Journal: Parasitology

Article Title: Identification of Leishmania infantum in blood donors from endemic regions for visceral leishmaniasis

doi: 10.1017/S0031182020001936

Figure Lengend Snippet: Results of the analysis of κ agreement between ELISA and qPCR and conventional PCR and qPCR for the diagnosis of asymptomatic visceral leishmaniasis

Article Snippet: ELISA rK39 and conventional polymerase chain reaction (PCR) ELISA rK39 (Kalazar Detect ELISA InBios ® Seattle, Washington, USA) was performed following the manufacturer's instructions, and conventional PCR was conducted as described by Ferreira-Silva et al . ( 2018 ).

Techniques: Enzyme-linked Immunosorbent Assay, Biomarker Discovery

Journal: Parasitology

Article Title: Identification of Leishmania infantum in blood donors from endemic regions for visceral leishmaniasis

doi: 10.1017/S0031182020001936

Figure Lengend Snippet: Comparative results between the techniques used in the 10 samples sequenced to identify infection by Leishmania infantum

Article Snippet: ELISA rK39 and conventional polymerase chain reaction (PCR) ELISA rK39 (Kalazar Detect ELISA InBios ® Seattle, Washington, USA) was performed following the manufacturer's instructions, and conventional PCR was conducted as described by Ferreira-Silva et al . ( 2018 ).

Techniques: Infection, Enzyme-linked Immunosorbent Assay, Sequencing

Journal: PLoS ONE

Article Title: Epigenetic Alterations in the Brain Associated with HIV-1 Infection and Methamphetamine Dependence

doi: 10.1371/journal.pone.0102555

Figure Lengend Snippet: The content of HIV DNA and cellular HIV RNA was profiled in the frontal cortex area of HIV seropositive individuals with and without history of METH use, by real-time PCR and normalized to Actin or GAPDH levels respectively. A. and B. HIV-1 DNA and cellular HIV RNA did not show significant changes among groups. C. HIV-1 RNA/DNA ratio (as a marker of average HIV transcription) was significantly higher in METH user group, suggesting increases on viral transcription in the brain. *p<0.05 by Mann Whitney pairs test.

Article Snippet: Bisulfite-converted DNA templates (40 ng) from the studied cases underwent PCR alongside with standards ranging from 0% to 100% methylation using MeltDoctor HRM Dye (Life Technologies).

Techniques: Real-time Polymerase Chain Reaction, Marker, MANN-WHITNEY

Journal: PLoS ONE

Article Title: Epigenetic Alterations in the Brain Associated with HIV-1 Infection and Methamphetamine Dependence

doi: 10.1371/journal.pone.0102555

Figure Lengend Snippet: A. Global methylation was determined by ELISA quantification of 5-mC on genomic DNA from frontal cortex samples. Significant increase in DNA methylation was observed in HIV seropositive METH users. *p<0.05 by Wilcoxon matched pairs test. B. Cellular HIV-1 RNA levels correlate with DNA methylation levels as determined by Spearman correlation between global DNA methylation and log HIV-1 RNA in the brain R = 0.527 with p = 0.01. C. Quantitative real-time PCR showed significant increase in DNMT1 transcript levels on HIV seropositive individuals who used METH. D. METH exposure did not alter mRNA levels of DNMT3B, a closely related family member reported to have redundant functions to DNMT1 in the brain. *p<0.05 by Wilcoxon matched pairs test. E. Western blot analysis of DNMT1 protein content in the nucleus showing representative HIV+METH− and HIV+METH+ cases. F. Image analysis showing integrated pixel intensity of Dnmt1 immunoreactivity. G. Immunofluorescence detection of DNMT1 on frontal cortex sections. Green fluorescent signal correspond to DNMT1 immunoreactivity and blue signal corresponds to DAPI nuclear staining. H. Image analysis showing average DNMT1 positive nuclear counts. Bar represents 10 µm. One way ANOVA was used to determine statistical significance, *p<0.05.

Article Snippet: Bisulfite-converted DNA templates (40 ng) from the studied cases underwent PCR alongside with standards ranging from 0% to 100% methylation using MeltDoctor HRM Dye (Life Technologies).

Techniques: Methylation, Enzyme-linked Immunosorbent Assay, DNA Methylation Assay, Real-time Polymerase Chain Reaction, Western Blot, Immunofluorescence, Staining

Journal: PLoS ONE

Article Title: Epigenetic Alterations in the Brain Associated with HIV-1 Infection and Methamphetamine Dependence

doi: 10.1371/journal.pone.0102555

Figure Lengend Snippet: Quantitative analysis of transcript abundance by real-time PCR showed significant increases of TGFBR3 ( A ) and NET1 ( C ) on the HIV+METH+ group in comparison to HIV+METH− subjects. *p<0.05 by Student’s t Test. Linear regression analysis of gene expression and DNA methylation showed positive correlations for TGFBR3 on HIV+METH− and on HIV+METH+ groups ( B ) and a significant correlation between gene expression and DNA methylation for NET1 ( D ) . **p<0.01 and ***p<0.001 by Linear regression analysis with a 95% confidence interval.

Article Snippet: Bisulfite-converted DNA templates (40 ng) from the studied cases underwent PCR alongside with standards ranging from 0% to 100% methylation using MeltDoctor HRM Dye (Life Technologies).

Techniques: Real-time Polymerase Chain Reaction, Expressing, DNA Methylation Assay

Journal: Journal of Virology

Article Title: Vectorial Release of Hepatitis E Virus in Polarized Human Hepatocytes

doi: 10.1128/JVI.01207-18

Figure Lengend Snippet: Polarization of HepG2/C3A subclone F2 cells on semipermeable collagen inserts. (A to D) Immunostaining of F2 cells on semipermeable collagen inserts. Nuclei were stained with DAPI (4′,6-diamidino-2-phenylindole). Bar =10 μm. (A and B) Maximum-intensity projections of x-y stacks (A) and x-z sections (B) for the tight-junction protein ZO-1. (C and D) x-y (C) and x-z (D) sections for DPP4. (E) Percentages of albumin exported from F2 cells. The albumin in the apical (black bars) and basolateral (gray bars) supernatants was quantified by ELISA. The data shown are means and standard deviations (SD) (n = 12) of the results of four experiments performed in triplicate. *, P < 0.05; ***, P < 0.001. (F) Total bile acids from 21-day cultures of F2 cells quantified by LC-MS. The results are expressed as percentages of exported bile acids in the apical (black bars) and basolateral (gray bars) supernatants. The data shown are means and SD (n = 4). *, P < 0.05. (G) Bile acids were quantified by LC-MS on day 21 postseeding. The amounts of each detected species in the apical (hatched bars) and basolateral (gray bar) supernatants are shown (n = 4). The bile acids quantified were allolithocholic acid (alloLCA), alpha-muricholic acid (aMCA), beta-muricholic acid (bMCA), cholic acid (CA), 3-sulfo-cholic acid (CA-3S), chenodeoxycholic acid (CDCA), 3-sulfo-cheno deoxycholic acid (CDCA-3S), deoxycholic acid (DCA), glycocholic acid (GCA), glycochenodeoxycholic acid (GCDCA), glycodeoxycholic acid (GDCA), glycolithocholic acid (GLCA), gamma-muricholic acid (gMCA), glycoursodeoxycholic acid (GUDCA), hyodeoxycholic acid (HDCA), isodeoxycholic acid (isoDCA), isolithocholic acid (isoLCA), lithocholic acid (LCA), tauroalfamuricholic acid (TaMCA), taurobetamuricholic acid (TbMCA), taurocholic acid (TCA), taurochenodeoxycholic acid (TCDCA), taurodeoxycholic acid (TDCA), taurolithocholic acid (TLCA), 3-sulfotaurolithocholic acid (TLCA-3S), ursodeoxycholic acid (UDCA), and omega muricholic acid (wMCA). For clarity, only detected bile acids are shown. (H) HepG2/C3A (circles) and F2 (squares) cells were infected with nHEV genotype 3 (1.35 × 106 HEV RNA copies/106 cells; white symbols) or eHEV genotype 3 (3.3 × 106 HEV RNA copies/106 cells; black symbols). Supernatants were collected every 2 days, and HEV RNA was quantified by RT-PCR. (I) HepG2/C3A (circles) and F2 (squares) cells were infected with eHEV genotype 3 (2.5 × 108 HEV RNA copies/106 cells). Supernatants were collected on day 15 postinfection, and HEV RNA was quantified by RT-PCR. The data shown are from three independent experiments performed in triplicate. The horizontal bars represent medians. *, P < 0.05. (J) Immunofluorescence of ORF2 protein in HepG2/C3A (white bars) and F2 (black bars) cells 21 to 30 days postinfection. Nuclei were stained with DAPI. The results are expressed as percentages of cells containing ORF2. The data shown are means and SD of the results of three independent experiments. ***, P < 0.001.

Article Snippet: Rabbit anti-CD26/DPP4 (Ab28340) was from Abcam (Paris, France).

Techniques: Immunostaining, Staining, Enzyme-linked Immunosorbent Assay, Liquid Chromatography with Mass Spectroscopy, Infection, Reverse Transcription Polymerase Chain Reaction, Immunofluorescence

Journal: Journal of Virology

Article Title: Vectorial Release of Hepatitis E Virus in Polarized Human Hepatocytes

doi: 10.1128/JVI.01207-18

Figure Lengend Snippet: Colocalization of ORF2 with Rab27A and DPP4 at the apical sides of hepatocytes. (A to E) Immunostaining of ORF2 protein (red) and Rab27a (green) in F2 cells grown on inserts for 14 days, infected with HEV genotype 3, and sampled 14 days postinfection. The arrows across the cells on the merged image indicate the paths of the 2-pixel-wide scans shown in panel E. (C and D) Stacks of 22 (x-y) sections were acquired, slices were separated, and Pearson’s (C) or Manders (D) coefficients were calculated on each section. The mean proportion of ORF2 intensities in Rab27a (red) and of Rab27a intensities in ORF2 (green) are shown. The error bars represent SD; n = 10. (F to J) Immunostaining of ORF2 protein (red) and DPP4 (green) in F2 cells grown on inserts for 14 days, infected with HEV genotype 3, and sampled 14 days postinfection. The arrows across the cells in the merged image indicate the paths of the 2-pixel-wide line scans shown in panel J. (H and I) Stacks of 22 (x-y) sections were acquired, slices were separated, and Pearson’s (H) or Manders (I) coefficients were calculated on each section. The mean proportions of ORF2 intensities in DPP4 (red) and of DPP4 intensities in ORF2 (green) are shown. The error bars represent SD; n = 10. Scale bars = 10 μm.

Article Snippet: Rabbit anti-CD26/DPP4 (Ab28340) was from Abcam (Paris, France).

Techniques: Immunostaining, Infection

Journal: Cell Metabolism

Article Title: Imatinib and methazolamide ameliorate COVID-19-induced metabolic complications via elevating ACE2 enzymatic activity and inhibiting viral entry

doi: 10.1016/j.cmet.2022.01.008

Figure Lengend Snippet: ACE2 is a key molecule potentially linking COVID-19 to associated metabolic defects (A) Volcano plot of differentially expressed genes after in vitro infection of SARS-CoV-2 (MOI = 0.005) in HUVECs for 24 h. (B) DisGeNET pathway enrichment of differentially expressed genes after infection (combined 685 upregulated and 774 downregulated genes from A). (C–E) Bioinformatics analysis of the mutual target disease-associated genes of diabetes mellitus (DM), hypertension (HTN), diabetic nephropathy (DN), and atherosclerosis (AS) that were queried from DisGeNET and Open Target database. The overlapping 48 shared genes from DisGeNET (C), the 72 shared genes from Open Target (D), and 20 genes identified on both DisGeNET (left) and Open Target database (right) as mutual target disease-associated genes of four diseases (E) were shown. (F) The heatmap to represent the alterations of the above 20 genes upon SARS-CoV-2 infection. Data were shown as log 2 fold change. The cross mark represents the undetectable gene from the transcriptomic study. (G) ELISA quantification of plasma angiotensin II (Ang II) and angiotensin-(1–7) (Ang-(1–7)) in COVID-19 patients, shown in Ang II against Ang-(1–7) ratio. HC, healthy control; I, infection phase; R, recovery phase. Health control group, n = 10, mean age ± SD = 42.30 ± 7.35, male/female = 5/5; non-severe group, n = 18, mean age ± SD = 51.61 ± 10.13, male/female = 9/9; severe group, n = 12, mean age ± SD = 57.75 ± 14.55, male/female = 6/6. (H and I) HUVECs were infected with SARS-CoV-2 (MOI = 0.005) for 24 h and were subjected to real-time PCR of ACE2 (H) and immunoblotting (I, blot shown on the left, quantification on the right, n = 4). (J) HUVECs were infected with SARS-CoV-2 (MOI = 0.005) for 48 h, and then the medium was removed and 100 nM Ang II in 1 mL HEPES solution was applied for the cells. After 1 h treatment in the incubator at 37°C, the supernatant containing Ang II and cleaved Ang-(1–7) was examined (n = 6). (K and L) Sixteen-week-old human ACE2 transgenic mice were intranasally challenged with 4 × 10 4 FFU SARS-CoV-2 after 10-week high-fat-diet treatment. After 7 days post-infection, all mice were fasted for 6 h and sacrificed. Livers (K) and kidneys (L) were subjected to real-time PCR (n = 3–4). (M–O) HUVECs were treated with a “cocktail” of different inflammatory factors (IFs), namely the combination of 10 or 50 ng/mL of TNF-α, IL-4, IL-6, and IFN-γ or 1 μM DX600 for 48 h and were subjected to real-time PCR of ACE2 (M) (n = 6), immunoblotting (N, blot shown on the left, quantification on the right, n = 4), and enzymatic activity assay (O, data were shown as the area under the kinetic activity curves, n = 6). DX600 was used as a negative control. Error bars represent SEM; ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001. HUVECs, human umbilical vein endothelial cells; AU, arbitrary unit; CoV-2, SARS-CoV-2. See also Figure S1 .

Article Snippet: Human angiotensin-I converting enzyme 2 (ACE2) , Sino Biological Company , Cat. 10108-H05H.

Techniques: In Vitro, Infection, Enzyme-linked Immunosorbent Assay, Real-time Polymerase Chain Reaction, Western Blot, Transgenic Assay, Enzyme Activity Assay, Activity Assay, Negative Control

Journal: Cell Metabolism

Article Title: Imatinib and methazolamide ameliorate COVID-19-induced metabolic complications via elevating ACE2 enzymatic activity and inhibiting viral entry

doi: 10.1016/j.cmet.2022.01.008

Figure Lengend Snippet: ACE2 plays an important role in maintaining metabolic homeostasis (A) HUVECs were infected with 10 nM control siRNA (Con) or ACE2 siRNA (siACE2) for 24 h and subjected to real-time PCR (n = 6). (B and C) Eight-week-old male ob/ob mice were treated with intravenous injection of AAV9-CAG-hACE2-EGFP (ob/ob-ACE2) and corresponding control virus (ob/ob-Con) and their wild-type littermates with control virus (WT-Con). Glucose tolerance testing (GTT) was performed at 10 weeks (B), and insulin tolerance testing (ITT) was performed at 11 weeks (C) (n = 6). The significance of ob/ob-Con versus WT-Con was shown as ∗ ; ob/ob-ACE2 versus ob/ob-Con as #. (D–F) At 12 weeks, all mice were fasted for 6 h and sacrificed. Quantification of fasting blood glucose (D), plasma insulin (E), and homeostatic model assessment of insulin resistance (HOMA-IR) (F) were shown (n = 5). (G and H) Plasma TNF-α (G) and IL-6 (H) were measured (n = 4). (I and J) Livers were subjected to oil red O (ORO) staining (I, images shown on the left, quantification on the right; n = 4) and real-time PCR (n = 6) (J). (K) Plasma KIM-1 (n = 4). (L and M) Kidneys were subjected to periodic acid-Schiff (PAS) staining (L, images shown on the left, quantification on the right; n = 8) and real-time PCR (M) (n = 5). (N) Aortas were subjected to real-time PCR (n = 5). (O) Hearts were subjected to echocardiography (representative M-mode echocardiography images shown on the top, quantification of LVEF (%) and FS (%) on the bottom; n = 8). Error bars represent SEM, ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001; # p < 0.05, ## p < 0.01, and ### p < 0.001. AUC, area under the curve; KIM-1, kidney injury molecule-1; LVEF, left ventricular ejection fraction; FS, fractional shortening. See also Figure S2 .

Article Snippet: Human angiotensin-I converting enzyme 2 (ACE2) , Sino Biological Company , Cat. 10108-H05H.

Techniques: Infection, Real-time Polymerase Chain Reaction, Injection, Staining

Journal: Cell Metabolism

Article Title: Imatinib and methazolamide ameliorate COVID-19-induced metabolic complications via elevating ACE2 enzymatic activity and inhibiting viral entry

doi: 10.1016/j.cmet.2022.01.008

Figure Lengend Snippet: Imatinib, harpagoside, and methazolamide are identified as ACE2 activators (A) Flow chart depicting the process of in silico identification of potential ACE2 activators with Tianhe-2 supercomputer virtual screening and CMAP bioinformatic analysis (top) and detailed information of the overlapped compounds (bottom, # and ∗ indicated different overlapping strategy). (B) The docking site (gray) of ACE2 protein (PDB: 1R42 ). (C) Schematic of the in vitro experimental workflow to identify the final three compounds. (D–F) Real-time PCR of gene expression in HUVECs. Log 10 fold change was calculated based on treated samples against control samples. HUVECs were treated with 15 compounds for 16 h (D), inflammatory factors for 32 h (50 ng/mL TNF-α, IL-4, IL-6, and IFN-γ) following eight compounds for 16 h (E), or 10 nM control siRNA (siCon) and ACE2 siRNA (siACE2) for 8 h following six compounds for 16 h (F) (n = 3). The selected compounds based on the scoring system were highlighted in red and applied for the following screening. (G) The low (L), medium (M), and high (H) concentrations of 15 compounds applied in the above three experiments. See also Figure S3 .

Article Snippet: Human angiotensin-I converting enzyme 2 (ACE2) , Sino Biological Company , Cat. 10108-H05H.

Techniques: In Silico, In Vitro, Real-time Polymerase Chain Reaction, Expressing

Journal: Cell Metabolism

Article Title: Imatinib and methazolamide ameliorate COVID-19-induced metabolic complications via elevating ACE2 enzymatic activity and inhibiting viral entry

doi: 10.1016/j.cmet.2022.01.008

Figure Lengend Snippet: Imatinib, harpagoside, and methazolamide directly bind to and activate ACE2 (A–E) The direct binding was illustrated by surface plasmon resonance (SPR) assay of purified ACE2 protein with imatinib (Ima) (A), harpagoside (Har) (B), methazolamide (Met) (C), diminazene aceturate (DIZE) (D), or xanthone (Xan) (E). Xanthone was used as a negative control. The Kd values (equilibrium dissociation constant) of compounds binding to ACE2 protein were calculated based on the fitted curves. (F and G) The detailed binding between ACE2 protein (gray) and imatinib (F, blue) and methazolamide (G, blue) was simulated with molecular dynamics simulation by GROMACS. Residues involved in binding were marked in green. (H) The HUVECs were treated with 25 μM imatinib, 100 μM harpagoside, 100 μM methazolamide, or 1 μM DX600 for 16 h, then subjected to ACE2 enzymatic activity assay. Data shown as the area under the kinetic activity curves. The ACE2 enzymatic inhibitor DX600 was used as a negative control (n = 3). (I–L) The HUVEC lysates were prepared and treated with 1.5 × 10 −6 M imatinib, 1.5 × 10 −6 M harpagoside, 1.5 × 10 −6 M methazolamide, or 1.0 × 10 −6 M DX600 for 30 min, then subjected to ACE2 enzymatic activity assay in a kinetics model. The kinetic activity curves were shown (I) (n = 3). The effects of imatinib (J) at concentrations of 1.5 × 10 −7 M (Ima-1) and 1.5 × 10 −6 M (Ima-2), harpagoside (K) at concentrations of 0.5 × 10 −7 M (Har-1) and 1.5 × 10 −6 M (Har-2), or methazolamide (L) at concentrations of 1.5 × 10 −7 M (Met-1) and 1.5 × 10 −6 M (Met-2) on the enzymatic activity of ACE2 in HUVEC lysates were shown as the area under the kinetic activity curves (n = 3). Error bars represent SEM, ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001. See also Figure S4 .

Article Snippet: Human angiotensin-I converting enzyme 2 (ACE2) , Sino Biological Company , Cat. 10108-H05H.

Techniques: Binding Assay, SPR Assay, Purification, Negative Control, Enzyme Activity Assay, Activity Assay

Journal: Cell Metabolism

Article Title: Imatinib and methazolamide ameliorate COVID-19-induced metabolic complications via elevating ACE2 enzymatic activity and inhibiting viral entry

doi: 10.1016/j.cmet.2022.01.008

Figure Lengend Snippet: Imatinib and methazolamide ameliorate metabolic defects in insulin-resistant mice via ACE2 (A–I) Twenty-eight-week-old male mice with 23-week high-fat-diet treatment (DIO) and controlled lean mice (Lean) were treated with vehicle, 250 mg/kg imatinib (DIO + Ima), or 100 mg/kg methazolamide (DIO + Met) through gavage once each day for 4 weeks. (A and B) Glucose tolerance testing (GTT) was performed at 30 weeks (A); insulin tolerance testing (ITT) was performed at 31 weeks (B) (n = 6). The significance of lean versus DIO was shown as ∗ , DIO versus DIO + Ima as #, and DIO versus DIO + Met as $. ∗,#,$ p < 0.05, ∗∗,##,$$ p < 0.01, and ∗∗∗,###,$$$ p < 0.001. At 32 weeks, all mice were fasted for 6 h and sacrificed. (C–F) Fasting blood glucose (C), plasma insulin (D), homeostatic model assessment of insulin resistance (HOMA-IR) (E), and plasma TNF-α (F) (n = 6). (G and H) Livers were subjected to oil red O (ORO) staining (G, images shown on the left, quantification on the right) and real-time PCR (H) (n = 6). (I) Aortas were subjected to real-time PCR (n = 6). (J–P) For kidney conditional knockdown of ACE2 (ACE2 C-kd), 26-week-old male mice with 21-week high-fat-diet treatment (DIO) and controlled lean mice (Lean) were treated with transparenchymal renal pelvis injection of AAV9-CAG-mACE2shRNA-EGFP or control virus. After 2-week recovery, mice were given vehicle, 250 mg/kg imatinib (DIO + Ima and DIO + Ima + ACE2 C-kd), or 100 mg/kg methazolamide (DIO + Met and DIO + Met + ACE2 C-kd) through gavage once each day for 4 weeks. At 32 weeks, all mice were fasted for 6 h and sacrificed. (J and K) Plasma KIM-1 (J) and CREA (K) (n = 6). (L and M) Kidneys were subjected to periodic acid-Schiff (PAS) staining (L, images and quantification were shown; n = 5) and real-time PCR (M) (n = 6). (N) Quantification of plasma ratio of Ang II against Ang-(1–7) (n = 5). (O and P) The lysates of kidneys (O) and aortas (P) were subjected to ACE2 enzymatic activity assay, and data were shown as the area under the kinetic activity curves (n = 3). (Q–U) For A779 (MasR inhibitor) intervention, 28-week-old male mice with 23-week high-fat-diet treatment were given vehicle (DIO and DIO + A779), 250 mg/kg imatinib (DIO + Ima and DIO + Ima + A779), or 100 mg/kg methazolamide (DIO + Met and DIO + Met + A779) with or without 3 mg/kg A779 once each day for 4 weeks. (Q and R) Glucose tolerance testing (GTT) was performed at 30 weeks (Q), and insulin tolerance testing (ITT) was performed at 31 weeks (R) (n = 6). The significance of DIO versus DIO + Ima was shown as ∗ , DIO versus DIO + Met as #, DIO + Ima versus DIO + Ima + A779 as $ and DIO + Met versus DIO + Met + A779 as &. ∗,#,$,& p < 0.05, ∗∗,##,$$,&& p < 0.01, and ∗∗∗,###,$$$,&&& p < 0.001. At 32 weeks, all mice were fasted for 6 h and sacrificed. (S–U) Livers (S), kidneys (T), and aortas (U) were subjected to real-time PCR. Error bars represent SEM. ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001. CREA, creatinine. See also Figure S5 .

Article Snippet: Human angiotensin-I converting enzyme 2 (ACE2) , Sino Biological Company , Cat. 10108-H05H.

Techniques: Staining, Real-time Polymerase Chain Reaction, Injection, Enzyme Activity Assay, Activity Assay

Journal: Cell Metabolism

Article Title: Imatinib and methazolamide ameliorate COVID-19-induced metabolic complications via elevating ACE2 enzymatic activity and inhibiting viral entry

doi: 10.1016/j.cmet.2022.01.008

Figure Lengend Snippet: ACE2 enzymatic activators improve metabolic defects and inhibit virus entry upon SARS-CoV-2 infection (A–Q) Twelve-week-old human ACE2 transgenic mice were given vehicle (Mock, CoV-2), 250 mg/kg imatinib (CoV-2 + Ima), and 100 mg/kg methazolamide (CoV-2 + Met) through gavage once each day for 4 weeks after 6-week high-fat-diet treatment and were intranasally challenged with 4 × 10 4 FFU SARS-CoV-2. After 7 days of infection, all mice were fasted for 6 h and sacrificed. (A–H) Quantification of the ratio of Ang II against Ang-(1–7) in plasma (A), fasting blood glucose (B), plasma insulin (C), homeostatic model assessment of insulin resistance (HOMA-IR) (D), plasma triglyceride (E), plasma total cholesterol (F), plasma TNF-α (G), and plasma IL-6 (H) (n = 4). (I–K) Livers were subjected to lipid assay (I and J) and real-time PCR (K) (n = 3–4). (L) Plasma KIM-1 (n = 4–5). (M and N) Kidneys were subjected to periodic acid-Schiff (PAS) staining (M, images and quantification were shown; n = 4) and real-time PCR (N) (n = 4). (O–Q) Lungs were subjected to H&E staining (O, images and quantification were shown; n = 4), real-time PCR (P) (n = 4), and immunoblotting of viral nucleocapsid protein (NP) (Q, blot shown on the left, quantification on the right; n = 4). (R) Vero E6 cells were pretreated with 25 μM imatinib, 100 μM harpagoside, or 100 μM methazolamide for 6 h, followed by SARS-CoV-2 (MOI = 0.005) infection for 42 h; the supernatant was subjected to real-time PCR (n = 4). (S) HEK293T cells expressing hACE2 were pretreated with 25 μM imatinib, 100 μM harpagoside, or 100 μM methazolamide for 6 h, followed by pseudovirion treatment for 66 h, and were observed with fluorescence microscopy (images shown on the left, quantification on the right; n = 5). (T) The HEK293T cells overexpressing spike-FLAG and ACE2-HA were treated with 25 μM imatinib, 100 μM harpagoside, or 100 μM methazolamide for 48 h. Precipitated proteins were subjected to immunoblotting (blot shown on the left, quantification on the right; n = 4). Error bars represent SEM. ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001. See also Figure S6 .

Article Snippet: Human angiotensin-I converting enzyme 2 (ACE2) , Sino Biological Company , Cat. 10108-H05H.

Techniques: Infection, Transgenic Assay, Real-time Polymerase Chain Reaction, Staining, Western Blot, Expressing, Fluorescence, Microscopy

Journal: Cell Metabolism

Article Title: Imatinib and methazolamide ameliorate COVID-19-induced metabolic complications via elevating ACE2 enzymatic activity and inhibiting viral entry

doi: 10.1016/j.cmet.2022.01.008

Figure Lengend Snippet:

Article Snippet: Human angiotensin-I converting enzyme 2 (ACE2) , Sino Biological Company , Cat. 10108-H05H.

Techniques: Recombinant, Enzyme-linked Immunosorbent Assay, RNA Extraction, SYBR Green Assay, Activity Assay, Cholesterol Assay, Software

Journal: Scientific Reports

Article Title: Novel human neutralizing mAbs specific for Spike-RBD of SARS-CoV-2

doi: 10.1038/s41598-021-90348-7

Figure Lengend Snippet: Schematic representation of selection and screening strategies for identification of SARS-CoV-2 neutralizing mAbs. ( a ) Cartoon representing SARS-CoV-2 host cell attachment mediated via ACE2-Spike interaction. The ACE2-RBD interaction in the box is adapted from PDB 6M0J . ( b ) Phage display of scFv library was carried out on recombinant SARS-CoV-2 RBD-Fc protein; acidic or competitive elution were implemented. ( c ) Variable heavy chains of scFvs were extracted from sub-libraries and sequenced by MiSeq Illumina. ( d ) The trend of enrichment of given clones was evaluated within and between the selection cycles. ( e ) Potential binders (scFvs) were converted into fully human IgG4 mAbs in a high yield expressing cell line (HEK293ES_1). ( f ) Binding to rRBD and competition with rACE2 were evaluated in vitro with recombinant protein assay. Neutralization capacity of RBD-specific mAbs was further evaluated for blocking SARS-CoV-2 replication.

Article Snippet: The following human recombinant proteins were used: SARS-CoV-2 (2019-nCoV) chimeric Spike RBD-Fc protein, SARS-CoV-2 (2019-nCoV) Spike RBD-His protein and ACE2 His Protein (all from Sino Biological, 10108-H08H Düsseldorfer Eschborn, Germany); Recombinant human coronavirus SARS-CoV-2 Spike Glycoprotein S1 (from Abcam, ab273068, Cambridge UK); Recombinant ACE-2 Fc chimeric protein (Z03484 GenScript, Piscataway NJ); recombinant IgG1 Fc protein (R & D Systems, 110-HG Minneapolis MN).

Techniques: Selection, Cell Attachment Assay, Recombinant, Clone Assay, Expressing, Binding Assay, In Vitro, Neutralization, Blocking Assay

Journal: Scientific Reports

Article Title: Novel human neutralizing mAbs specific for Spike-RBD of SARS-CoV-2

doi: 10.1038/s41598-021-90348-7

Figure Lengend Snippet: Screening by ELISA and expression of anti-Spike scFvs for analysis of their binding to Spike RBD and their competition with ACE2. ( a ) Representative image of scFv-phages screening by ELISA to test their binding to Spike-RBD recombinant protein. ( b ) Screening of positive phage clones by ELISA assay on human Spike RBD-Fc recombinant protein (black bars) or human recombinant IgG Fc used as a negative control in parallel assays (grey bars). ( c ) Western blotting analysis with the anti-c-myc antibody of the periplasmic extracts of the cells transformed with the two selected positive clones, D3 and F12, expressed in the absence or in the presence of IPTG, used for induction (The blot was obtained by grouping two different parts of the same blot and the black line has been inserted to indicate the two distinct parts. The corresponding full-length blot has been inserted in Supplementary Data set as full-length blot of Figure . The samples were processed in parallel in the same experiment). ( d ) Representative image of soluble scFvs binding to Spike-RBD recombinant protein by ELISA ( e ) at two different concentrations: 45 nM (grey bars) or 90 nM (black bars). ( f ) Representative image of soluble scFvs interference in Spike-ACE2 interaction tested by ELISA ( g ). The binding of ACE2-His to immobilized Spike RBD protein in the absence (dark grey bars) or in the presence (light grey bars) of the indicated soluble scFvs (90 nM). ( h ) Competitive ELISA assay was performed by measuring the binding of Spike RBD-Fc protein on ACE2-positive VERO E6 cells in the absence or in the presence of D3 and F12 scFvs.

Article Snippet: The following human recombinant proteins were used: SARS-CoV-2 (2019-nCoV) chimeric Spike RBD-Fc protein, SARS-CoV-2 (2019-nCoV) Spike RBD-His protein and ACE2 His Protein (all from Sino Biological, 10108-H08H Düsseldorfer Eschborn, Germany); Recombinant human coronavirus SARS-CoV-2 Spike Glycoprotein S1 (from Abcam, ab273068, Cambridge UK); Recombinant ACE-2 Fc chimeric protein (Z03484 GenScript, Piscataway NJ); recombinant IgG1 Fc protein (R & D Systems, 110-HG Minneapolis MN).

Techniques: Enzyme-linked Immunosorbent Assay, Expressing, Binding Assay, Recombinant, Clone Assay, Negative Control, Western Blot, Transformation Assay, Competitive ELISA

Journal: Scientific Reports

Article Title: Novel human neutralizing mAbs specific for Spike-RBD of SARS-CoV-2

doi: 10.1038/s41598-021-90348-7

Figure Lengend Snippet: Binding affinity of D3 and F12 mAbs for Spike RBD and their competition with ACE2. ( a ) The binding affinity of ACE2 receptor to immobilized SARS-CoV-2 RBD recombinant protein was tested by ELISA as a positive control. ( b , c ) ELISA assays were performed by testing D3 ( b ) and F12 ( c ) mAbs at increasing concentrations (0.5–100 nM) on human Spike RBD-Fc chimeric protein (black curves). In parallel the Fc domain (grey curves) was used as a negative control. ( d , e ) Competitive ELISA assays were performed by measuring the binding of ACE2-His protein to Spike RBD in the absence or in the presence of D3 ( d ) and F12 ( e ) mAb used at a concentration of 100 nM. ( f ) Competitive ELISA assays were performed by measuring the binding of ACE2-His to RBD in the absence (striped bar) or in the presence of the indicated mAbs used alone (gray bars) or in combination (black bars) at a concentration of 100 nM. The binding values were reported as the mean of at least three determinations obtained in three independent experiments. Error bars depicted means ± SD .

Article Snippet: The following human recombinant proteins were used: SARS-CoV-2 (2019-nCoV) chimeric Spike RBD-Fc protein, SARS-CoV-2 (2019-nCoV) Spike RBD-His protein and ACE2 His Protein (all from Sino Biological, 10108-H08H Düsseldorfer Eschborn, Germany); Recombinant human coronavirus SARS-CoV-2 Spike Glycoprotein S1 (from Abcam, ab273068, Cambridge UK); Recombinant ACE-2 Fc chimeric protein (Z03484 GenScript, Piscataway NJ); recombinant IgG1 Fc protein (R & D Systems, 110-HG Minneapolis MN).

Techniques: Binding Assay, Recombinant, Enzyme-linked Immunosorbent Assay, Positive Control, Negative Control, Competitive ELISA, Concentration Assay

Journal: Scientific Reports

Article Title: Novel human neutralizing mAbs specific for Spike-RBD of SARS-CoV-2

doi: 10.1038/s41598-021-90348-7

Figure Lengend Snippet: ELISA assays on Spike RBD to test the binding affinity of S96 and AC2 mAbs and their competition with ACE-2. ( a ) The binding affinity and specificity for Spike RBD of S96 and AC2 mAbs was evaluated by testing each mAb at increasing concentrations (0.5–100 nM) on Spike RBD-Fc chimeric protein or Fc, used as a negative control. ( b ) Competitive ELISA assays were performed by measuring the binding of ACE2-His to RBD in the absence (white bars) or in the presence of the indicated mAbs (gray and black bars) at a concentration of 100 nM. ( c ) Competitive ELISA assays to determine the epitope binning were performed by measuring the binding of Biotinylated F12 (F12-B) mAb to RBD, pre-incubated in the absence (white bars) or in the presence of the indicated mAbs (grey and black bars). The binding values were reported as the mean of at least three determinations obtained in three independent experiments. Error bars depicted means ± SD .

Article Snippet: The following human recombinant proteins were used: SARS-CoV-2 (2019-nCoV) chimeric Spike RBD-Fc protein, SARS-CoV-2 (2019-nCoV) Spike RBD-His protein and ACE2 His Protein (all from Sino Biological, 10108-H08H Düsseldorfer Eschborn, Germany); Recombinant human coronavirus SARS-CoV-2 Spike Glycoprotein S1 (from Abcam, ab273068, Cambridge UK); Recombinant ACE-2 Fc chimeric protein (Z03484 GenScript, Piscataway NJ); recombinant IgG1 Fc protein (R & D Systems, 110-HG Minneapolis MN).

Techniques: Enzyme-linked Immunosorbent Assay, Binding Assay, Negative Control, Competitive ELISA, Concentration Assay, Incubation

Journal: Scientific Reports

Article Title: Novel human neutralizing mAbs specific for Spike-RBD of SARS-CoV-2

doi: 10.1038/s41598-021-90348-7

Figure Lengend Snippet: Analysis of human primary cells, infected with the VOC 202012/01 lineage B.1.1.7 variant in the absence or presence of D3, by immunofluorescence assays and RT-PCR. ( a ) The cells untreated or infected with the VOC 202012/01 variant of SARS-CoV-2, in the absence or presence of D3 for 72 h at 37 °C, were fixed, washed and permeabilised, as described in the Methods. After blocking, the slides were incubated with the relevant primary antibodies overnight at 4 °C: anti-ACE2 (1:100; ab15348; Abcam) or anti-SARS-CoV-2 Nucleoprotein (N) Antibody (1:100; No. 35-579, ProSci), followed by the relevant secondary anti-mouse Alexa Fluor 488 (1:200; ab150113; Abcam) and anti-Rabbit Alexa Fluor 546 (1:200; A-11035; ThermoFisher), respectively. DNA was stained with DAPI (1:5000; #62254; Thermo Fisher). Microscopy images were obtained with the Elyra 7 platform (Zeiss) with the optical Lattice SIM technology, using the 63 × oil immersion objective. ( b ) Quantification of the mean fluorescence intensity was performed by using the ZEN software (Zeiss, black edition). ( c ) Cell extracts were analyzed by RT-PCR for measuring the expression of N1 viral gene and the levels of the indicated cytokines. In parallel, the extract of uninfected cells was used as negative control.

Article Snippet: The following human recombinant proteins were used: SARS-CoV-2 (2019-nCoV) chimeric Spike RBD-Fc protein, SARS-CoV-2 (2019-nCoV) Spike RBD-His protein and ACE2 His Protein (all from Sino Biological, 10108-H08H Düsseldorfer Eschborn, Germany); Recombinant human coronavirus SARS-CoV-2 Spike Glycoprotein S1 (from Abcam, ab273068, Cambridge UK); Recombinant ACE-2 Fc chimeric protein (Z03484 GenScript, Piscataway NJ); recombinant IgG1 Fc protein (R & D Systems, 110-HG Minneapolis MN).

Techniques: Infection, Variant Assay, Immunofluorescence, Reverse Transcription Polymerase Chain Reaction, Blocking Assay, Incubation, Staining, Microscopy, Fluorescence, Software, Expressing, Negative Control

Journal: Journal of Thoracic Disease

Article Title: Inhibition of macrophage pyroptosis protects against sepsis-induced injury to the alveolar epithelial barrier

doi: 10.21037/jtd-2025-aw-2292

Figure Lengend Snippet: MCC950 inhibits LPS-induced macrophage pyroptosis. Macrophages were pretreated for 2 h with MCC950 (20 µM), followed by stimulation with 1 µg/mL LPS for 12 h. (A-E) RT-qPCR analysis of mRNA expression levels for NLRP3, caspase-1, ASC, IL-1β, and IL-18. (F) Caspase-1 activity was measured using a specific activity assay. (G-J) Protein levels of NLRP3, caspase-1 and cleaved caspase-1, and ASC, were assessed by Western blotting. (K,L) ELISA was used to quantify the concentrations of IL-1β and IL-18 in the culture supernatant (n=9). Data are presented as mean ± standard deviation. *, P<0.05; **, P<0.01. ASC, apoptosis-associated speck-like protein containing a CARD; ELISA, enzyme-linked immunosorbent assay; LPS, lipopolysaccharide; RT-qPCR, reverse transcription quantitative polymerase chain reaction.

Article Snippet: Samples were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to polyvinylidene fluoride membranes, and blocked for 10 min. Membranes were incubated overnight at 4 °C with antibodies against NLRP3 (15101, CST, Danvers, MA, USA), caspase-1 (24232, CST), cleaved caspase-1 (E2G2I, CST), ASC (13833S, CST), GAPDH (4970S, CST), Occludin (DF7504, Affinity Biosciences, Cincinnati, OH, USA), and ZO-1 (21773-1-AP, Proteintech, Wuhan, China).

Techniques: Quantitative RT-PCR, Expressing, Activity Assay, Western Blot, Enzyme-linked Immunosorbent Assay, Standard Deviation, Reverse Transcription, Real-time Polymerase Chain Reaction

Journal: PLoS ONE

Article Title: Rickettsia australis Activates Inflammasome in Human and Murine Macrophages

doi: 10.1371/journal.pone.0157231

Figure Lengend Snippet: BMMs of WT and ASC -/- mice were isolated, cultivated, and infected with R . australis at an MOI of 10 or treated with LPS plus ATP as described in Materials and Methods. At 24 h p.i., the secretion of IL-1β (A), IL-18 (B) and IL-10 (C) was assayed by ELISA. D, Activation of caspase-1 was determined by immunoblotting detection of the active unit p10 in the processed supernatant of infected WT and ASC -/- BMMs at 24 h p.i.. Data represent two independent experiments with consistent results. Each experiment included at least 4 replicates. *, p <0.05.

Article Snippet: The cell lysates and concentrated supernatant were separated by SDS-PAGE, transferred to a polyvinylidene difluoride (PVDF) membrane and probed with anti-caspase-1 p20 antibody (EMD Millipore, MA, 06-503-I) for lysates, and anti-caspase-1 p10 antibody (sc-514, Santa Cruz Biotechnology) for concentrated supernatants.

Techniques: Isolation, Infection, Enzyme-linked Immunosorbent Assay, Activation Assay, Western Blot

Journal: PLoS ONE

Article Title: Rickettsia australis Activates Inflammasome in Human and Murine Macrophages

doi: 10.1371/journal.pone.0157231

Figure Lengend Snippet: WT and NLRP3 -/- BMMs were isolated, cultivated, and infected with R . australis at MOIs of 6 and 2. The transcriptional levels of NLRP3 in WT BMMs at different time intervals of infection (at MOI of 6) were determined by RT-PCR as described in Materials and Methods (A). At 12 h (B) p.i., the secretion levels of IL-1β by WT and NLRP3 -/- BMMs were determined by ELISA. The cleavage of caspase-1 was determined by detection of the active unit p10 in the processed supernatant at 12 h p.i. (C). Data represent mean ± SD for at least 3 replicates each group. *, p <0.05 for a significant difference between WT and NLRP3 -/- mice; ns, not significantly different.

Article Snippet: The cell lysates and concentrated supernatant were separated by SDS-PAGE, transferred to a polyvinylidene difluoride (PVDF) membrane and probed with anti-caspase-1 p20 antibody (EMD Millipore, MA, 06-503-I) for lysates, and anti-caspase-1 p10 antibody (sc-514, Santa Cruz Biotechnology) for concentrated supernatants.

Techniques: Isolation, Infection, Reverse Transcription Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay

Journal: Journal of Diabetes Research

Article Title: Huajuxiaoji Formula Alleviates Phenyl Sulfate-Induced Diabetic Kidney Disease by Inhibiting NLRP3 Inflammasome Activation and Pyroptosis

doi: 10.1155/2024/8772009

Figure Lengend Snippet: HJXJ reverses PS-induced related protein expressions in db/db mice. Western blotting is used to measure the expressions of (a) podocin, nephrin, (b) NLRP3, Caspase-1 p20, GSDMD-N in kidney tissues, and (c) ZO-1 and occludin in colon tissues of db/m (control) and db/db mice with PS in the absence or presence of HJXJ. Values are presented as mean ± SD. n = 6/group. ∗∗∗ P < 0.001 versus control. # P < 0.05, ## P < 0.01, ### P < 0.001 versus PS.

Article Snippet: Thereafter, the membranes were blocked with 5% skim milk for 1 h and probed with specific primary antibodies at 4°C for 12 h. The antibodies used were specific for podocin (ab181143), nephrin (ab216341), NLRP3 (ab263899), gasdermin D-N-terminal (GSDMD-N) (ab215203), ZO-1 (ab216880), occludin (ab216327) (Abcam, Cambridge, UK), Caspase-1 p20 (sc-398715, Santa Cruz Biotechnology, California, USA), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH, #5174, CST, USA) at 4°C for 12 h, followed by secondary antibodies for 2 h. An enhanced chemiluminescence system was used to detect protein expression.

Techniques: Western Blot, Control

Journal: Journal of Diabetes Research

Article Title: Huajuxiaoji Formula Alleviates Phenyl Sulfate-Induced Diabetic Kidney Disease by Inhibiting NLRP3 Inflammasome Activation and Pyroptosis

doi: 10.1155/2024/8772009

Figure Lengend Snippet: PS induces reactive oxygen species (ROS) production, pyroptosis, and NLRP3 inflammasome activation in MPC5 cells. MPC5 cells are treated with different PS concentrations. Flow cytometry is used to evaluate (a) ROS production and (b, c) pyroptosis level. Western blotting is used to measure (d) expressions of NLRP3, Caspase-1 p20, and GSDMD-N. ELISA kits are used to measure (e) release of IL-1 β and IL-18. ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001 versus control.

Article Snippet: Thereafter, the membranes were blocked with 5% skim milk for 1 h and probed with specific primary antibodies at 4°C for 12 h. The antibodies used were specific for podocin (ab181143), nephrin (ab216341), NLRP3 (ab263899), gasdermin D-N-terminal (GSDMD-N) (ab215203), ZO-1 (ab216880), occludin (ab216327) (Abcam, Cambridge, UK), Caspase-1 p20 (sc-398715, Santa Cruz Biotechnology, California, USA), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH, #5174, CST, USA) at 4°C for 12 h, followed by secondary antibodies for 2 h. An enhanced chemiluminescence system was used to detect protein expression.

Techniques: Activation Assay, Flow Cytometry, Western Blot, Enzyme-linked Immunosorbent Assay, Control

Journal: Journal of Diabetes Research

Article Title: Huajuxiaoji Formula Alleviates Phenyl Sulfate-Induced Diabetic Kidney Disease by Inhibiting NLRP3 Inflammasome Activation and Pyroptosis

doi: 10.1155/2024/8772009

Figure Lengend Snippet: HJXJ alleviates PS-induced ROS production, pyroptosis, and NLRP3 inflammasome activation in MPC5 cells. MPC5 cells are treated with 1 mM PS in the absence or presence of different HJXJ concentrations. Flow cytometry was used to evaluate (a) ROS production and (b, c) pyroptosis. Western blotting is used to measure expressions of NLRP3, Caspase-1 p20, GSDMD-N, podocin, and nephrin (d, e). ELISA kits are used to measure (f) release of IL-1 β and IL-18. ∗∗∗ P < 0.001 versus control. # P < 0.05, ## P < 0.01, ### P < 0.001 versus PS.

Article Snippet: Thereafter, the membranes were blocked with 5% skim milk for 1 h and probed with specific primary antibodies at 4°C for 12 h. The antibodies used were specific for podocin (ab181143), nephrin (ab216341), NLRP3 (ab263899), gasdermin D-N-terminal (GSDMD-N) (ab215203), ZO-1 (ab216880), occludin (ab216327) (Abcam, Cambridge, UK), Caspase-1 p20 (sc-398715, Santa Cruz Biotechnology, California, USA), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH, #5174, CST, USA) at 4°C for 12 h, followed by secondary antibodies for 2 h. An enhanced chemiluminescence system was used to detect protein expression.

Techniques: Activation Assay, Flow Cytometry, Western Blot, Enzyme-linked Immunosorbent Assay, Control

Journal: Journal of Diabetes Research

Article Title: Huajuxiaoji Formula Alleviates Phenyl Sulfate-Induced Diabetic Kidney Disease by Inhibiting NLRP3 Inflammasome Activation and Pyroptosis

doi: 10.1155/2024/8772009

Figure Lengend Snippet: HJXJ alleviates Nlrp3 overexpression-induced pyroptosis and NLRP3 inflammasome activation in MPC5 cells. MPC5 cells are transduced with Nlrp3 expression vector or blank vector. (a) The NLRP3 mRNA expression is measured using quantitative real-time PCR, and (b) protein expression is measured using western blotting. MPC5 cells are pretreated with 10% HJXJ and transduced with Nlrp3 expression vector. Flow cytometry is used to evaluate (c, d) pyroptosis level. Western blotting is used to measure (e, f) expressions of NLRP3, Caspase-1 p20, and GSDMD-N. ELISA kits are used to measure (g) release of IL-1 β and IL-18 .∗∗∗ P < 0.001 versus vector. # P < 0.05, ## P < 0.01, ### P < 0.001 versus oeNlrp3.

Article Snippet: Thereafter, the membranes were blocked with 5% skim milk for 1 h and probed with specific primary antibodies at 4°C for 12 h. The antibodies used were specific for podocin (ab181143), nephrin (ab216341), NLRP3 (ab263899), gasdermin D-N-terminal (GSDMD-N) (ab215203), ZO-1 (ab216880), occludin (ab216327) (Abcam, Cambridge, UK), Caspase-1 p20 (sc-398715, Santa Cruz Biotechnology, California, USA), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH, #5174, CST, USA) at 4°C for 12 h, followed by secondary antibodies for 2 h. An enhanced chemiluminescence system was used to detect protein expression.

Techniques: Over Expression, Activation Assay, Transduction, Expressing, Plasmid Preparation, Real-time Polymerase Chain Reaction, Western Blot, Flow Cytometry, Enzyme-linked Immunosorbent Assay