Journal: Cell reports

Article Title: Activator of KAT3 histone acetyltransferase family ameliorates a neurodevelopmental disorder phenotype in the syntaxin 1A ablated mouse model.

doi: 10.1016/j.celrep.2024.114101

Figure Lengend Snippet: Figure 2. Identification of the KAT3B function binding to OL4 (120 to 88) region (A) DNA affinity purification for MS analyses. Proteins associating with the biotinylated OL4 DNA were affinity purified from PC12 nuclear extracts by avidin beads and separated with 5%SDS-PAGE. OL4 DNA affinity-purified protein band (AP1) is indicated with an asterisk. (B) Scheme showing a putative KAT3B (P300) motif and two identified Sp/GC motifs in the CPR and the oligonucleotide probes (OL1–8) used for supershift assay. (C) Supershift assay of the OL4-protein complex. KAT3 monoclonal antibody was incubated with PC12 nuclear extract before addition of the biotinylated OL4 probe. The DNA-protein-antibody complex supershift band (SS1) is indicated with an asterisk. The enlarged image is shown in the right rectangle. (D) Effect of KAT3B suppression on promoter activity in neuronal PC12 cells transfected with stx1a CPR (204 to +2) construct. Cells were transfected by electroporation with stx1a CPR plasmids and pcDNA3 constructs expressing rat control shRNA or KAT3B shRNA. Values indicate means (n = 18 wells; two-tailed Mann-Whitney test) from six independent experiments. **p < 0.01 compared to control shRNA. (E) Effect of KAT3B suppression on STX1A protein expression in neuronal PC12 cells. Whole-cell extracts from PC12 were immunoblotted using antibodies against STX1A, KAT3, and TUBA. (F) Effect of a KAT3 inhibitor, anacardic acid (Ana), on reporter activity in PC12 cells. The pGL-CPR (204 to +2) constructs were transfected into PC12 cells, and cells were treated with 0–50 mM Ana for 24 h. Reporter activity was measured and normalized against activity of 0 mM (0.1%) DMSO. Values indicate means (n = 9 wells; Kruskal-Wallis Dunn’s post hoc test) from three independent experiments. *p < 0.05, **p < 0.01 compared to 0 mM. (G) Effect of KAT3 inhibitor, Ana, on STX1A protein expression in neuronal PC12 cells. Whole-cell extracts from PC12 were immunoblotted using antibodies against STX1A and TUBA. (H) Effect of a KAT3 activator, TTK21, on reporter activity in PC12 cells. The pGL-CPR (204 to +2) constructs were transfected into PC12 cells, and cells were treated with 0–100 mM TTK21 for 24 h. Reporter activity was measured and normalized against activity of 0 mM (0.1% DMSO). Values indicate means (n = 9 wells; Kruskal-Wallis Dunn’s post hoc test) from three independent experiments. **p < 0.01 compared to 0 mM. (I) Effect of KAT3 activator, TTK21, on STX1A protein expression in neuronal PC12 cells. Whole-cell extracts from PC12 were immunoblotted using antibodies against STX1A and TUBA. (J) Effect of a KAT3 activator, TTK21, on [3H] 5-HT release in PC12 cells. Values indicate means (n = 7 wells; two-tailed unpaired t test) from three independent experiments. **p < 0.01 compared to 0 mM.

Article Snippet: Transfection and luciferase reporter assays For the knockdown experiment in PC12 cells, KAT3B (P300) short hairpin RNA (shRNA) constructs and negative control (15 mg) (Santa Cruz Biotechnology) were cotransfected with pGL3-(-204 +1) (10 mg) and phRG-TK reporter (1 mg) into 1 3 108 cells using a super electroporator, CUY21 EDIT-II (BEX).

Techniques: Binding Assay, Avidin-Biotin Assay, SDS Page, Incubation, Activity Assay, Transfection, Construct, Electroporation, Expressing, Control, shRNA, Two Tailed Test, MANN-WHITNEY

Journal: Cell reports

Article Title: Activator of KAT3 histone acetyltransferase family ameliorates a neurodevelopmental disorder phenotype in the syntaxin 1A ablated mouse model.

doi: 10.1016/j.celrep.2024.114101

Figure Lengend Snippet: Figure 3. KAT3B associates to stx1a CPR in cell/tissue expressing stx1a (A and B) ChIP assays using KAT3 antibody in PC12 (A) or FRSK (B) cells treated with TSA (+) or untreated (). Primers for the stx1a CPR were used to amplify isolated DNA. The data shown are representative images. Values indicate means (n = 3 wells; two-tailed unpaired t test) from three independent experiments.KAT3 binds to the CPR in PC12 cells, which express stx1a, but not in FRSK cells, which do not. **p < 0.01 compared to the untreated control. The KAT3B association to theCPRisledbyTSAtreatmentinFRSKcellsnotendogenouslyexpressingstx1a. (C and D) ChIP assays using KAT3 antibody in tissues of brain (C) or lung (D). Primers for the stx1a CPR were used to amplify isolated DNA. The data shown are representative images. Values indicate means (n = 3 wells; two-tailed unpaired t test) from three independent experiments. The KAT3B associates with the CPR in the brain tissue endogenously expressing stx1a, but not in the lung tissue not expressing stx1a. *p < 0.05 compared to the untreated control.

Article Snippet: Transfection and luciferase reporter assays For the knockdown experiment in PC12 cells, KAT3B (P300) short hairpin RNA (shRNA) constructs and negative control (15 mg) (Santa Cruz Biotechnology) were cotransfected with pGL3-(-204 +1) (10 mg) and phRG-TK reporter (1 mg) into 1 3 108 cells using a super electroporator, CUY21 EDIT-II (BEX).

Techniques: Expressing, Isolation, Two Tailed Test, Control

Journal: Cell reports

Article Title: Activator of KAT3 histone acetyltransferase family ameliorates a neurodevelopmental disorder phenotype in the syntaxin 1A ablated mouse model.

doi: 10.1016/j.celrep.2024.114101

Figure Lengend Snippet: Figure 7. A scheme of the mechanism of stx1a transcription and rescue of low LI The neuron-specific transcription of stx1a is pre- cisely regulated via the epigenetic regulation mechanism represented by histone modification by class 1-HDACs (HDAC1, 2, 8). The expressional intensity of stx1a in neuron is modulated by a histone acetyltransferase, KAT3B. Therefore, the abnormal low LI (LLI) behavior caused by attenu- ation of neuronal stx1a is ameliorated via the elevation of stx1a expression and 5-HT release by the KAT3 activator.

Article Snippet: Transfection and luciferase reporter assays For the knockdown experiment in PC12 cells, KAT3B (P300) short hairpin RNA (shRNA) constructs and negative control (15 mg) (Santa Cruz Biotechnology) were cotransfected with pGL3-(-204 +1) (10 mg) and phRG-TK reporter (1 mg) into 1 3 108 cells using a super electroporator, CUY21 EDIT-II (BEX).

Techniques: Expressing

Journal: Infection and Immunity

Article Title: Aerobic glycolysis of bronchial epithelial cells rewires Mycoplasma pneumoniae pneumonia and promotes bacterial elimination

doi: 10.1128/iai.00248-23

Figure Lengend Snippet: Overview of differentially expressed genes in M. pneumoniae- infected BEAS-2B cells. The human bronchial epithelial cell line BEAS-2B was subjected to M. pneumoniae infection for 16 h. The upregulated genes were subjected to gene ontology (GO) enrichment analysis (A), and the top 20 pathways with the highest KEGG pathway enrichment levels were displayed according to the amount and enrichment level of total differentially expressed genes annotated in the control vs M. pneumoniae comparison (B). TNF, tumor necrosis factor.

Article Snippet: M. pneumoniae (ATCC 29432) was cultured in mycoplasma broth supplemented with 20% supplement as described previously ( 32 ).

Techniques: Infection, Control, Comparison

Journal: Infection and Immunity

Article Title: Aerobic glycolysis of bronchial epithelial cells rewires Mycoplasma pneumoniae pneumonia and promotes bacterial elimination

doi: 10.1128/iai.00248-23

Figure Lengend Snippet: M. pneumoniae-induced aerobic glycolysis in bronchial epithelial cells. HBE cells were cultured in 96-well plates and infected with M. pneumoniae at an MOI of 100 for 24 h. Thereafter, the ECAR and OCR were detected using fluorescence luminescence. (A and B) HBE cells were infected with various MOIs of M. pneumoniae for 24 h, and the expressions of HK1, PFKM, and pyruvate kinase M2 (PKM2) mRNA (C–E) or protein (F) were measured using quantitative PCR and western blot analysis. Data shown are one experiment representative of three independent experiments with three duplicate samples for each group. *P <0.05, as compared with the untreated group (C–E).

Article Snippet: M. pneumoniae (ATCC 29432) was cultured in mycoplasma broth supplemented with 20% supplement as described previously ( 32 ).

Techniques: Cell Culture, Infection, Fluorescence, Real-time Polymerase Chain Reaction, Western Blot

Journal: Infection and Immunity

Article Title: Aerobic glycolysis of bronchial epithelial cells rewires Mycoplasma pneumoniae pneumonia and promotes bacterial elimination

doi: 10.1128/iai.00248-23

Figure Lengend Snippet: M. pneumoniae enhances glucose uptake in bronchial epithelial cells. HBE cells were seeded in 24-well plates and infected with various MOIs of M. pneumoniae for 24 h or with MOI 100 for 0, 8, 12 and 24 h. Glucose and lactate concentrations were detected using the glucose oxidase method and enzyme colorimetry, respectively (A–D); (E) HBE cells were pre-treated with the glycolysis inhibitor 2-DG (1 and 5 mM) for 3 h, and subsequently infected with 100 MOI of M. pneumoniae for 24 h. The concentrations of lactate in the supernatant was detected; (F–H) HBE cells were infected with M. pneumoniae for various time intervals. GLUT1 expression was detected using quantitative PCR or western blot analysis. Data represent at least three independent experiments with similar results. *P <0.05, as compared with the control (A, C, F, and G) or specified groups (B, D, and E).

Article Snippet: M. pneumoniae (ATCC 29432) was cultured in mycoplasma broth supplemented with 20% supplement as described previously ( 32 ).

Techniques: Infection, Colorimetric Assay, Expressing, Real-time Polymerase Chain Reaction, Western Blot, Control

Journal: Infection and Immunity

Article Title: Aerobic glycolysis of bronchial epithelial cells rewires Mycoplasma pneumoniae pneumonia and promotes bacterial elimination

doi: 10.1128/iai.00248-23

Figure Lengend Snippet: Metabolic shift in response to transfection of dominant negative MyD88 and TRAM. BEAS-2B cells were transfected with the dominant negative MyD88 or TRAM plasmid before infection with M. pneumoniae (MOI = 100) for 24 h. ECAR (A and C), OCR (B and D), glucose uptake (E), lactate production (F), and GLUT1 expression (G) were measured. Data shown are one experiment representative of three independent experiments, and n = 3 per group for each experiment. *P <0.05, compared with the indicated groups.

Article Snippet: M. pneumoniae (ATCC 29432) was cultured in mycoplasma broth supplemented with 20% supplement as described previously ( 32 ).

Techniques: Transfection, Dominant Negative Mutation, Plasmid Preparation, Infection, Expressing

Journal: Infection and Immunity

Article Title: Aerobic glycolysis of bronchial epithelial cells rewires Mycoplasma pneumoniae pneumonia and promotes bacterial elimination

doi: 10.1128/iai.00248-23

Figure Lengend Snippet: M. pneumoniae induces the bronchial epithelial cell secretion of eATP. HBE cells were seeded on 24-well plates and infected with varying doses of M. pneumoniae for specified time intervals. The concentration of eATP was measured using luminescence ( A and B ). (C–G ) Cells were pre-treated with glycolysis inhibitor (5 mM 2-DG) or ATP channel inhibitors (10- and 50 -µM CBX, 10- and 50 -µM 18α-GA, 20- and 100 -µM N-ethylmaleimide (NEM), and 50- and 100 -µM FFA), and subsequently infected with 100 MOI of M. pneumoniae for 4 h . eATP concentration was measured using luminescence. Data shown are one experiment representative of three independent experiments, and n = 3 per group for each experiment. *P <0.05, as compared with the uninfected ( A and B ) or indicated groups ( C–G ). ns, no significant difference.

Article Snippet: M. pneumoniae (ATCC 29432) was cultured in mycoplasma broth supplemented with 20% supplement as described previously ( 32 ).

Techniques: Infection, Concentration Assay

Journal: Infection and Immunity

Article Title: Aerobic glycolysis of bronchial epithelial cells rewires Mycoplasma pneumoniae pneumonia and promotes bacterial elimination

doi: 10.1128/iai.00248-23

Figure Lengend Snippet: eATP enhances the pro-inflammatory effect of PBMCs via the P2 X7 receptor. PBMCs were infected with M. pneumoniae (MOI = 100) for 4 h and stimulated with 1:1 diluted CM from infected bronchial epithelial cells for 24 h , and the concentrations of IL-1β (A) and IL-18 (B) were measured using enzyme-linked immunosorbent assay (ELISA). ( C and D ) PBMCs were infected with M. pneumoniae for 4 h and treated with the P2 X7 receptor inhibitor A740003 (10 µM ) or oxATP (300 µM ) before CM stimulation, and the concentrations of cytokines were detected using ELISA. (E) HBE cells were infected with M. pneumoniae at an MOI of 100 for 24 h , and the expression of P2X7 mRNA was detected using quantitative PCR. (F) PBMCs were infected with M. pneumoniae for 4 h and subsequently co-incubated with CM collected from HBE cells pre-treated with 2-DG before M. pneumoniae infection. IL-1β and IL-18 levels in the supernatant were measured 24 h later using ELISA. Data are derived from the results of one experiment representative of three independent experiments performed with n = 3 for each group. *P <0.05 as compared with the indicated groups ( A–D and F ) or uninfected group (E). CTRL, control.

Article Snippet: M. pneumoniae (ATCC 29432) was cultured in mycoplasma broth supplemented with 20% supplement as described previously ( 32 ).

Techniques: Infection, Enzyme-linked Immunosorbent Assay, Expressing, Real-time Polymerase Chain Reaction, Incubation, Derivative Assay, Control

Journal: Infection and Immunity

Article Title: Aerobic glycolysis of bronchial epithelial cells rewires Mycoplasma pneumoniae pneumonia and promotes bacterial elimination

doi: 10.1128/iai.00248-23

Figure Lengend Snippet: Histological assessment and pathogen burden after eATP treatment. C57BL/6 mice were intratracheally infected with 107 M. pneumoniae for 2 h. Thereafter, 40 µL of CM (or 200-µmol ATP) was nasally instilled (once daily for 3 days) or mice were intraperitoneally administered oxATP (6mg/kg) (30 min prior to CM administration). The mice were euthanized, and their lung tissues were subsequently analyzed using histological techniques (A); the BALF was examined for IL-1β and IL-18 concentrations (B).(C and D) M. pneumoniae burden in lung and CFUs in BALF were quantified using quantitative PCR (qPCR) for the Mp P1-adhesin gene relative to GAPDH and cultured on solid pleuropneumonia-like organism (PPLO) agar plates, respectively. (E and F) Mice were nasally instilled 107 M. pneumoniae plus 0.25-µmol 2-DG. The tissue was obtained for qPCR or cultured on PPLO agar 72 h after infection. Data are derived from the results of one experiment representative of three independent experiments performed with n = 4–6 mice per group. *P < 0.05, as compared with indicated groups.

Article Snippet: M. pneumoniae (ATCC 29432) was cultured in mycoplasma broth supplemented with 20% supplement as described previously ( 32 ).

Techniques: Infection, Real-time Polymerase Chain Reaction, Cell Culture, Derivative Assay

Journal: Experimental & molecular medicine

Article Title: Oncogenic KRAS mutation confers chemoresistance by upregulating SIRT1 in non-small cell lung cancer.

doi: 10.1038/s12276-023-01091-0

Figure Lengend Snippet: Fig. 5 KRASMut lysine 104 is acetylated by the p300 acetyltransferase. A HEK293T cells were transfected with KRASWT, Flag-E.V., and Flag- p300 plasmids. Cell lysates were immunoprecipitated with IgG and an anti-KRAS antibody and then immunoblotted with anti-acetyl-lysine, anti-Flag, and anti-KRAS antibodies. B, C Myc-His-KRASG12C, Flag-SBP-E.V., Flag-SBP-p300, siCon, and/or sip300 were overexpressed in HEK293T cells; after 48 h, cell lysates were separated into nuclear and cytoplasmic fractions, which were subjected to immunoprecipitation with an anti-KRAS antibody and immunoblotting with anti-acetylated lysine and anti-KRAS antibodies. TATA-binding protein was used as a nuclear marker, and tubulin was used as a cytoplasmic marker to verify that nuclear separation was successful. D HEK293T cells were transfected with GFP-E.V., GFP-KRAS (all lysine residues between a.a. 101 and 147 except for K104 mutated to arginine), Flag-SBP-E.V., Flag-SBP- p300, siCon, and/or sip300. Protein lysates were immunoprecipitated with an anti-GFP antibody and then immunoblotted with anti-acetylated lysine, anti-KRAS, and anti-GFP antibodies. E The recombinant KRASG12C protein and the p300 C.D. (catalytic domain, aa 1284–1674, 45.1 kDa) were incubated in the acetylation assay reaction mixture at 32 °C for 4 h, and lysine-acetylated peptides were then identified using an anti- acetyl-lysine antibody. F Lysine-acetylated KRASG12C peptides were also incubated with recombinant SIRT1 protein in the deacetylation assay reaction mixture for 4 h. Deacetylation was quantified using an anti-acetyl-lysine antibody.

Article Snippet: Small interfering RNAs (siRNAs), including siKRAS (sc35731 and sc-43874), siSIRT1 (sc-40986), siSIRT2 (sc-40988), siHDAC6 (sc35544), sip300 (sc-29431), sic-Myc (sc-29226), siADAM17 (sc-36604), and shSIRT1 (sc-40986), and antibodies, including antibodies specific for SIRT1 (sc-15454), SIRT2 (sc-20966), KRAS (F234, sc-30), Tubulin (sc-32293), PARP (sc-8007), cleaved PARP (sc-56196), and p300 (sc-32244), were purchased from Santa Cruz Biotechnology Inc. (Dallas, TX, USA).

Techniques: Transfection, Immunoprecipitation, Western Blot, Binding Assay, Marker, Recombinant, Incubation, Acetylation Assay