Fig 1: PMA reverses the effect of ROCK2 silencing on apoptosis related protein expression in LPS-induced HK-2 cells. (A) Protein expression of Bcl-2, Bax, cleaved caspase 3/caspase 3 and cleaved PARP/PARP was measured using western blotting. (B) Protein expression levels of KIM-1 and NGAL was measured using western blotting. ***P<0.001 vs. control; $$$P<0.001 vs. LPS; #P<0.05, ##P<0.01 and ###P<0.001 vs. LPS + shRNA-ROCK2; +P<0.05, ++P<0.01 and +++P<0.001 vs. DMSO + LPS + shRNA-ROCK2. ROCK2, Rho-associated protein kinase 2; LPS, lipopolysaccharide; sh, short hairpin; PARP, poly (ADP-ribose) polymerase; KIM-1, kidney injury molecule-1; NGAL, neutrophil gelatinase-associated lipocalin; PMA, phorbol 12-myristate 13-acetate.
Fig 2: Mel reduced expression of kidney injury markers. (a) Immunofluorescence staining of NGAL (red). Magnification: 200x. (b) Immunofluorescence staining of KIM-1 (green). Magnification: 200x. (c) The expression of NGAL and KIM-1 was detected by western blot analysis. ****p < 0.0001 vs. control and ####p < 0.0001 vs. LPS, n = 3.
Fig 3: Patients with NPHPL and compound heterozygous mutations of XPNPEP3(A) Pedigrees of Family A and Family B. Family A harbors variants c.634G>C and c.761G>T; Family B harbors variants c.-87C>T and c.1261C>G. Arrows indicate nucleotide changes. Square for males; circles for females; solid for patients with NPHPL1; slash for deceased individuals.(B) Ultrasound image of patient A-II-1. Asterisks for medulla.(C) Abdominal CT image of patient B-II-1, showing the left kidney cyst (left panel) and left ilium cystic foci (right panel). Arrowheads indicate the foci.(D) Immunofluorescence analysis of KIM1 and NGAL in tubular epithelial cells. AQP1, aquaporin 1, a water channel protein located in the kidney proximal tubule. Bar: 5μm.(E) Schematic of XPNPEP3 gene variants. The XPNPEP3 gene is located on chromosome 22q. Both the c.634G>C and c.761G>T variants in Family A are located in exon 4, while the c.-87C>T and c.1261C>G variants in Family B are distributed in the noncoding region and exon 9, respectively.(F) Conservation analysis of XPNPEP3 proteins. Linear domain organization of the XPNPEP3 protein showing the p.A212 residue located in the AMP_N (aminopeptidase) domain and the p.R254 residue and p.H421 residue in the prolidase domain. MTS, mitochondrial target signal.
Fig 4: RBCEV-mediated viral neutralization is dependent on TIM-1 expression. (a) Mean fluorescent intensity of GPA signals indicating degree of EV binding for Vero E6 and A549-ACE2 cells following transfection with a negative control (NC) siRNA or TIM-1 siRNA. (b) Relative infection of the SARS-CoV-2 spike (w/t) pseudotyped virus in Vero E6 and A549-ACE2 cells following TIM-1 knockdown. (c) Percentage neutralization of SARS-CoV-2 spike (w/t) pseudotyped virus in Vero E6 and A549-ACE2 cells in the presence of RBCEVs following TIM-1 knockdown. (d) Effect of TIM-1 knockdown on the relative infection of Vero E6 and A549-ACE2 cells by authentic SARS-CoV-2. (e) Effect of knocking down TIM-1 on the ability of RBCEVs to neutralize authentic SARS-CoV-2 (w/t) infection in Vero E6 and A549-ACE2 cells. (f) Mean GPA signals indicative of relative EV binding following the transfection of 293T-ACE2 cells with a control plasmid or TIM-1 plasmid. (g) Relative infection of 293T-ACE2 cells with SARS-CoV-2 spike (w/t) pseudotyped virus following transfection with a control or TIM-1 plasmid. (h) Percentage neutralization of SARS-CoV-2 spike (w/t) pseudotyped virus infection in 293T-ACE2 cells using RBCEVs following TIM-1 overexpression. (i) Effect of TIM-1 overexpression on the relative infection of authentic SARS-CoV-2 (w/t) infection. (j) Effect of TIM-1 overexpression in 293T-ACE2 cells on the ability of RBCEVs to neutralize authentic SARS-CoV-2 (w/t) infection. (k) Representative bioluminescent images depicting the relative infection of NSG mice with VSV.G pseudotyped virus carrying a firefly luciferase reporter upon coadministration with RBCEVs or a vehicle control of PBS. Viruses and EVs were administered intratracheally. Mice were administered with 50 μL of undiluted VSV.G pseudotyped virus. An RBCEV concentration of 2 μg/μL was utilized for the RBCEV+PV condition. (l) Luciferase signals from mice administered with VSV.G pseudotyped virus carrying a firefly luciferase reporter with or without coadministration of RBCEVs as described in (k) (n = 4 mice). For (a–j), an RBCEV concentration of 0.2 μg/μL was utilized, and each replicate represents data obtained from 3 to 8 biological replicates performed using EVs from 3 separate donors or cells from separate passages. The graphs represent the mean ± standard deviation and data were compared using Student’s one-tailed t test such that *P < 0.05, **P < 0.01, and ***P < 0.001.
Fig 5: RBCEVs compete for binding to TIM-1 and inhibit viral entry. (a) The expression of TIM-1 in Vero E6, A549-ACE2, HuH-7, and 293T-ACE2 cells determined using flow cytometric analysis. (b) Relative levels of binding of RBCEVs to the cell lines are shown in (a). RBCEV binding was assessed using flow cytometric analysis staining for GPA. (c) Effect of the presence or absence of a TIM-1 blocking antibody on the relative amount of RBCEV binding to each of the cell lines shown in (a), assessed using flow cytometry. (d) Effect of the TIM-1 blocking antibody or RBCEVs (at a range of doses) on the binding of SARS-CoV-2 spike (w/t) pseudotyped virus to Vero E6, A549-ACE2, HuH-7, and 293T-ACE2 cells (n = 3 biological repeats). The graphs represent the mean ± standard deviation and data were compared using Student’s one-tailed t test such that *P < 0.05, **P < 0.01, and ***P < 0.001.
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