NFκB1 genes and eGFP or mCherry reporters, with promoters EF1a and IRES2, respectively; A bicistronic lenti-plasmid expresses both NFκB2 and RELB controlled by EF1a and IRES promoters, respectively; Six new transgenic cell lines overexpressing NFκB family transgenes in leukemia blast MV4-11 were generated, including: NFκB2 - eGFP -MV411, NFκB1-mCherry -MV411, RELB - mCherry -MV411, NFκB2 - RELB - eGFP -MV411, NFκB2 -eGFP/ RELB-mCherry -MV411, NFκB2 -eGFP/ NFKB1-mCherry -MV411; and GFP -MV411 will be the vector control without transgene ORF insert; ( B ) All new cell lines were purified by FACS-sorting (see Materials and Methods); A representative FC plot shows multiple populations with eGFP and/or mCherry expression which were identified as NFκB2 - eGFP -MV411, RELB - mCherry -MV411, NFκB2-eGFP/RELB-mCherry -MV411 before purification; ( C ) Gene expressions of NFκB2, NRF1, TFAM and TFB2M were analyzed by qPCR. Data of mRNA expressions show the fold change (normalized to β-actin ) of genes; ( D ) Representative FC plots show protein expressions of TFAM and/or TFB2M in NFκB2-eGFP -MV411 and GFP -MV411 (vector control); Right table: Cumulative percentage data of viable TFAM+/TFB2M+ cells in different groups; ( E ) Gene expressions of NFκB2 and TFAM after shRNA knockdown of NFκB2 in MV4-11 cells, analyzed by qPCR. Data of mRNA expressions show the fold change (normalized to β-actin ) of NFκB2 and TFAM in different treatment groups; Where applicable, data are means ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.005.

Article Snippet: MV4-11 cells were grown in 6-well plates and transduced with human shRNA- NFκB2 lentiviral particles, which contain 4 sets of unique 29mer target-specific shRNA targeting NFκB2 ( P100/P52 ) (Catalog#: TL311187V, OriGene, Rockville, MD, USA).

Techniques: Transgenic Assay, Over Expression, In Vitro, Expressing, Construct, Plasmid Preparation, Generated, Control, Purification, shRNA, Knockdown

Journal: International Journal of Molecular Sciences

Article Title: Discovery of NFκB2-Coordinated Dual Regulation of Mitochondrial and Nuclear Genomes Leads to an Effective Therapy for Acute Myeloid Leukemia

doi: 10.3390/ijms25158532

Figure Lengend Snippet: Visualization of mitochondrial NFκB2 in MV4-11 blasts. ( N = 3 ) . ( A ) Generation of P100-eGFP (fusion)-MV4-11 cell line through the lentiviral system; Consistent with P100-eGFP (fusion)-HEK-293T cells, P100-eGFP was localized in cytoplasm of MV4-11. The immunocytochemistry (representative fluorescent images) was performed to show the co-localization of MitoView™633-stained mitochondria and GFP+ mitochondria at low magnification with a scale bar of 10 µm. ( B , C ) The white box of ( A ) was magnified to show many GFP+ mitochondria by a GFP-fluorescent image (indicated by white arrows, ( B ) and by a Phase-bright image of mitochondrial morphology (indicated by black arrows, ( C ) in these P100-eGFP (fusion)-MV4-11 cells with a scale bar of 20 µm.

Article Snippet: MV4-11 cells were grown in 6-well plates and transduced with human shRNA- NFκB2 lentiviral particles, which contain 4 sets of unique 29mer target-specific shRNA targeting NFκB2 ( P100/P52 ) (Catalog#: TL311187V, OriGene, Rockville, MD, USA).

Techniques: Immunocytochemistry, Staining

Journal: International Journal of Molecular Sciences

Article Title: Discovery of NFκB2-Coordinated Dual Regulation of Mitochondrial and Nuclear Genomes Leads to an Effective Therapy for Acute Myeloid Leukemia

doi: 10.3390/ijms25158532

Figure Lengend Snippet: NFκB2 activated promoters of dual mitochondrial-nuclear genomes in vitro. ( N = 3 ) . ( A ) Sequence information of lentiviral vector constructs (custom-built by GeneCopoeia) of mitochondrial LSP promoter (blue sequence), HSP1 promoter (red sequence) or HSP1/HSP2 promoter (underlined sequence) and a NFκB2-binding sequence (green sequence and named as Enhancer); ( B , C ) Promoter assays of mitochondrial genome ( B ) and nuclear genome ( C ) in blasts were performed (see details in the Materials and Methods); representative live images show luciferase activity in the supernatants of different experimental groups; ( D ) A schematic diagram illustrating the NFκB2-binding sequence on the D-loop of human mitochondrial DNA genome; Under pathologic or physiologic condition, NFκB2 may activate mitochondrial LSP promoter, HSP1/2 promoters and nuclear TFAM promoter to initiate mitochondrial biogenesis.

Article Snippet: MV4-11 cells were grown in 6-well plates and transduced with human shRNA- NFκB2 lentiviral particles, which contain 4 sets of unique 29mer target-specific shRNA targeting NFκB2 ( P100/P52 ) (Catalog#: TL311187V, OriGene, Rockville, MD, USA).

Techniques: In Vitro, Sequencing, Plasmid Preparation, Construct, Binding Assay, Luciferase, Activity Assay

Journal: International Journal of Molecular Sciences

Article Title: Discovery of NFκB2-Coordinated Dual Regulation of Mitochondrial and Nuclear Genomes Leads to an Effective Therapy for Acute Myeloid Leukemia

doi: 10.3390/ijms25158532

Figure Lengend Snippet: Novel therapies eliminated AML blasts in vitro by inhibiting their mitochondrial biogenesis and functions. ( N = 3 ). ( A ) Representative FC plots of different experimental groups of MV4-11 cells with NO TX, 80 nM GILT, 100 nM oligomycin (OA), 15 µM SN52 (NFκB2-I), or 15 µM BAY11-7082 (NFκB-I), GILT + OA, GILT + SN52, GILT + SN52 + OA and GILT + SN52 + OA + BAY, which were analyzed by cell death biomarkers including Annexin-V (apoptosis), Propidium Iodide (PI, necrosis) and Annexin-V+/PI+ (dead); The blue arrows indicate Annexin-V+/PI- cells (early apoptotic); The orange arrows indicate Annexin-V+/PI+ cells (late-stage dying or dead cells); ( B ) Upper panel: Cumulative percentage data of viable MV4-11 blasts in different treatment groups (both Annexin-V negative /PI negative populations in the FC plots); Lower panel: Cumulative percentage data of Annexin-V+/PI- cells (blue column and indicated by blue arrows in plots of ( A )), and Annexin-V+/PI+ cells (orange column and indicated by orange arrows in plots of ( A )) in different treatment groups; ( C ) Gene expressions of TFAM were analyzed by qPCR. Data of mRNA expressions show the fold change (normalized to β-actin ) of TFAM in different treatment groups; ( D ) ATP concentration was measured in MV411 cells of different treatment groups; ( E ) Representative FC histograms show expression of MitoView™633 in different treatment groups with NO TX, GILT + SN52 + OA and GILT + SN52 + BAY + OA; Right table: Cumulative MFI (mean fluorescence intensity) data of MitoView™633 expression in different treatment groups; Where applicable, data are means ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.005.

Article Snippet: MV4-11 cells were grown in 6-well plates and transduced with human shRNA- NFκB2 lentiviral particles, which contain 4 sets of unique 29mer target-specific shRNA targeting NFκB2 ( P100/P52 ) (Catalog#: TL311187V, OriGene, Rockville, MD, USA).

Techniques: In Vitro, Concentration Assay, Expressing, Fluorescence

Journal: International Journal of Molecular Sciences

Article Title: Discovery of NFκB2-Coordinated Dual Regulation of Mitochondrial and Nuclear Genomes Leads to an Effective Therapy for Acute Myeloid Leukemia

doi: 10.3390/ijms25158532

Figure Lengend Snippet: Overview of inhibiting NFκB2-mediated mitochondrial-nuclear transcriptional adaptation to overcome AML relapse. ( A ) Mitochondria-targeted drugs such as oligomycin, an ATP synthase blocker (Complex V) can improve the therapeutic efficacy of TKIs; however, mitochondrial damage and stress result in the activation of pro-survival transcription factors of noncanonical NFκB2 in refractory blasts. ( B ) Notably, increased NFκB2 can promote the nuclear gene (nDNA) expression of TFAM , TFB2M , and NRF1 , which are known to be essential for mitochondrial biogenesis and metabolic plasticity, in addition to its activation of a group of pro-inflammatory and pro-survival factors such as CD44/cytokines/receptors (as we reported previously), thus leading to a large cascading effect on homeostatic adaptation to support blast survival and AML relapse energetically and metabolically. Increased NFKB2 enters the mitochondria and binds specific “TTGGGGGGTG” region of D-loop to promote biogenesis and biosynthesis to support the respiratory chain for ATP/metabolites production. ( C ) In this regard, we developed a novel triplet strategy blocking FLT3 signaling, targeting blast mitochondrial energy conversion, and inhibiting NFκB2 simultaneously, which can effectively promote the terminal death of AML blasts and prevent AML relapse.

Article Snippet: MV4-11 cells were grown in 6-well plates and transduced with human shRNA- NFκB2 lentiviral particles, which contain 4 sets of unique 29mer target-specific shRNA targeting NFκB2 ( P100/P52 ) (Catalog#: TL311187V, OriGene, Rockville, MD, USA).

Techniques: Activation Assay, Expressing, Metabolic Labelling, Blocking Assay

Journal: iScience

Article Title: Cytokine profile of anti-spike CD4 + T cells predicts humoral and CD8 + T cell responses after anti-SARS-CoV-2 mRNA vaccination

doi: 10.1016/j.isci.2024.110441

Figure Lengend Snippet: Percentage of vaccine-generated anti-spike CD4 + T cell response depending on the cytokine measured A total of 128 patients, including 76 who were pre-infected and 52 who were uninfected, received vaccination. Non-pre-infected volunteers received BNT162b2 vaccine (30 μg) at V1 (D0) and V2 (D29), while pre-infected volunteers received only one dose of vaccine at V1. At V3, which is one month after the second vaccination or 2 months after the only 1 st vaccination depending on their infection status, the patients' CD4 + T cells were sorted and sensitized in vitro with a megapool of overlapping peptides covering the S1 protein and another pool for the S2 protein. An ELIspot (ELI) IFNγ assay and a 27-cytokine Luminex assay, were then performed after 24 or 48 h of incubation, respectively. The Luminex assay was used with supernatants of ELIspot IFNγ not coated with anti-IFNγ antibodies. The frequency of vaccine response for each cytokine, as determined by the V3/V1 ratio ≥2, and a concentration of the cytokine ≥10 pg/mL (after background subtraction when cells were sensitized with medium) is shown. The threshold for vaccine response detection for a given cytokine is indicated by the dotted line at 10% frequency.

Article Snippet: Elispot IFNg , Diaclone , 856.051.020P.

Techniques: Generated, Infection, In Vitro, Enzyme-linked Immunospot, Luminex, Incubation, Concentration Assay

Journal: iScience

Article Title: Cytokine profile of anti-spike CD4 + T cells predicts humoral and CD8 + T cell responses after anti-SARS-CoV-2 mRNA vaccination

doi: 10.1016/j.isci.2024.110441

Figure Lengend Snippet: Difference in the induction of CD4 + T cell vaccine response and the detection of T cell responses based on cytokine assays (A) Anti-spike multicytokine CD4 + T cell responses were measured prior to vaccination (V1) in pre-infected (PI) ( n = 76) and non-pre-infected (NPI) ( n = 52) volunteers. (B) The absolute value of ELISpot IFNγ and cytokines assay were measured one month after the second BNT162b2 vaccination (V3) for non-pre-infected volunteers and after only one dose of vaccination for pre-infected participants at the same time. (C) The vaccine response based on CD4 + T cell cytokines profile and the V3/V1 ratio was calculated for the same set of volunteers. (D) The ratio between V3 and V1 is shown regardless of infection status. Statistical differences, determined using the Wilcoxon test with FDR correction, are shown between the pre-infected and non-infected groups for each cytokine. Data are represented as mean ± SEM ∗: p ≤ 0.05; ∗∗: p ≤ 0.01; ∗∗∗: p ≤ 0.001.

Article Snippet: Elispot IFNg , Diaclone , 856.051.020P.

Techniques: Infection, Enzyme-linked Immunospot

Journal: iScience

Article Title: Cytokine profile of anti-spike CD4 + T cells predicts humoral and CD8 + T cell responses after anti-SARS-CoV-2 mRNA vaccination

doi: 10.1016/j.isci.2024.110441

Figure Lengend Snippet: Enhancement in sensitivity of the anti-spike CD4 + T cell assay through multiple cytokines detection A comparison of the CD4 + T cell vaccine responses (V3/V1) was performed using either a positive ELISpot IFNγ assay alone (without cytokine) or in combination with the detection of IL-2 or 3 cytokines (IL-2, IP-10, TNFα), or 18 cytokines via Luminex ( n = 128). The respective sensitivity of the different tests is shown on the histograms.

Article Snippet: Elispot IFNg , Diaclone , 856.051.020P.

Techniques: Comparison, Enzyme-linked Immunospot, Luminex

Journal: iScience

Article Title: Cytokine profile of anti-spike CD4 + T cells predicts humoral and CD8 + T cell responses after anti-SARS-CoV-2 mRNA vaccination

doi: 10.1016/j.isci.2024.110441

Figure Lengend Snippet: Correlation between IL-21 produced by anti-spike CD4 + T cell and spike serology IL-21 ELISpot assay against S1 (A), or S1+S2 (B) peptide MP on sorted CD4 + T cells was performed in a cohort of patients vaccinated with the BNT162b2 vaccine ( n = 108). The ELISpot results are expressed as the number of spots/10 5 cells. These results were correlated with spike serology performed at the same time point as the IL-21 ELISpot results.

Article Snippet: Elispot IFNg , Diaclone , 856.051.020P.

Techniques: Produced, Enzyme-linked Immunospot

Journal: iScience

Article Title: Cytokine profile of anti-spike CD4 + T cells predicts humoral and CD8 + T cell responses after anti-SARS-CoV-2 mRNA vaccination

doi: 10.1016/j.isci.2024.110441

Figure Lengend Snippet: Profile of cytokines produced by CD4 + T cells could predict the vaccine induced CD8 + T cell response using a machine learning approach Correlation via Pearson’s Chi-Squared test was sought between the positivity of the ELISpot IFNγ performed on CD4 + T cells against S1 or S2 after vaccination (D57) in non-pre-infected ( n = 108) (A) or pre-infected ( n = 104) (B) volunteers and the parallel induction of CD8 + T cells against S1 or S2. (C) Correlation was sought between the positivity for the V3/V1 ratio criteria of each cytokine produced by CD4 + T cells sensitized by S1 and S2 after vaccination (D57) in non-pre-infected volunteers and the parallel induction of CD8 + T cells against S1 or S2 (detected by ELISpot IFNγ) using the statistical Cox test. (D) An algorithm was established using machine learning by training 80% of the Pfizer cohort with a Gradient Boosting algorithm (XGB). The model was validated on 20% of volunteers from the unused Pfizer cohort using 5-fold cross-validation to demonstrate the stability of the model. Boxplots representing the resulting AUC for the resulting models are shown. (E) ROC curve calculated on the mRNA-vaccinated Moderna cohort dataset ( n = 68). The dashed diagonal line represents random classification. AUC and p -values are shown. (F) The confusion matrix generated by the model summarizes the model’s performance. The matrix’s diagonal elements represent the number of corrected predictions (True Negative [TN] and True Positive [TP]), while the off-diagonal elements represent incorrect predictions (False positive [FP] and False negative [FN]). The Fisher exact test was used to determine the p value.

Article Snippet: Elispot IFNg , Diaclone , 856.051.020P.

Techniques: Produced, Enzyme-linked Immunospot, Infection, Generated

Journal: iScience

Article Title: Cytokine profile of anti-spike CD4 + T cells predicts humoral and CD8 + T cell responses after anti-SARS-CoV-2 mRNA vaccination

doi: 10.1016/j.isci.2024.110441

Figure Lengend Snippet:

Article Snippet: Elispot IFNg , Diaclone , 856.051.020P.

Techniques: Blocking Assay, Recombinant, Staining, Enzyme-linked Immunospot, Multiplex Assay, Selection, Software, Gentle