Journal: medRxiv

Article Title: Empagliflozin Improves Mitochondrial Biogenesis and Ameliorates Experimental Pulmonary Vascular Remodeling, But May Not Benefit Patients with Pulmonary Arterial Hypertension

doi: 10.1101/2025.01.27.25321226

Figure Lengend Snippet: A through C , Representative western blot and quantification of SGLT2 and PGC-1α of PAH MVECs treated with empagliflozin (EMPA) or DMSO. D and E , Representative western blot and quantification of SIRT1 of PAH MVECs treated with EMPA or DMSO, normalized by GAPDH. F and G , Representative western blot and quantification of p-AMPK of PAH MVECs treated with EMPA or DMSO, normalized by t-AMPK. H through I , Representative western blot and quantification of BMPR2, p-Smad1/5/9 and p-Smad2/3 of PAH MVECs treated with EMPA or DMSO, normalized by GAPDH. J and K , Representative western blot and quantification of SIRT1 of PAH MVECs treated with EMPA or DMSO, normalized by GAPDH. L and M , Representative western blot and quantification of p-AMPK of PAH MVECs treated with EMPA or DMSO, normalized by t-AMPK. Values are mean ± SEM. Difference was tested by using Paired t-test. *p<0.05, **p<0.01.

Article Snippet: After starvation for 4 hours with 1% Fetal Bovine Serum (FBS), MVECs were treated for 1 hour by either 1 μM empagliflozin (HY-15409, MedChem Express, Monmouth Junction, NJ, USA) dissolved in dimethyl sulfoxide (DMSO) or DMSO without empagliflozin.

Techniques: Western Blot

Journal: medRxiv

Article Title: Empagliflozin Improves Mitochondrial Biogenesis and Ameliorates Experimental Pulmonary Vascular Remodeling, But May Not Benefit Patients with Pulmonary Arterial Hypertension

doi: 10.1101/2025.01.27.25321226

Figure Lengend Snippet: A through C , Quantification of gene level of COXIII, NADH dehydrogenase and Cyt B of PAH MVECs treated by EMPA or DMSO, normalized by RLP27. D and E , Oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) of PAH MVECs treated by EMPA or DMSO. F and G , Representative image of using MitoTracker to detect the mitochondrial reactive oxygen species (ROS) and quantification of PAH MVECs treated with empagliflozin (EMPA) or DMSO. H and I , Representative western blot and quantification of PCNA of PAH MVECs treated with EMPA or DMSO, normalized by Vinculin. J, Evaluation of cell proliferation using MTT assay in PAH MVECs. K through M , Representative western blot and quantification of BMPR2, p-Smad1/5/9 and p-Smad2/3 of PAH MVECs treated with EMPA or DMSO, normalized by GAPDH. Values are mean ± SEM. Difference was tested by using Paired t-test. *p<0.05, **p<0.01.

Article Snippet: After starvation for 4 hours with 1% Fetal Bovine Serum (FBS), MVECs were treated for 1 hour by either 1 μM empagliflozin (HY-15409, MedChem Express, Monmouth Junction, NJ, USA) dissolved in dimethyl sulfoxide (DMSO) or DMSO without empagliflozin.

Techniques: Western Blot, MTT Assay

Journal: Infection and Immunity

Article Title: Rickettsia disrupts and reduces endothelial tight junction protein zonula occludens-1 in association with inflammasome activation

doi: 10.1128/iai.00468-24

Figure Lengend Snippet: Rickettsia conorii induced discontinuous immunofluorescence staining of the junctional protein ZO-1 and diminished the expression levels of ZO-1 in cultured monolayers of microvascular endothelial cells. MVECs were cultured and plated as monolayers and then infected with R. conorii at an MOI of 2. Mock infected cells served as negative controls. At 48 h post-infection, the expression of endothelial tight junction protein ZO-1 (red) and nuclei (DAPI, blue) was determined by confocal immunofluorescence microscopy. The representative image obtained from the randomly selected field is shown ( A ). Quantification of the mean fluorescence intensity signal in confocal images of mock and R. conorii -infected MVECs is shown. The mean fluorescence intensity of the data from 12 randomly selected regions from three independent cultures is shown ( B ). Tight junctions are represented by histograms of ZO-1 fluorescence intensity as indicated by a white line across the two cell-cell contacts ( C ). R. conorii in infected MVECs was detected by confocal immunofluorescence microscopy. Rickettsiae are depicted in green, and nuclei (DAPI) are shown in blue ( D ). The expression levels of ZO-1 in the cell lysates were determined by immunoblotting using a specific antibody against ZO-1 ( E ). By densitometry analysis, the representative normalized ratios (fold changes) of ZO-1 to loading control GAPDH and to uninfected controls are shown ( F ). Representative results are shown from three independent experiments. Data represent mean ± SD, n = 3. ** P < 0.01, *** P < 0.001. Scale bar = 100 µm. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Article Snippet: To evaluate whether rickettsiae activate inflammasomes in endothelial cells, uninfected and infected MVECs were collected and lysed in Cell Lysis Buffer (Cell Signaling, 9803) with phenylmethanesulfonyl fluoride (Cell Signaling, 8553) to inhibit serine proteases.

Techniques: Immunofluorescence, Staining, Expressing, Cell Culture, Infection, Microscopy, Fluorescence, Western Blot, Control

Journal: Infection and Immunity

Article Title: Rickettsia disrupts and reduces endothelial tight junction protein zonula occludens-1 in association with inflammasome activation

doi: 10.1128/iai.00468-24

Figure Lengend Snippet: Rickettsia conorii activated inflammasome in association with upregulated expression levels of NLRP3 in endothelial cells. MVECs were infected with R. conorii at an MOI of 2 (+) or 10 (++). At 6 h p.i., cells were collected. Activation of inflammasome by R. conorii was determined by cleavage of pro-caspase-1 ( A and B ) and pro-IL-1β ( C and D ) into their activated forms by immunoblotting, respectively. The expression levels of NLRP3 ( E and F ) and ZO-1 ( G and H ) were determined using specific antibodies against NLRP3 and ZO-1, respectively. GAPDH served as loading control. The densitometry data show the representative fold changes as normalized ratios of activated caspase-1 ( B ), activated IL-1β ( D ), NLRP3 ( F ), and ZO-1 ( H ) to GAPDH and to controls. Data are expressed as means ± SD of three independent experiments. * P < 0.05, ** P <0.01 (compared with the control group). ns, not statistically significant.

Article Snippet: To evaluate whether rickettsiae activate inflammasomes in endothelial cells, uninfected and infected MVECs were collected and lysed in Cell Lysis Buffer (Cell Signaling, 9803) with phenylmethanesulfonyl fluoride (Cell Signaling, 8553) to inhibit serine proteases.

Techniques: Expressing, Infection, Activation Assay, Western Blot, Control

Journal: Infection and Immunity

Article Title: Rickettsia disrupts and reduces endothelial tight junction protein zonula occludens-1 in association with inflammasome activation

doi: 10.1128/iai.00468-24

Figure Lengend Snippet: Selective inhibition of NLRP3 ameliorated the discontinuous immunostaining of ZO-1 and disrupted intercellular junctions caused by R. conorii infection in endothelial cell monolayers. MVECs were pre-treated for 3 h with MCC950 (10 µM) and infected with R. conorii at an MOI of 2 (+) or 10 (++). At 6 h p.i., cell lysates were collected, and the cleavage of pro-caspase-1 was determined by immunoblotting ( A ). At 48 h p.i., the continuous expression of endothelial tight junction protein ZO-1 in infected MVECs treated with or without MCC950 was determined by immunofluorescence microscopy ( B ). Tight junctions were represented by histograms of ZO-1 fluorescence intensity as indicated by a white line across the two cell-cell contacts ( C ). The expression levels of ZO-1 ( D and E ) and NLRP3 ( F and G ) at 48 h p.i. were determined by immunoblotting. The densitometry data show the representative fold changes as normalized ratios of ZO-1 ( E ) and NLRP3 ( G ) to GAPDH and to controls. Data are expressed as means ± SD of three independent experiments. ** P <0.01, *** P <0.001 (compared with the control group). Scale bar = 20 µm. ns, not statistically significant.

Article Snippet: To evaluate whether rickettsiae activate inflammasomes in endothelial cells, uninfected and infected MVECs were collected and lysed in Cell Lysis Buffer (Cell Signaling, 9803) with phenylmethanesulfonyl fluoride (Cell Signaling, 8553) to inhibit serine proteases.

Techniques: Inhibition, Immunostaining, Infection, Western Blot, Expressing, Immunofluorescence, Microscopy, Fluorescence, Control

Journal: Infection and Immunity

Article Title: Rickettsia disrupts and reduces endothelial tight junction protein zonula occludens-1 in association with inflammasome activation

doi: 10.1128/iai.00468-24

Figure Lengend Snippet: MCC950 mitigated the integrity of endothelial barrier function interrupted by R. conorii infection. MVECs were cultured in Transwell chambers and infected with R. conorii , with or without MCC950 (10 µM). The passage of 40-kDa FITC-dextran was quantified through the endothelial monolayer after 48 h. MVECs treated with DMSO vehicle, medium, or MCC950 served as negative controls. Recombinant TNF-alpha (200 ng/mL)-stimulated MVECs served as positive controls ( A ). At 48 h p.i., the concentrations of R. conorii in MVECs treated with MCC950 were determined by quantitative real-time PCR amplifying citrate synthase gene normalized to the amount of genomic DNA ( B ). The cytotoxicity of R. conorii -infected MVECs treated with or without MCC950 at 48 h p.i. was determined by LDH assay ( C ). Values represent mean ± SD of three replicates. * P < 0.05. ns, not statistically significant.

Article Snippet: To evaluate whether rickettsiae activate inflammasomes in endothelial cells, uninfected and infected MVECs were collected and lysed in Cell Lysis Buffer (Cell Signaling, 9803) with phenylmethanesulfonyl fluoride (Cell Signaling, 8553) to inhibit serine proteases.

Techniques: Infection, Cell Culture, Recombinant, Real-time Polymerase Chain Reaction, Lactate Dehydrogenase Assay

Journal: Infection and Immunity

Article Title: Rickettsia disrupts and reduces endothelial tight junction protein zonula occludens-1 in association with inflammasome activation

doi: 10.1128/iai.00468-24

Figure Lengend Snippet: Schematic diagram of the proposed model of disruption of interendothelial tight junction ZO-1 by R. conorii. Rickettsia conorii invades microvascular endothelial cells and replicates in their cytoplasm. These obligately intracellular bacteria trigger activation of NLRP3 inflammasome ( A ). Assembly of NLRP3 multiprotein complex activates caspase-1, which induces cleavage of pro-IL-1β into IL-1β ( B ). Activation of NLRP3 inflammasome subsequently reduces and interrupts the cytoplasmic tight junction protein ZO-1 ( C ). These signaling pathways may open the tight junction complex, potentially increasing microvascular hyperpermeability, which results in transmigration of plasma fluid and/or infiltration of inflammatory cells from blood into peripheral tissues.

Article Snippet: To evaluate whether rickettsiae activate inflammasomes in endothelial cells, uninfected and infected MVECs were collected and lysed in Cell Lysis Buffer (Cell Signaling, 9803) with phenylmethanesulfonyl fluoride (Cell Signaling, 8553) to inhibit serine proteases.

Techniques: Disruption, Bacteria, Activation Assay, Protein-Protein interactions, Transmigration Assay, Clinical Proteomics