Journal:

Article Title: Activation of Protein Tyrosine Kinases by Coxiella burnetii : Role in Actin Cytoskeleton Reorganization and Bacterial Phagocytosis

doi: 10.1128/IAI.69.4.2520-2526.2001

Figure Lengend Snippet: Tyrosine phosphorylations stimulated by C. burnetii. THP-1 cells were incubated with virulent (A) or avirulent C. burnetii (B) at a bacterium-to-cell ratio of 200:1 for different periods at 37°C. Tyrosine phosphorylations were revealed by using antiphosphotyrosine MAb, peroxidase-conjugated secondary Ab, and an enhanced chemiluminescence kit. The molecular masses of phosphoproteins were determined using standards of known molecular mass (right margin, in kilodaltons). Arrowheads at the left indicate positions of phosphorylated proteins. Panels A and B are representative of five distinct experiments. (C) Tyrosine phosphorylation levels of 55-, 66-, and 120-kDa proteins were assessed by densitometric scanning. Results are expressed as relative increase over the unstimulated value and are the mean ± SE of five distinct experiments.

Article Snippet: THP-1 cells (4 × 10 6 per assay) were stimulated with C. burnetii for different periods, and cell homogenates were incubated overnight with 1 μg of rabbit affinity-purified antibody (Ab) (Santa Cruz Biotechnology) directed against Hck, Lyn, or Fgr in lysis buffer.

Techniques: Incubation

Journal:

Article Title: Activation of Protein Tyrosine Kinases by Coxiella burnetii : Role in Actin Cytoskeleton Reorganization and Bacterial Phagocytosis

doi: 10.1128/IAI.69.4.2520-2526.2001

Figure Lengend Snippet: Src-related kinases in C. burnetii-stimulated cells. THP-1 cells were stimulated with virulent or avirulent C. burnetii for different periods, homogenized, and incubated with 1 μg of anti-Hck, -Lyn, or -Fgr MAb. Proteins were immunoprecipitated (IP), electrophoresed, and transferred onto nitrocellulose sheets. Immunoblotting was performed with antiphosphotyrosine MAb (PY). Membranes were stripped and reprobed with anti-Hck, -Lyn, and -Fgr MAbs, respectively. Each blot is representative of three distinct experiments.

Article Snippet: THP-1 cells (4 × 10 6 per assay) were stimulated with C. burnetii for different periods, and cell homogenates were incubated overnight with 1 μg of rabbit affinity-purified antibody (Ab) (Santa Cruz Biotechnology) directed against Hck, Lyn, or Fgr in lysis buffer.

Techniques: Incubation, Immunoprecipitation, Western Blot

Journal:

Article Title: Activation of Protein Tyrosine Kinases by Coxiella burnetii : Role in Actin Cytoskeleton Reorganization and Bacterial Phagocytosis

doi: 10.1128/IAI.69.4.2520-2526.2001

Figure Lengend Snippet: Colocalization of tyrosine phosphoproteins and F-actin. THP-1 cells were stimulated with virulent (A) or avirulent (B) C. burnetii for the times (minutes) indicated. Tyrosine phosphoproteins and F-actin were labeled with antiphosphotyrosine MAb and rhodamine-conjugated F(ab′)2 anti-mouse IgG and with bodipy phallacidin, respectively. Cells were examined with a laser scanning confocal fluorescence microscope, and representative cells are shown. The colocalization of tyrosine phosphoproteins and F-actin appears in yellow.

Article Snippet: THP-1 cells (4 × 10 6 per assay) were stimulated with C. burnetii for different periods, and cell homogenates were incubated overnight with 1 μg of rabbit affinity-purified antibody (Ab) (Santa Cruz Biotechnology) directed against Hck, Lyn, or Fgr in lysis buffer.

Techniques: Labeling, Fluorescence, Microscopy

Journal:

Article Title: Activation of Protein Tyrosine Kinases by Coxiella burnetii : Role in Actin Cytoskeleton Reorganization and Bacterial Phagocytosis

doi: 10.1128/IAI.69.4.2520-2526.2001

Figure Lengend Snippet: Distribution of PTK activity in cell fractions. THP-1 cells were pretreated with cytochalasin D (1 μg ml−1) or not pretreated and stimulated by virulent (A) or avirulent (B) C. burnetii for 10 min. Cells were then lysed by 1% Triton X-100. Triton-soluble and -insoluble fractions were incubated with poly(Glu, Tyr) and 1 μCi of [γ-32P]ATP. Radioactivity was measured with a scintillation counter; cpm in Triton-soluble fraction before stimulation = 29,500 ± 5,870; cpm in Triton-insoluble fraction before stimulation = 25,660 ± 5,110. Results are expressed as the ratio of counts after stimulation to counts before stimulation and represent the mean ± SE of five distinct experiments. ∗, P < 0.05.

Article Snippet: THP-1 cells (4 × 10 6 per assay) were stimulated with C. burnetii for different periods, and cell homogenates were incubated overnight with 1 μg of rabbit affinity-purified antibody (Ab) (Santa Cruz Biotechnology) directed against Hck, Lyn, or Fgr in lysis buffer.

Techniques: Activity Assay, Incubation, Radioactivity

Journal:

Article Title: Activation of Protein Tyrosine Kinases by Coxiella burnetii : Role in Actin Cytoskeleton Reorganization and Bacterial Phagocytosis

doi: 10.1128/IAI.69.4.2520-2526.2001

Figure Lengend Snippet: Effect of PTK inhibitors on F-actin organization. (A) THP-1 cells were pretreated with 10 μM PTK inhibitors for 30 min before stimulation with virulent C. burnetii for 10 min. F-actin was labeled with bodipy phallacidin, and its reorganization was examined with fluorescence microscopy. a, control cells; b, C. burnetii-stimulated cells; c, cells preincubated with lavendustin A; d, cells preincubated with lavendustin A and stimulated by C. burnetii; e, cells preincubated with PP1; f, cells preincubated with PP1 and stimulated by C. burnetii. (B) Percentage of cells with protrusions rich in F-actin.

Article Snippet: THP-1 cells (4 × 10 6 per assay) were stimulated with C. burnetii for different periods, and cell homogenates were incubated overnight with 1 μg of rabbit affinity-purified antibody (Ab) (Santa Cruz Biotechnology) directed against Hck, Lyn, or Fgr in lysis buffer.

Techniques: Labeling, Fluorescence, Microscopy

Journal: bioRxiv

Article Title: Chloride Homeostasis Regulates cGAS-STING Signaling

doi: 10.1101/2024.04.08.588475

Figure Lengend Snippet: ( a ) Schematic diagram illustrating the mechanism for monitoring cGAS-STING pathway activation via IRF3 reporter cells. Activation of IRF3 induces the production of secreted reporter enzyme. The reporter enzyme is secreted into cell culture supernatant and IRF 3 activation can be determined by reporter activity using ( b − e ) colorimetric (OD at 655nm) or ( f − g ) luminescent methods. ( b-e ) THP1-Blue ISG cells were pretreated with ( b ) 50-300 μM NPPB, ( c ) 50-200 μM DIDS, ( d ) 50-200 μM IAA-94, ( e ) 50-200 μM FFA, and 2.5 μM H-151 (STING inhibitor) for 1 h then transfected with 2×10 −2 μg/µL HT-DNA overnight. Error bars indicate the mean ± standard error of the mean (s.e.m.) of three independent measurements. * P < 0.05; ** P < 0.01; **** P < 0.0001. One-way analysis of variance (ANOVA) followed by Dunnett’s test for multiple comparison. ns, not significant. ( f-g ) RAW-Dual cells were pretreated with 100 μM NPPB, 150 μM DIDS, and 15 μM H-151 for 1 h then transfected with 2×10 −2 μg/µL HT-DNA for ( f ) overnight, ( g )1-24 h. Error bars indicate the mean ± standard error of the mean (s.e.m.) of three independent measurements. **** P < 0.0001. One-way analysis of variance (ANOVA) followed by Dunnett’s test for multiple comparison. (h) IFN-β mRNA expression levels in THP1 cells pretreated with 100 μM NPPB,150 μM DIDS,15μM H-151 for overnight and then transfected with 2×10 −2 µg/µL HT-DNA overnight. mRNA levels were measured by the RT-qPCR. Error bars indicate the mean ± standard error of the mean (s.e.m.) of three independent measurements. *** P < 0.001; **** P < 0.0001. One-way analysis of variance (ANOVA) followed by Dunnett’s test for multiple comparison. (i) Western blotting to measure the protein expression level of p-IRF 3 , IRF 3 and β-actin in THP-1 cells that were pretreated with 100 μM NPPB,150 μM DIDS, and 15 μM H-151 overnight and then transfected with 2×10 −2 µg/µL HT-DNA overnight. Experiments were performed in three biological replicates. (j) Primary human dermal fibroblasts (HDF) were pretreated with 100 μM NPPB, 150 μM DIDS for 18 h and then transfected with 2×10 −2 µg/µL HT-DNA for 6 h. Cells were fixed using paraformaldehyde and stained with anti-STING antibodies. Experiments were performed in three biological replicates.

Article Snippet: THP1-Blue™ ISG cells were purchased from InvivoGen (San Diego, CA, USA).

Techniques: Activation Assay, Cell Culture, Activity Assay, Transfection, Comparison, Expressing, Quantitative RT-PCR, Western Blot, Staining

Journal: bioRxiv

Article Title: Chloride Homeostasis Regulates cGAS-STING Signaling

doi: 10.1101/2024.04.08.588475

Figure Lengend Snippet: (a) RAW-Dual cells were pretreated with 100 µM 2,3-cGAMP for 30 min, followed by washing with PBS, and then incubated with 100 μM NPPB and 15 μM H-151 overnight. (b) RAW-Dual cells were pretreated with 100 µM DMXAA or 100 µM 2,3-cGAMP for 30 min, followed by washing with PBS, and then incubated with 150 μM DIDS, and 15 μM H-151 overnight. (c) THP1-Blue ISG cells were pretreated with 100 μM NPPB, 200 µM IAA-94, 100 μM FFA, and 15 μM H-151 for 1 h and then stimulated with 30 µM MSA-2 overnight. (d) IFN-β mRNA expression levels were measured in THP1 cells pretreated with 100 μM NPPB, and 150 μM DIDS overnight and stimulated with 100 µM 2,3-cGAMP overnight. mRNA levels were measured using the RT-qPCR. (e) IFN-β mRNA expression levels were measured in THP1 cells pretreated with 100 μM NPPB, 150 μM DIDS and 15 μM H-151 overnight and stimulated with 30 µM MSA-2 overnight. mRNA levels were measured using the RT-qPCR. (a-e) Error bars indicate the mean ± standard error of the mean (s.e.m.) of three independent measurements. **** P < 0.0001. One-way analysis of variance (ANOVA) followed by Dunnett’s test for multiple comparison. ns, not significant. (f-g) Western blotting to measure the protein expression level of p-IRF 3 , IRF 3 and β-actin in THP-1 cells that were pretreated with ( f ) 100 μM NPPB, ( g ) 150 μM DIDS, and 15 μM H-151 overnight and then stimulated with 100 µM 2,3-cGAMP overnight. Experiments were performed in three biological replicates. (h) Immunofluorescent analysis of HDF cells pretreated with 100 μM NPPB, and 150 μM DIDS overnight and then stimulated with 30 µM MSA-2 for 6 h. (i) Percentage of cells with STING puncta correlating to ( h ). Experiments were performed in three biological replicates (>125 cells per trial). Error bars indicate the mean ± standard error of the mean (s.e.m.) of three independent measurements. **** P < 0.0001. One-way analysis of variance (ANOVA) followed by Dunnett’s test for multiple comparison.

Article Snippet: THP1-Blue™ ISG cells were purchased from InvivoGen (San Diego, CA, USA).

Techniques: Incubation, Expressing, Quantitative RT-PCR, Comparison, Western Blot

Journal: Communications Biology

Article Title: Lysoptosis is an evolutionarily conserved cell death pathway moderated by intracellular serpins

doi: 10.1038/s42003-021-02953-x

Figure Lengend Snippet: a HT3 B3-WT (blue) or HT3 B3-KO (red) cells were subjected to nucleofection (nuc, +) in the absence (−) or presence (+) of 5 ug/ml LPS-EK (LPS), caspase-1 inhibitor, VX765, or lysosomal cysteine protease inhibitor, E64. An aliquot of cells were also not nucleofected (nuc, −). After 3 h, cells were stained with SG and Hoescht 33342 and imaged. Quantification: (# Sytox positive nuclei/# of blue nuclei) × 100. Means ± SD were compared using a two-tailed t -test (* P < 0.05). b Immunoblot of gasdermin D (GSDMD) and gasdermin E (GSDME) cleavage in THP1 cells treated with α-hemolysin (HlA); positive control. HT3 B3-WT /HT3 B3-KO were nucleofected with 0, 1, or 5 µg of LPS and left to recover for 1 h. Open arrowheads: full-length GSDMD and GSDME based on molecular mass. Black arrowheads: cleaved GSDMD. Dashed box: area contrast enhanced for clarity. Note, the positive control for GSDME cleavage is provided in Fig. . c , d Flow cytometry analysis of HT3 B3-WT /HT3 B3-KO cells treated with a lysosomotropic agent, LLOMe ( c ) or 1 µg/ml LPS ( d ). Cells were stained with the lysosomotropic dye, acridine orange (Y-axis), and the cell viability dye, Sytox™ blue (X-axis). Lines indicate the threshold fluorescence levels (gate) to determine each quadrant. Numbers indicate cell percentage in each quadrant. e Schematic representation of each quadrant. Quadrant 1 (Q1) contained live cells positive for lysosomal staining (acridine orange positive, Sytox blue negative). Quadrant 2 (Q2) contained dead cells with positive lysosomal staining (acridine orange positive, Sytox blue positive). Quadrant 3 (Q3) contained dead cells negative for lysosomal staining (acridine orange negative, Sytox blue positive). Quadrant 4 (Q4) contained live cells negative for lysosomal staining (acridine orange negative, Sytox blue negative). Arrows indicate the timing of fluorescence loss for different cell death pathways. If the lysosomal loss occurred prior to plasma membrane permeabilization, then the percentage of cells will increase from Q1 to Q4 to Q3 over time. If the lysosomal loss occurred after plasma membrane loss, then the percentage of cells will increase from Q1 to Q2 to Q3 over time. f , g Graphical representation of the average of three separate experiments indicating the percentage of cells treated with LLOMe ( f ) or nucleofected LPS ( g ) in HT3 B3-WT (blue) and HT3 B3-KO (red) cells. Error bars represent means ± SD. Uncropped immunoblots can be found in Supplementary Fig. .

Article Snippet: The human monocyte cell line THP1-HMGB1-Lucia™ (thp-gb1lc) was obtained from InvivoGen.

Techniques: Protease Inhibitor, Staining, Two Tailed Test, Western Blot, Positive Control, Flow Cytometry, Fluorescence, Membrane