Journal: Cell reports

Article Title: Heat shock factor 1 (HSF1) specifically potentiates c-MYC-mediated transcription independently of the canonical heat shock response

doi: 10.1016/j.celrep.2023.112557

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: NEB ® 5-α Competent E. coli (High Efficiency) , New England Biolabs , Cat# C2987.

Techniques: Control, In Situ, Virus, Recombinant, Protease Inhibitor, Transfection, SYBR Green Assay, Luciferase, Chromatin Immunoprecipitation, Quantitation Assay, Western Blot, Microscopy, Mutagenesis, shRNA, Plasmid Preparation, Software, Expressing, Magnetic Beads, Blocking Assay, Membrane

Journal: Life Science Alliance

Article Title: Divergent roles of RIPK3 and MLKL in high-fat diet–induced obesity and MAFLD in mice

doi: 10.26508/lsa.202302446

Figure Lengend Snippet: (A, B, C, D) WT, Mlkl −/− , Ripk3 −/− and Ripk3 −/− Casp8 −/− BMDMs were primed with or without LPS (50 ng/ml) for 3 h and treated with 600 μM PA-BSA or BSA alone, as indicated, for 18–20 h. (A) Cell lysates and supernatants were analyzed by immunoblot for specified proteins. The results shown are representative of two independent biological experiments. (B) IL-1β and (C) TNF levels were measured in cell supernatants by ELISA. Data shown are the mean ± SEM of n = 3 biological replicates and are representative of at least four independent experiments. One-way ANOVA followed by Tukey’s multiple comparison test, **** P < 0.0001. (D) Cell viability was assessed by PI uptake and flow cytometric analysis. Data shown are the mean ± SEM of n = 3 biological replicates and are representative of at least four independent experiments. One-way ANOVA followed by Tukey’s multiple comparison test, **** P < 0.0001. Source data are available for this figure.

Article Snippet: RIPK3 expression was then induced with 100 ng/ml DOX for at least 3 h, and cells were treated, as indicated in the figure legends, with recombinant human TNF (100 ng/ml; R&D) and birinapant (1 μM; kindly provided by TetraLogic Pharmaceuticals) alone or in combination with Q-VD-OPh (40 μM; MedChemExpress) for 12–16 h. RIPK3 induction efficiency was confirmed by immunoblot analysis, as detailed below, and functional effects on cell death were measured by flow cytometric analysis of PI (2 μg/ml) uptake on an LSRFortessa instrument using FACSDiva software.

Techniques: Western Blot, Enzyme-linked Immunosorbent Assay, Comparison

Journal: Life Science Alliance

Article Title: Divergent roles of RIPK3 and MLKL in high-fat diet–induced obesity and MAFLD in mice

doi: 10.26508/lsa.202302446

Figure Lengend Snippet: (A) WT, Mlkl −/− , Ripk3 −/− , and Ripk3 −/− Casp8 −/− BMDMs were primed with or without with LPS (50 ng/ml) for 3 h and treated with 600 μM PA-BSA or BSA alone, as indicated, for 18–20 h. Cell lysates were subjected to immunoblot for RIPK3, caspase-8, and MLKL. Results shown are representative of one experiment. (B, C) WT, Mlkl −/− , Ripk3 −/− , and Ripk3 −/− Casp8 −/− BMDMs were primed with LPS (50 ng/ml) for 3 h and treated with nigericin (10 μM) for ∼45 min (B) IL-1β levels were measured in cell supernatants by ELISA. (C) Cell viability was assessed by PI incorporation and flow cytometric analysis. Data shown are the mean ± SEM of n = 3 biological replicates and are representative of at least four independent experiments. One-way ANOVA followed by Tukey’s multiple comparison test, ** P < 0.01. (D, E, F, G) WT, Mlkl −/− , Ripk3 −/− , and Ripk3 −/− Casp8 −/− BMDMs were primed with or without Pam3Cys (500 ng/ml) for 3 h and treated with 600 μM PA-BSA or BSA alone, as indicated, for 18–20 h. (D) IL-1β and (F) TNF levels were measured in cell supernatants by ELISA. Data are the mean ± SEM of n = 3 biological replicates and are representative of at least four independent experiments. One-way ANOVA followed by Tukey’s multiple comparison test, * P < 0.05, *** P < 0.001, **** P < 0.0001. (E) Cell lysates and supernatants were analyzed by immunoblot for specified proteins. The results shown are representative of two independent biological experiments. (G) Cell viability was assessed by PI uptake and flow cytometric analysis. Data shown are the mean ± SEM of n = 3 biological replicates and are representative of at least three independent experiments. One-way ANOVA followed by Tukey’s multiple comparison test, **** P < 0.0001.

Article Snippet: RIPK3 expression was then induced with 100 ng/ml DOX for at least 3 h, and cells were treated, as indicated in the figure legends, with recombinant human TNF (100 ng/ml; R&D) and birinapant (1 μM; kindly provided by TetraLogic Pharmaceuticals) alone or in combination with Q-VD-OPh (40 μM; MedChemExpress) for 12–16 h. RIPK3 induction efficiency was confirmed by immunoblot analysis, as detailed below, and functional effects on cell death were measured by flow cytometric analysis of PI (2 μg/ml) uptake on an LSRFortessa instrument using FACSDiva software.

Techniques: Western Blot, Enzyme-linked Immunosorbent Assay, Comparison

Journal: Life Science Alliance

Article Title: Divergent roles of RIPK3 and MLKL in high-fat diet–induced obesity and MAFLD in mice

doi: 10.26508/lsa.202302446

Figure Lengend Snippet: (A, B) Body weights were measured weekly in Casp8 lox/lox control (WT), Casp8 lox/lox Ripk3 −/− , and Casp8 LysMcre Ripk3 −/− mice fed a (A) normal chow diet (ND) or (B) high-fat diet (HFD) for ∼25 wk. Data shown are the mean ± SEM, n ≥ 11 ND-fed mice per group and n ≥ 13 HFD-fed mice per group pooled from three independent experiments. One-way ANOVA of the AUC, ** P < 0.01. (C) End-stage organ weights in HFD-fed Casp8 lox/lox control, Casp8 lox/lox Ripk3 −/− , and Casp8 LysMcre Ripk3 −/− mice. Data shown are the mean ± SEM, n ≥ 11 mice per group pooled from three independent experiments. One-way ANOVA followed by Tukey’s multiple comparison test, * P < 0.05. Gray boxes in (C) show the mean ± SEM from Casp8 lox/lox , Casp8 lox/lox Ripk3 −/− , and Casp8 LysMcre Ripk3 −/− ND-fed mice for comparisons. (D, E) Glucose tolerance was measured in (D) ND- and (E) HFD-challenged Casp8 lox/lox control, Casp8 lox/lox Ripk3 −/− , and Casp8 LysMcre Ripk3 −/− mice by intraperitoneal glucose tolerance tests (IP-GTT; 1.5 g/kg) at 8 wk. Data shown are the mean ± SEM, n = 3–6 mice per group representative of 2–3 independent experiments. One-way ANOVA of the AUC, * P < 0.05. (F) Representative microscopy images of F4/80-immunostained VAT sections from ND- and HFD-fed Casp8 lox/lox control, Casp8 lox/lox Ripk3 −/− , and Casp8 LysMcre Ripk3 −/− mice at 25 wk. Arrows point to macrophage crown-like structures. The scale bar is 200 μm. (G) Mean adipocyte size in HFD-fed Casp8 lox/lox control, Casp8 lox/lox Ripk3 −/− , and Casp8 LysMcre Ripk3 −/− VAT was quantified using an automated algorithm on H&E-stained sections . Data are the mean ± SEM, n ≥ 11 mice per group pooled from three independent experiments. One-way ANOVA followed by Tukey’s multiple comparison test. (H) Numbers of neutrophils, inflammatory monocytes, and macrophages were quantified in the VAT of HFD-fed Casp8 lox/lox control, Casp8 lox/lox Ripk3 −/− , and Casp8 LysMcre Ripk3 −/− mice by flow cytometric analysis. Data shown are the mean ± SEM, n = 7–8 mice per group pooled from two independent experiments. One-way ANOVA followed by Tukey’s multiple comparison test, * P < 0.05, ** P < 0.01, *** P < 0.001. (I) VAT from HFD-fed Casp8 lox/lox control, Casp8 lox/lox Ripk3 −/− , and Casp8 LysMcre Ripk3 −/− mice was cultured ex vivo with and without LPS (50 ng/ml) overnight, and IL-1β and TNF were measured in the supernatants by ELISA. Data shown are the mean ± SEM, n ≥ 12 mice per group pooled from three independent experiments. One-way ANOVA followed by Dunnett’s multiple comparison test, * P < 0.05, ** P < 0.01. (J) Representative microscopy images of H&E-stained liver sections. The scale bar is 100 μm. (K) Histopathological evaluation of MAFLD in Casp8 lox/lox control, Casp8 lox/lox Ripk3 −/− , and Casp8 LysMcre Ripk3 −/− ND-fed and HFD-fed mice after 25 wk of challenge. Data shown are the mean ± SEM, n ≥ 9 mice per group pooled from three independent experiments. One-way ANOVA followed by Tukey’s multiple comparison test, * P < 0.05, ** P < 0.01. Source data are available for this figure.

Article Snippet: RIPK3 expression was then induced with 100 ng/ml DOX for at least 3 h, and cells were treated, as indicated in the figure legends, with recombinant human TNF (100 ng/ml; R&D) and birinapant (1 μM; kindly provided by TetraLogic Pharmaceuticals) alone or in combination with Q-VD-OPh (40 μM; MedChemExpress) for 12–16 h. RIPK3 induction efficiency was confirmed by immunoblot analysis, as detailed below, and functional effects on cell death were measured by flow cytometric analysis of PI (2 μg/ml) uptake on an LSRFortessa instrument using FACSDiva software.

Techniques: Control, Comparison, Microscopy, Staining, Cell Culture, Ex Vivo, Enzyme-linked Immunosorbent Assay

Journal: Life Science Alliance

Article Title: Divergent roles of RIPK3 and MLKL in high-fat diet–induced obesity and MAFLD in mice

doi: 10.26508/lsa.202302446

Figure Lengend Snippet: (A, B) % Weight gain over time in Casp8 lox/lox control (WT), Casp8 lox/lox Ripk3 −/− , and Casp8 LysMcre Ripk3 −/− mice fed a (A) normal chow diet (ND) or a (B) high-fat diet (HFD). Data shown are the mean ± SEM, n ≥ 11 mice per group pooled from three independent experiments. One-way ANOVA of the AUC, * P < 0.05, ** P < 0.01, *** P < 0.001. (C) ND and HFD food intake and output per mouse per day were calculated from weekly measurements in Casp8 lox/lox control, Casp8 lox/lox Ripk3 −/− , and Casp8 LysMcre Ripk3 −/− mice. Data shown are the mean + SD, n ≥ 8 mice per group from two pooled independent experiments, one-way ANOVA. (D, E, F) End-stage (25 wk of diet) organ weights and (G) organ weights expressed as a % of total body weight in Casp8 lox/lox control, Casp8 lox/lox Ripk3 −/− , and Casp8 LysMcre Ripk3 −/− mice. Data shown are the mean ± SEM, n ≥ 11 mice/group pooled from three independent experiments. One-way ANOVA followed by Tukey’s multiple comparison test, * P < 0.05, ** P < 0.01. Gray boxes in (F) show the mean ± SEM from Casp8 lox/lox control, Casp8 lox/lox Ripk3 −/− , and Casp8 LysMcre Ripk3 −/− ND-fed mice (E). (H) Weight gain and (I) end-stage organ weights were measured in Casp8 lox/lox control and Casp8 LysMcre mice fed a HFD for 26 wk. Data shown are the mean ± SEM, n = 3–4 mice from one experiment.

Article Snippet: RIPK3 expression was then induced with 100 ng/ml DOX for at least 3 h, and cells were treated, as indicated in the figure legends, with recombinant human TNF (100 ng/ml; R&D) and birinapant (1 μM; kindly provided by TetraLogic Pharmaceuticals) alone or in combination with Q-VD-OPh (40 μM; MedChemExpress) for 12–16 h. RIPK3 induction efficiency was confirmed by immunoblot analysis, as detailed below, and functional effects on cell death were measured by flow cytometric analysis of PI (2 μg/ml) uptake on an LSRFortessa instrument using FACSDiva software.

Techniques: Control, Comparison

Journal: Life Science Alliance

Article Title: Divergent roles of RIPK3 and MLKL in high-fat diet–induced obesity and MAFLD in mice

doi: 10.26508/lsa.202302446

Figure Lengend Snippet: (A, B, C) Intraperitoneal glucose tolerance (GTT, 1.5 g/kg) and (D) insulin tolerance tests (ITT, 0.75 U/kg) were performed, as indicated, in ND- and HFD-fed Casp8 lox/lox control, Casp8 lox/lox Ripk3 −/− , and Casp8 LysMcre Ripk3 −/− mice, and blood glucose levels were monitored over time. Data shown are the mean ± SEM, n = 5–6 mice per group and are representative of one of two to three experiments. For ITT, blood glucose changes are normalized to baseline measurements. One-way ANOVA of the AUC, * P < 0.05. (E, F, G, H, I, J) Fasting serum, (E) glucose, (F) insulin, (G) triglyceride, (H) cholesterol, (I) ALT, and (J) AST levels in Casp8 lox/lox control, Casp8 lox/lox Ripk3 −/− , and Casp8 LysMcre Ripk3 −/− ND- and HFD-fed mice after 16–18 (diamonds) or 25 (triangles/circles) wk. Data shown are the mean ± SEM, n ≥ 9 mice per group pooled from at least three experiments. One-way ANOVA followed by Tukey’s multiple comparison test, * P < 0.05, ** P < 0.01. The dotted line shows the mean values of control Casp8 lox/lox ND-fed mice extrapolated for comparison.

Article Snippet: RIPK3 expression was then induced with 100 ng/ml DOX for at least 3 h, and cells were treated, as indicated in the figure legends, with recombinant human TNF (100 ng/ml; R&D) and birinapant (1 μM; kindly provided by TetraLogic Pharmaceuticals) alone or in combination with Q-VD-OPh (40 μM; MedChemExpress) for 12–16 h. RIPK3 induction efficiency was confirmed by immunoblot analysis, as detailed below, and functional effects on cell death were measured by flow cytometric analysis of PI (2 μg/ml) uptake on an LSRFortessa instrument using FACSDiva software.

Techniques: Control, Comparison

Journal: Life Science Alliance

Article Title: Divergent roles of RIPK3 and MLKL in high-fat diet–induced obesity and MAFLD in mice

doi: 10.26508/lsa.202302446

Figure Lengend Snippet: (A) Representative microscopy images of VAT sections from Casp8 lox/lox control, Casp8 lox/lox Ripk3 −/− , and Casp8 LysMcre Ripk3 −/− mice fed a ND or HFD for 25 wk stained with H&E (top row; the scale bar is 200 μm) or cleaved caspase-3 immunohistochemistry (bottom row; the scale bar is 100 μm). (B) Mean adipocyte size as quantified on VAT H&E sections from Casp8 lox/lox control, Casp8 lox/lox Ripk3 −/− , and Casp8 LysMcre Ripk3 −/− mice fed a ND. Data shown are the mean ± SEM, n = 6–12 mice pooled from three independent experiments, one-way ANOVA. (C) Flow cytometric gating strategy for analyzing live CD45 + PI − myeloid populations in the VAT of HFD mice after 25 wk. (D) Representative microscopy images of Sirius Red–stained liver tissue sections showing fibrosis in ND- and HFD-fed Casp8 lox/lox , Casp8 lox/lox Ripk3 −/− , and Casp8 LysMcre Ripk3 −/− mice. The scale bar is 200 μm. (E) Flow cytometric gating strategy for analyzing live CD45 + PI − myeloid populations in the liver of mice fed a HFD for 25 wk. (F, G) Flow cytometric analysis showing the (F) % of CD45 + leukocytes and (G) proportion of neutrophils and monocyte/macrophages in the livers of Casp8 lox/lox control, Casp8 lox/lox Ripk3 −/− , and Casp8 LysMcre Ripk3 −/− HFD mice. Data shown are the mean ± SEM, n = 6–8 mice per group pooled from two independent experiments. One-way ANOVA followed by Tukey’s multiple comparison test. (H) qRT–PCR measurement of the relative expression of Casp8 , Tnf , Nlrp3 , Casp1 , and Il1b mRNA in the livers of Casp8 lox/lox , Casp8 lox/lox Ripk3 −/− , and Casp8 LysMcre Ripk3 −/− HFD mice after 25 wk of diet (fold change over Casp8 lox/lox ND). Data shown are the mean ± SEM, n ≥ 5 mice per group pooled from three experiments. One-way ANOVA followed by Tukey’s multiple comparison test, * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: RIPK3 expression was then induced with 100 ng/ml DOX for at least 3 h, and cells were treated, as indicated in the figure legends, with recombinant human TNF (100 ng/ml; R&D) and birinapant (1 μM; kindly provided by TetraLogic Pharmaceuticals) alone or in combination with Q-VD-OPh (40 μM; MedChemExpress) for 12–16 h. RIPK3 induction efficiency was confirmed by immunoblot analysis, as detailed below, and functional effects on cell death were measured by flow cytometric analysis of PI (2 μg/ml) uptake on an LSRFortessa instrument using FACSDiva software.

Techniques: Microscopy, Control, Staining, Immunohistochemistry, Comparison, Quantitative RT-PCR, Expressing

Journal: Life Science Alliance

Article Title: Divergent roles of RIPK3 and MLKL in high-fat diet–induced obesity and MAFLD in mice

doi: 10.26508/lsa.202302446

Figure Lengend Snippet: (A) Liver lysates from WT and Mlkl −/− mice fed a ND and HFD for ∼25 wk were subjected to immunoblot for the indicated proteins. n = 3 mice per genotype and diet; each lane represents an individual mouse. Relevant protein levels were analyzed by densitometry and normalized to GAPDH and expressed as a fold change over WT ND liver lysates. Results are presented as the mean ± SEM. One-way ANOVA followed by Tukey’s multiple comparison test, ** P < 0.01, **** P < 0.0001. (B) Representative microscopy images of RIPK3 immunostaining in liver and VAT sections from WT and Mlkl −/− ND- and HFD-fed mice. n ≥ 13 mice per group pooled from two to three experiments. The scale bar is 100 μm.

Article Snippet: RIPK3 expression was then induced with 100 ng/ml DOX for at least 3 h, and cells were treated, as indicated in the figure legends, with recombinant human TNF (100 ng/ml; R&D) and birinapant (1 μM; kindly provided by TetraLogic Pharmaceuticals) alone or in combination with Q-VD-OPh (40 μM; MedChemExpress) for 12–16 h. RIPK3 induction efficiency was confirmed by immunoblot analysis, as detailed below, and functional effects on cell death were measured by flow cytometric analysis of PI (2 μg/ml) uptake on an LSRFortessa instrument using FACSDiva software.

Techniques: Western Blot, Comparison, Microscopy, Immunostaining

Journal: Life Science Alliance

Article Title: Divergent roles of RIPK3 and MLKL in high-fat diet–induced obesity and MAFLD in mice

doi: 10.26508/lsa.202302446

Figure Lengend Snippet: (A, B) WT and Mlkl −/− Hepa1-6 cells (Clone #1 and #3) were treated with doxycycline (DOX, 100 ng/ml) for 3 h, as indicated, to induce RIPK3 expression and then treated with TNF (T, 100 ng/ml), Smac-mimetic 711 (S, 1 μM), and pan-apoptotic caspase inhibitor Q-VD-OPh (Q, 40 μM) for 12–16 h. (A) Immunoblots were performed on cell lysates for relevant proteins. The results are representative of 2 independent experiments. < Reprobe of MLKL membrane, * nonspecific bands. (B) Cell death was measured via flow cytometric analysis of PI uptake. Data shown are the mean ± SD, n = 3 replicates from one of three experiments. One-way ANOVA followed by Tukey’s multiple comparison test, **** P < 0.0001. (C, D, E) WT and Mlkl −/− Hepa1-6 cells were treated with DOX (100 ng/ml) for 3 h to induce RIPK3, as indicated, and media were replaced. Cells were then stimulated with 100–300 μM palmitate conjugated to BSA (BSA-PA) or BSA (equivalent to highest BSA-PA) for 16–24 h. (C) Cell lysates were subjected to immunoblot for relevant proteins. Representative of two independent experiments. < Reprobe of MLKL membrane. (D) Cell death was measured via flow cytometric analysis of PI uptake. Data shown are the mean ± SD, n = 3 replicates from one of three experiments. One-way ANOVA followed by Tukey’s multiple comparison test, ** P < 0.01, **** P < 0.0001. (E) Lipid accumulation was measured by BODIPY staining and flow cytometric analysis. Data are presented as the mean ± SD, n = 4 technical replicates pooled from two independent experiments. One-way ANOVA followed by Tukey’s multiple comparison test, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. (F, G, H, I, J, K, L) WT and Mlkl −/− Hepa1-6 cells were treated with BSA and BSA-PA (200 μM) for 16 h, and relative levels of lipid metabolism–related genes were measured by qRT–PCR. Data shown are the mean ± SD, n = 4 replicates representative of one of two independent experiments. One-way ANOVA followed by Tukey’s multiple comparison test, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: RIPK3 expression was then induced with 100 ng/ml DOX for at least 3 h, and cells were treated, as indicated in the figure legends, with recombinant human TNF (100 ng/ml; R&D) and birinapant (1 μM; kindly provided by TetraLogic Pharmaceuticals) alone or in combination with Q-VD-OPh (40 μM; MedChemExpress) for 12–16 h. RIPK3 induction efficiency was confirmed by immunoblot analysis, as detailed below, and functional effects on cell death were measured by flow cytometric analysis of PI (2 μg/ml) uptake on an LSRFortessa instrument using FACSDiva software.

Techniques: Expressing, Western Blot, Membrane, Comparison, Staining, Quantitative RT-PCR

Journal: Cell Host & Microbe

Article Title: Lassa virus glycoprotein nanoparticles elicit neutralizing antibody responses and protection

doi: 10.1016/j.chom.2022.10.018

Figure Lengend Snippet:

Article Snippet: NEB 5-alpha Competent E. coli (High Efficiency) , New England Biolabs , Cat# C2987H.

Techniques: Virus, Recombinant, Mass Spectrometry, Sequencing, Lysis, Emulsion, Adjuvant, Plasmid Preparation, Molecular Weight, Marker, Electron Microscopy, Luciferase, Software, Chromatography, Random Hexamer Labeling, Luminex, Expressing, Modification, Nano Differential Scanning Fluorimetry

Journal: The EMBO Journal

Article Title: PUFFFIN: an ultra-bright, customisable, single-plasmid system for labelling cell neighbourhoods

doi: 10.1038/s44318-024-00154-w

Figure Lengend Snippet: Reagents and tools table

Article Snippet: NEB® 10-beta Competent E. coli (High efficiency) , New England Biolabs , #C3019I.

Techniques: Subcloning, Recombinant, Plasmid Preparation, Modification, Saline, Produced, Transfection, Cell Culture, SYBR Green Assay, Staining, PCR Cloning, Software, High Content Screening, Microscopy

Journal: iScience

Article Title: Host ZCCHC3 blocks HIV-1 infection and production through a dual mechanism

doi: 10.1016/j.isci.2024.109107

Figure Lengend Snippet:

Article Snippet: NEB 5-alpha F′Iq Competent E. coli (High Efficiency) , NEB, Ipswich , C2992H.

Techniques: Virus, Plasmid Preparation, Clone Assay, Construct, Luciferase, Recombinant, Modification, Transfection, Western Blot, Protease Inhibitor, Staining, DNA Ligation, Cell Isolation, Activation Assay, Stripping Membranes, Cell Viability Assay, In Vitro, Membrane, Isolation, Software, Laser-Scanning Microscopy, Flow Cytometry, Cytometry, Real-time Polymerase Chain Reaction, Mass Spectrometry

Journal: Molecular Systems Biology

Article Title: Oncogenic PIK3CA corrupts growth factor signaling specificity

doi: 10.1038/s44320-024-00078-x

Figure Lengend Snippet: Reagents and tools table

Article Snippet: Next, 2 µl of the ligation reaction were heat-shock transformed into high-efficiency 5-alpha competent E. coli (NEB #C2987I), followed by conventional colony picking and bacterial culture expansion for subsequent plasmid DNA extraction with the Maxi Plus kit from Qiagen (#12964).

Techniques: Knock-Out, Recombinant, Western Blot, Mass Cytometry, Sequencing, Clone Assay, Mutagenesis, Cell Culture, Membrane, Transfection, Lysis, Extraction, Protease Inhibitor, Sterility, Gel Extraction, Reverse Transcription, SYBR Green Assay, Blocking Assay, Staining, Software, Cytometry, Real-time Polymerase Chain Reaction, Microscopy

Journal: iScience

Article Title: Spns1-dependent endocardial lysosomal function drives valve morphogenesis through Notch1-signaling

doi: 10.1016/j.isci.2024.111406

Figure Lengend Snippet:

Article Snippet: For gRNA selection, cutting efficiency was determined by T7-endonuclease assay (T7 Endonuclease I, New England Biolabs, M0302) according to the manufacturer’s protocol.

Techniques: Virus, Recombinant, Electron Microscopy, Isolation, CRISPR, Reverse Transcription, Plasmid Preparation, Saline, SYBR Green Assay, Transgenic Assay, Software

Journal: Scientific Reports

Article Title: Genetic variation, structural analysis, and virulence implications of BimA and BimC in clinical isolates of Burkholderia pseudomallei in Thailand

doi: 10.1038/s41598-024-74922-3

Figure Lengend Snippet: Plaque-forming efficiencies of representative strains harboring different BimA Bp types in A549 cells. ( a ) Photographic representation of plaques. ( b ) Plaque-forming efficiencies of B. pseudomallei isolates harboring the different BimA Bp types in A549 cells. The cells were infected with B. pseudomallei strains representative of BimA Bp type 1 ( B. pseudomallei K96243, classified as type 1 was used as the reference strain), type 2 (DR10025A, DR20021A and DR40130A), type 4 (DR40111A, DR80025A and DR90085A), type 6 (DR10008A, DR40025A and DR50053A), type 9 (DR50173A, DR70003A and DR90006A), and type 10 (DR20062A, DR50003A and DR60054A) at MOI of 0.1:1. Plaques were stained with 2% (w/v) crystal violet at 24 h post-infection. Plaque-forming efficiency (PFU/ml) was counted as the number of plaques (plaque-forming units: PFU) divided by the CFU (colony-forming units) of bacteria added per well (CFU/ml). Error bars represent means ± standard deviation of data from three independent experiments (one-way ANOVA; P < 0.05).

Article Snippet: The plaque-forming efficiency was evaluated using A549 epithelial lung cells (CCL-185, American Type Culture Collection, MD, USA) as described previously with some modifications for 16 representative strains of the BimA Bp type 1 ( B. pseudomallei K96243), type 2 (DR10025A, DR20021A and DR40130A), type 4 (DR40111A, DR80025A and DR90085A), type 6 (DR10008A, DR40025A and DR50053A), type 9 (DR50173A, DR70003A and DR90006A) and type 10 (DR20062A, DR50003A and DR60054A).

Techniques: Infection, Staining, Bacteria, Standard Deviation

Journal: Scientific Reports

Article Title: Genetic variation, structural analysis, and virulence implications of BimA and BimC in clinical isolates of Burkholderia pseudomallei in Thailand

doi: 10.1038/s41598-024-74922-3

Figure Lengend Snippet: Confocal microscopy images of A549 cells infected with B. pseudomallei isolates with different BimA Bp types. The representative strains of B. pseudomallei ( a ) BimA Bp type 1 ( B. pseudomallei K96243, classified as type 1 was used as the reference strain), ( b ) BimA Bp type 2 (DR40130A), ( c ) BimA Bp type 4 (DR40111A), ( d ) BimA Bp type 6 (DR10008A), ( e ) BimA Bp type 9 (DR50173A), and ( f ) BimA Bp type 10 (DR50003A) were used to infect A549 cells ( g ) at MOI of 30:1. Immunofluorescence staining was performed at 8 h post-infection using 4B11 monoclonal antibody specific to B. pseudomallei capsular polysaccharide to visualize the bacteria in green; phalloidin for F-actin in red; and Hoechst 33258 for the host DNA in blue. ( h ) The length of actin tails (white arrow) was determined using Zen Zeiss 3.0 SR (black) software tools. The mean lengths were: 3.1605 μm, BimA Bp type 1; 1.988 μm, BimA Bp type 2; 2.043 μm, BimA Bp type 4; 1.807 μm, BimA Bp type 6; 1.465 μm, BimA Bp type 9; and 1.603 μm, BimA Bp type 10 (one-way ANOVA; P < 0.05). Scale bar, 10 μm.

Article Snippet: The plaque-forming efficiency was evaluated using A549 epithelial lung cells (CCL-185, American Type Culture Collection, MD, USA) as described previously with some modifications for 16 representative strains of the BimA Bp type 1 ( B. pseudomallei K96243), type 2 (DR10025A, DR20021A and DR40130A), type 4 (DR40111A, DR80025A and DR90085A), type 6 (DR10008A, DR40025A and DR50053A), type 9 (DR50173A, DR70003A and DR90006A) and type 10 (DR20062A, DR50003A and DR60054A).

Techniques: Confocal Microscopy, Infection, Immunofluorescence, Staining, Bacteria, Software