Journal: Journal of Virology

Article Title: Kidney organoids reveal redundancy in viral entry pathways during ACE2-dependent SARS-CoV-2 infection

doi: 10.1128/jvi.01802-23

Figure Lengend Snippet: Drug inhibition assays revealing dual viral entry pathway usage by SARS-CoV-2. ( A ) Bar graph depicting the viral titres (log 10 TCID 50 /mL) of culture media sampled from PT-enhanced organoids 6 days post-infection, treated with either protease inhibitors (Camostat and E64d, alone or in combination; dark/light red bars), drug reconstitution reagent (DMSO controls; dark/light blue bars), or remaining untreated (no drug controls; light/dark green bars). PT-enhanced organoids were infected with WA1 SARS-CoV-2. (icSARS-CoV-2-GFP). LOD and dotted line represent lower limit of detection. Error bars represent SEM from three independent experiment, with three (drug inhibition tests and no drug controls) or two (DMSO controls) biological replicates per timepoint. Significance was calculated using a one-way ANOVA with Tukey’s multiple comparisons test. Asterisks represent two-tailed P values (* P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001). NS, not significant. ( B ) scRNAseq DotPlot of PT-enhanced organoids (day 14 of organoid culture) depicting the expression of TMPRSS genes within the highest ACE2 -expressing proximal tubule clusters and distal (connecting segment) nephron cluster. The proximal tubule cluster most enriched with ACE2 expression is outlined in gray. Dot size represents the percentage of cells expressing a gene within each cluster, while shade intensity correlates with gene expression level. ( C and D ) Confocal immunofluorescence of TMPRSS10 (C, red) and corresponding rat isotype control (D, red), co-stained with markers of proximal tubule (LTL, blue) and nephron epithelium (EPCAM, green). Arrows indicate region shown at higher magnification in inset images. Scale bars represent 50 µm. ( E ) Confocal immunofluorescence of SARS-CoV-2-infected and uninfected (control) PT-enhanced organoids, depicting virus (dsRNA, red) within TMPRSS10-expressing (green) proximal tubules (marked with LTL, blue) within infected samples and lack of dsRNA detection in uninfected control. Arrows depict examples of dsRNA staining. Scale bars represent 50 µm.

Article Snippet: For inhibition of proteases involved in viral infection, organoids were treated with either 10 μM of Camostat mesylate (Sigma Aldrich, MO, USA, cat. SML0057), 10 μM of E64d (Selleck Chem, TX, USA, cat. S7393), Camostat + E64 d combined (10 μM each), or an equivalent concentration of the drug reconstitution reagent, DMSO (Sigma Aldrich).

Techniques: Inhibition, Infection, Two Tailed Test, Expressing, Gene Expression, Immunofluorescence, Control, Staining, Virus

Journal: Cellular and Molecular Life Sciences

Article Title: The secreted inhibitor of invasive cell growth CREG1 is negatively regulated by cathepsin proteases

doi: 10.1007/s00018-020-03528-5

Figure Lengend Snippet: CREG1 abundance is post-translationally modulated by CTSB inhibition and CTSB induction. a The abundance of CREG1 is increased in PyMT cells treated with the broad spectrum CTSB inhibitor E64d (10 µM). b CREG1 abundance can be reduced upon doxycycline induction (1 µM) of CTSB in a PyMT Ctsb −/− cell line carrying a CTSB doxycycline-inducible vector. c No significant changes of CREG1 at the mRNA level (qRT-PCR) were observed in tumor-cell co-cultures carrying Mϕ lacking both Ctsb and Ctsz compared to wild-type Mϕ (dashed line). d Inhibition of CTSB in PyMT cells with E64d (10 µM) or with CA-074 (10 µM) lead to no significant changes of CREG1 at the mRNA level compared to control (dashed line). n.s. non-significant change, Mϕ macrophages, indCTSB doxycycline-inducible CTSB, DOX doxycycline, CTSB human CTSB

Article Snippet: Cysteine CTSB protease was inhibited by addition of E64d (10 μM) or CA-074 (10 μM) (both Bachem, Bubendorf, Switzerland), using DMSO as solvent control, as reported [ ].

Techniques: Inhibition, Plasmid Preparation, Quantitative RT-PCR, Control

Journal: International Journal of Molecular Sciences

Article Title: Investigations on Primary Cilia of Nthy-ori 3-1 Cells upon Cysteine Cathepsin Inhibition or Thyrotropin Stimulation

doi: 10.3390/ijms24119292

Figure Lengend Snippet: Staining and morphological analysis of cilia upon E64 and E64d inhibition in Nthy-ori 3-1 cells . Confocal LSM micrographs of non-treated controls ( A ), DMSO solvent controls ( B ), and Nthy-ori 3-1 cells treated for 5 h with 10 μM E64d ( C ) and with 10 μM E64 ( D ). ( A – D ) Merged and single channel micrographs are shown as indicated for Arl13b-stained cilia (arrows, green, white) and Draq5™-stained nuclei (N) (red, grey). ( A 1 – D 1 ) denote Arl13b-positive structures (cilia). ( A 2 – D 2 ) denote nuclei depicted in inverted contrast. Arrows in ( A – D ) and ( A 1 – D 1 ) point to cilia. The average cilia frequencies per cell ( E ), lengths ( F ), and perimeters ( G ) representative of the Arl13b signal were measured using CellProfiler software in non-treated controls, solvent controls, and E64d- and E64-treated cells as indicated. For each condition, 10 images were retrieved from two coverslips of the same experimental setup. ( E – G ) display the data retrieved by using bar charts, where means are represented by the height of each bar, and individual data points are indicated by symbols. The number of cells probed without inhibitor was n = 365; solvent control, n = 250; E64d inhibitor, n = 363; E64 inhibitor, n = 529. Nuclei were counterstained with Draq5™. Scale bars represent 20 µm.

Article Snippet: Cysteine peptidase activities were inhibited in confluently grown Nthy-ori 3-1 cells via incubation with 10 μM E64 (Carl Roth GmbH, Karlsruhe, Germany, #2935) or 10 μM E64d (Enzo Life Sciences, Lörrach, Germany, #BML-p1107-0001) for 5 h. As a vehicle of control for E64 or E64d, DMSO (Carl Roth GmbH, Karlsruhe, Germany, #4720.1) (0.1%, final concentration) was used.

Techniques: Staining, Inhibition, Software