Journal: eLife

Article Title: SARS-CoV-2 entry into human airway organoids is serine protease-mediated and facilitated by the multibasic cleavage site

doi: 10.7554/eLife.64508

Figure Lengend Snippet:

Article Snippet: Aloxistatin , E64D , MedChemExpress , Cat# HY-100229 , .

Techniques: Virus, Membrane, Sterility, Concentration Assay, Low Protein Binding, Recombinant, Plasmid Preparation, Silver Staining, Mass Spectrometry, Microscopy, Software

Journal: Virologica Sinica

Article Title: Construction of Non-infectious SARS-CoV-2 Replicons and Their Application in Drug Evaluation

doi: 10.1007/s12250-021-00369-9

Figure Lengend Snippet: Assembly of a SARS-CoV-2-GFP replicon. A Genome structure of SARS-CoV-2 and workflow of fragment generation to assemble a SARS-CoV-2-GFP replicon. The open reading frames (ORFs) of viral genes and sequences of untranslated regions (UTRs) are indicated. B Strategy for transformation-associated recombination (TAR) of SARS-CoV-2-GFP replicon fragments in yeast. Relevant sequences are indicated. CMVp: cytomegalovirus promoter; ORF1ab ΔNSP1: ORF of viral 1ab gene deleted with coding region for NSP1; tGFP-BlaR: GFP-blasticidin deaminase fusion gene; HDVRZ: hepatitis delta virus ribozyme; BGH: bovine growth hormone termination and polyadenylation sequences.

Article Snippet: A , B 293 T cells were transfected with SARS-CoV-2-GFP replicon, E64-D and remdesivir were added to culture media 10 h post transfection or just before transfection respectively, the green fluorescence intensity was determined thirty-six hours post-transfection, and dose responsive curves were drawn and EC 50 value of E64-D ( A ) and remdesivir ( B ) was calculated by four-parameter nonlinear regression respectively.

Techniques: Transformation Assay

Journal: Virologica Sinica

Article Title: Construction of Non-infectious SARS-CoV-2 Replicons and Their Application in Drug Evaluation

doi: 10.1007/s12250-021-00369-9

Figure Lengend Snippet: Sequence analysis of SARS-CoV-2-GFP replicon.

Article Snippet: A , B 293 T cells were transfected with SARS-CoV-2-GFP replicon, E64-D and remdesivir were added to culture media 10 h post transfection or just before transfection respectively, the green fluorescence intensity was determined thirty-six hours post-transfection, and dose responsive curves were drawn and EC 50 value of E64-D ( A ) and remdesivir ( B ) was calculated by four-parameter nonlinear regression respectively.

Techniques: Sequencing

Journal: Virologica Sinica

Article Title: Construction of Non-infectious SARS-CoV-2 Replicons and Their Application in Drug Evaluation

doi: 10.1007/s12250-021-00369-9

Figure Lengend Snippet: Verification of a SARS-CoV-2-GFP replicon plasmid. A Recombinant yeast colonies on CSM-Trp agar plates. B Transformed yeast clone was tested for fragments F12–F13, F18–F19, and F23–F25 by yeast colony PCR. C E. coli clones transformed with yeast plasmid were tested for F16–F17 by PCR. D Recombinant SARS-CoV-2-GFP replicon plasmid was tested for all nine sub-genomic fragments used in yeast homologous recombination by PCR.

Article Snippet: A , B 293 T cells were transfected with SARS-CoV-2-GFP replicon, E64-D and remdesivir were added to culture media 10 h post transfection or just before transfection respectively, the green fluorescence intensity was determined thirty-six hours post-transfection, and dose responsive curves were drawn and EC 50 value of E64-D ( A ) and remdesivir ( B ) was calculated by four-parameter nonlinear regression respectively.

Techniques: Plasmid Preparation, Recombinant, Transformation Assay, Clone Assay, Homologous Recombination

Journal: Virologica Sinica

Article Title: Construction of Non-infectious SARS-CoV-2 Replicons and Their Application in Drug Evaluation

doi: 10.1007/s12250-021-00369-9

Figure Lengend Snippet: SARS-CoV-2-GFP replicon transfection assay. A Fluorescence microscopy monitoring tGFP expression. 293 T cells were transfected or nontransfected (BLANK) with SARS-CoV-2-GFP replicon, and 36 h post-transfection, cells were fixed, observed and photographed, the nuclei were stained with Hoechst dye, scale bar: 100 µm. B 293 T cells were transfected or nontransfected (BLANK) with SARS-CoV-2-GFP replicon, and 36 h post-transfection, cells were lysed followed by Western blot to monitor the expression of tGFP-BlaR, Nucleocapsid protein (N), and Actin was used as a loading control. C Kinetics of tGFP-BlaR expression, 293 T cells were transfected with SARS-CoV-2-GFP replicon and were lysed at indicated hours post-transfection (hpt) and tGFP-BlaR expression was monitored by Western blot, Actin was used as a loading control.

Article Snippet: A , B 293 T cells were transfected with SARS-CoV-2-GFP replicon, E64-D and remdesivir were added to culture media 10 h post transfection or just before transfection respectively, the green fluorescence intensity was determined thirty-six hours post-transfection, and dose responsive curves were drawn and EC 50 value of E64-D ( A ) and remdesivir ( B ) was calculated by four-parameter nonlinear regression respectively.

Techniques: Transfection, Fluorescence, Microscopy, Expressing, Staining, Western Blot

Journal: Virologica Sinica

Article Title: Construction of Non-infectious SARS-CoV-2 Replicons and Their Application in Drug Evaluation

doi: 10.1007/s12250-021-00369-9

Figure Lengend Snippet: Inhibitory effect of E64-D and remdesivir on a SARS-CoV-2-GFP replicon. A , B 293 T cells were transfected with SARS-CoV-2-GFP replicon, and ten hours post-transfection, cells were treated or nontreated (BLANK) with E64-D at indicated concentrations. Thirty-six hours post-transfection, cells were observed and photographed by fluorescence microscopy, the nuclei were stained with Hoechst, scale bar: 100 µm ( A ) or lysed for Western blot monitored the expression of tGFP-BlaR, actin was used as a loading control ( B ). C , D 293 T cells were transfected SARS-CoV-2-GFP replicon, and cells were treated or nontreated (BLANK) with remdesivir at indicated concentrations just before transfection. Thirty-six hours post-transfection, cells were observed and photographed using fluorescence microscopy, the nuclei were stained with Hoechst, scale bar: 100 µm ( C ), or lysed for Western blot for tGFP-BlaR, Actin was used as a loading control ( D ).

Article Snippet: A , B 293 T cells were transfected with SARS-CoV-2-GFP replicon, E64-D and remdesivir were added to culture media 10 h post transfection or just before transfection respectively, the green fluorescence intensity was determined thirty-six hours post-transfection, and dose responsive curves were drawn and EC 50 value of E64-D ( A ) and remdesivir ( B ) was calculated by four-parameter nonlinear regression respectively.

Techniques: Transfection, Fluorescence, Microscopy, Staining, Western Blot, Expressing

Journal: Virologica Sinica

Article Title: Construction of Non-infectious SARS-CoV-2 Replicons and Their Application in Drug Evaluation

doi: 10.1007/s12250-021-00369-9

Figure Lengend Snippet: Determining the EC 50 of E64-D and remdesivir using a SARS-CoV-2-GFP replicon. A , B 293 T cells were transfected with SARS-CoV-2-GFP replicon, E64-D and remdesivir were added to culture media 10 h post transfection or just before transfection respectively, the green fluorescence intensity was determined thirty-six hours post-transfection, and dose responsive curves were drawn and EC 50 value of E64-D ( A ) and remdesivir ( B ) was calculated by four-parameter nonlinear regression respectively. Cell viability was determined by CCK8 assay in parallel on 293 T cells. The experiments were done in triplicates. C – F Replicon transfection and drug treatment were performed as mentioned in ( A , B ), thirty-six hours post-transfection, Western blot for tGFP-BlaR expression was carried out with E64-D ( C ) and remdesivir treated cells ( E ). EC 50 value of E64-D ( F ) and remdesivir ( G ) as determined by dose response curves using the ratio of tGFP-BlaR/β-actin gray values with different concentration and calculated by four-parameter nonlinear regression. The experiments were done at least two times.

Article Snippet: A , B 293 T cells were transfected with SARS-CoV-2-GFP replicon, E64-D and remdesivir were added to culture media 10 h post transfection or just before transfection respectively, the green fluorescence intensity was determined thirty-six hours post-transfection, and dose responsive curves were drawn and EC 50 value of E64-D ( A ) and remdesivir ( B ) was calculated by four-parameter nonlinear regression respectively.

Techniques: Transfection, Fluorescence, CCK-8 Assay, Western Blot, Expressing, Concentration Assay

Journal: Virologica Sinica

Article Title: Construction of Non-infectious SARS-CoV-2 Replicons and Their Application in Drug Evaluation

doi: 10.1007/s12250-021-00369-9

Figure Lengend Snippet: Construction a SARS-CoV-2-GFP-FLuc replicon and determination of the EC 50 of E64-D. A Diagram of SARS-CoV-2-GFP-FLuc replicon. B Verification of a SARS-CoV-2-GFP-FLuc replicon plasmid. Recombinant SARS-CoV-2-GFP-FLuc replicon plasmid was tested for all sub-genomic fragments used in yeast homologous recombination by PCR. C Luciferase activity in nontransfected (BLANK) or SARS-CoV-2-GFP-FLuc replicon-transfected or 293 T cells, the luciferase activity was determined 36 h post transfection. D Kinetics study of luciferase activity after SARS-CoV-2-GFP-FLuc replicon transfection, 293 T cells were transfected with SARS-CoV-2-GFP-Fluc replicon and luciferase activity was determined at indicated hours post-transfection (hpt). E EC 50 value of E-64D was determined using a dose response curve drawn based on luciferase activity in SARS-CoV-2-GFP-FLuc-transfected 293 T cells using four-parameter nonlinear regression. F EC 50 value of E-64D was determined using a dose response curve drawn based on the ratio of tGFP-BlaR/β-actin gray values with different concentration and calculated by four-parameter nonlinear regression, the experiments were done at least two times.

Article Snippet: A , B 293 T cells were transfected with SARS-CoV-2-GFP replicon, E64-D and remdesivir were added to culture media 10 h post transfection or just before transfection respectively, the green fluorescence intensity was determined thirty-six hours post-transfection, and dose responsive curves were drawn and EC 50 value of E64-D ( A ) and remdesivir ( B ) was calculated by four-parameter nonlinear regression respectively.

Techniques: Plasmid Preparation, Recombinant, Homologous Recombination, Luciferase, Activity Assay, Transfection, Concentration Assay

Journal: Cell reports

Article Title: HLS1 interacts with ATG8 to negatively regulate the ABS3-mediated plant senescence pathway.

doi: 10.1016/j.celrep.2025.115507

Figure Lengend Snippet: Figure 6. C-deprivation induces HLS1 degradation (A) Seedlings of hls1-1 p35S:GFP-HLS1 were subjected to C-deprivation treatment for the indicated time periods in liquid medium containing MG132, E-64d, or an equal amount of DMSO. GFP-HLS1 was detected by immunoblotting using an anti-GFP antibody. Anti-PBA1 served as the loading control. (B) Quantification of GFP-HLS1 levels in (A). GFP-HLS1 band intensities were normalized to the levels of PBA1. The GFP-HLS1 level at 0 h of C-deprivation in each treatment was defined as 100%. Data are mean ± SD. n = 3. (C) Seedlings of hls1-1 p35S:GFP-HLS1 lines were subjected to C-deprivation treatment as described in (A) for 6 h. GFP-HLS1 signals in root tips were imaged with fluorescence microscopy. (D) Quantification of GFP-HLS1 signals shown in (C). (E) Seedlings of hls1-1 p35S:GFP-HLS1 lines were subjected to C-deprivation treatment as described in (A) for 2 h. GFP-HLS1 signals in root epidermal cells were imaged with confocal microscopy. (F) Quantification of GFP-HLS1 signals on the plasma membrane and in the nucleus shown in (E). In (D) and (F), data are median ± interquartile range. Each dot represents one seedling (D) or one cell (F). n is listed in parentheses. Two tailed t test, ****p < 0.0001. Scale bars, 100 mm (C) and 10 mm (E).

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies anti-GFP Takara Bio Cat#632381; RRID: AB_2313808 anti-HA Roche Cat#11867431001; RRID: AB_390919 anti-MBP Abcam Cat#ab65; RRID: AB_308711 anti-His Abcam Cat#ab18184; RRID: AB_444306 anti-GST Abcam Cat#ab19256; RRID: AB_444809 anti-PEPC1 PhytoAB Cat#PHY2037S anti-PBA1 Abcam Cat#ab98861; RRID: AB_10672453 anti-H3 Abcam Cat#ab1791; RRID: AB_302613 Biological samples Agrobacterium tumefaciens GV3101 TSINGKE Cat#TSC-A01 Escherichia coli strain BL21 (DE3) TSINGKE Cat#TSC-E01 Chemicals, peptides, and recombinant proteins Murashige and Skoog (MS) medium PhytoTechnology Laboratories Cat#M153 Bacto agar BD Cat#214010 Trizol Reagent Thermo Fisher Scientific Cat#15596018 Ni Sepharose 6 Fast Flow resin GE Healthcare Cat#17-5318-02 Amylose resin New England Biolabs Cat#E8021 Glutathione Sepharose 4B resin GE Healthcare Cat#17-0756-01 GFP-Trap ChromoTek Cat#gtma-20 MG132 GLPBIO Cat#GC10383 Protease inhibitor cocktail Roche Cat#4693132001 E-64d Selleckchem Cat#S7393 DMSO Sigma-Aldrich Cat#D8418 Triton X-100 Amresco Cat#0694 BSA VWR Cat#0332 MBP-HLS1 this study N/A His-ATG8e this study N/A Critical commercial assays UEIris RT mix with DNase (All-in-One) US EVERBRIGHT Cat#R2020 ChamQ SYBR qPCR Master Mix Vazyme Cat#Q311-02 Gibson Assembly Master mix New England BioLabs Cat#E2611 ZymoPURETM Plasmid Maxiprep Kit ZYMO Research Cat#D4203 EndoFree Maxi Plasmid Kit TIANGEN Cat#DP117 Q5 High-Fidelity DNA Polymerase New England BioLabs Cat#M0491L PrimeSTAR GXL DNA Polymerase Takara Cat#TKR-R050B Experimental models: Organisms/strains Columbia-0 (Col-0) ABRC CS70000 hls1-1 ABRC CS3073 hls1-102 ABRC SALK_136528C hls1-101 this study N/A p35S:ABS3-GFP Wang et al., 20157 N/A pUBQ10:mCherry-ARA7 Geldner et al., 200933 Wave_2R pUBQ10:mCherry-VTI12 Geldner et al., 200933 Wave_13R (Continued on next page) 14 Cell Reports 44, 115507, April 22, 2025

Techniques: Western Blot, Control, Microscopy, Confocal Microscopy, Clinical Proteomics, Membrane, Two Tailed Test

Journal: Nature microbiology

Article Title: Spatiotemporal proteomics uncovers cathepsin-dependent macrophage cell death during Salmonella infection

doi: 10.1038/s41564-020-0736-7

Figure Lengend Snippet: a ) Nuclei were extracted from RAW264.7 cells treated with DCG04-Boclicky-TAMRA (5 μM) for 2 hours prior to harvest at 20 hpi with wildtype (Wt), a SPI-2 mutant (Δ ssaV ) or heat killed wildtype bacteria (Wt HK) (MOI = 100:1). Formaldehyde fixed nuclei were subsequently counterstained with Hoechst 33342 and analysed by flow cytometry. Cells in G1, S or G2 phase of the cell cycle are separated by Hoechst 33342 staining on the x-axis. Cathepsin activity (DCG04-Boclicky-TAMRA) is indicated by the % of total DCG04-Boclicky-TAMRA positive nuclei in either sub-G1 or G1/S/G2 or combined total of sub-G1 and G1/2/G2. Data combining all biological replicates from two independent experiments can be seen in Extended Data Fig. 4. b ) Pyroptotic cell death was assessed by quantifying LDH release into culture supernatants of RAW264.7 cells infected with wildtype, SPI-2 (Δ ssaV ) and the effector deletion (Δ sifA) mutants in the presence of 25 μM CA-074-Me or DMSO solvent control at 19 hpi. The Δ sifA mutant has a strong replication defect and readily escapes into the cytoplasm, hyperactivating Caspase-11 dependent cell death . (n) denotes biologically independent samples combined from three independent experiments (batches); each batch contained a minimum of 4 biological replicate wells per condition. Data represents the % LDH release per condition relative maximum LDH release (see Methods). Box plots are depicted as in Fig. 3C. An unpaired t-test (two-sided) was used to calculate p . c ) BMDMs were infected with wild-type S Tm (MOI 100:1), followed by incubation in the presence of cathepsin inhibitor CA-074-Me at the indicated concentrations. At the indicated hpi’s, cell death was measured as the % of LDH released into culture supernatants. Data points represent the mean and error bars indicate the 95% CI. (n) denotes biologically independent samples. Time points 0, 10 and 14 hours are derived from three biological replicates per condition (n=3 per condition) from a single batch, whereas the 18-hour time point contains combined data from 3 or more independent experiments (batches), each batch containing 3-4 biological replicates per condition (DMSO n=30; CA-074-Me (12.5 μM) n=28; CA-074-Me (25 μM) n=13). d ) related to a ), wildtype, caspase-11/1 -/-, caspase-11 -/-, NLRP3 -/-, NLRC4 -/- BMDMs were infected with wildtype S Tm (MOI 100:1), followed by incubation in the presence of cathepsin inhibitor CA-074-Me (12.5 μM) for 18 hpi. The % LDH released into culture supernatants was measured 18 hours post-infection. n denotes biologically independent samples combined from >3 (wildtype) and 3 (mutant genotypes) independent experiments (batches), each batch containing 2-4 biological replicates per condition. A two-sided unpaired t-test was used to calculate p . e ) RAW264.7 cell were transfected with LPS with Fugene (Promega) for 20 hours. Pyroptotic cell death was assessed by quantifying LDH release into culture supernatants in the presence of 25 μM CA-074-Me relative to a DMSO solvent control. (n) denotes biologically independent samples combined from three independent experiments (batches), each batch consisting of a minimum of 3 biological replicate wells per condition. Boxplots are depicted as in Fig. 3C. A two-sided unpaired t-test was used to calculate p .

Article Snippet: In brief, 10 U/100 μL of recombinant Caspase-11 (mouse, Enzo life sciences; BML-SE155-5000) in caspase activity buffer (200 mM NaCl, 50 mM HEPES pH 8.0, 50 mM KCl, 10 mM DTT) supplemented with 100 μMAcLEHD-afc (Santa Cruz; sc-311277) was incubated in the presence of CA-074-Me, E64d (Sigma; E8640) and the Caspase-9/11 inhibitor Z-LEHD-FMK (Abcam; ab142026) or a DMSO solvent control as the indicated concentrations on the figure legend.

Techniques: Mutagenesis, Flow Cytometry, Staining, Activity Assay, Infection, Incubation, Derivative Assay, Transfection

Journal: Nature microbiology

Article Title: Spatiotemporal proteomics uncovers cathepsin-dependent macrophage cell death during Salmonella infection

doi: 10.1038/s41564-020-0736-7

Figure Lengend Snippet: CA-074-Me inhibits cathepsins and reduces expression of proinflammatory proteins. a) 2D-TPP of RAW264.7 cells infected with wildtype S Tm 14028s for 20 hours in the presence of increasing concentrations of CA-074-Me (1-100 μM). To assess CA-074-Me specificity, we applied 2-dimensional Thermal Proteome Profiling (2D-TPP). 2D-TPP enables proteome assessment of protein ligand binding based on the principle that ligand-bound proteins are more thermally stable than proteins not bound to a ligand 20,21 , and in parallel provides information of proteome-wide protein abundance. We subjected RAW264.7 cells infected with wildtype S Tm to increasing doses of CA-074-Me and compared proteome-wide thermostabilisation relative to solvent controlled cells (see methods). Increased (blue) or decreased fold changes are calculated by normalising the abundance of each protein relative to the abundance in the DMSO vehicle per temperature. Protein stabilization (red framed) is demonstrated by increasing (blue) fold changes with respect to increasing temperature (top to bottom) as well as with increasing drug concentrations (left to right). Changes in protein expression (green framed) can be detected if abundance changes are already evident from low temperatures i.e. before proteins melt. All cathepsins detected by 2D-TPP are displayed. CtsA and CtsD are serine and aspartic-acid proteases, respectively, and are therefore not targeted by CA-074-Me and serve as negative controls. Contrary to evidence demonstrating CA-074-Me targets CtsL 35 , we failed to detect its stabilisation in our experiment. Cathepsins are arranged in the top row, followed by caspases and other cellular proteases in descending order. Note Caspase-1 stability was not affected by CA-074-Me, in line with previous reports 22 . Additionally, CA-074-Me reduced IL-1b expression as previously shown 35,36 . 2D-TPP data can be found in . b) Caspase-11-like proteolysis activity was assessed using purified active Caspase-11 and measuring cleavage of the fluorescent substrate AcLEHD-afc as previously described 37 . Caspase-11-like proteolytic activity was assessed in the presence of indicated concentrations of CA-074-Me and the Caspase-11 inhibitor Z-LEHD-FMK as a positive control. Activity is expressed as the % of AcLEHD-afc cleavage relative to the DMSO solvent control. Data is combined from n = 3 biologically independent experiments; error bars depict standard deviation and the center value denotes the mean. c) 2D-TPP profiles of inflammation related proteins reduced in abundance upon CA-074-Me treatment. Data presented as in (a).

Article Snippet: In brief, 10 U/100 μL of recombinant Caspase-11 (mouse, Enzo life sciences; BML-SE155-5000) in caspase activity buffer (200 mM NaCl, 50 mM HEPES pH 8.0, 50 mM KCl, 10 mM DTT) supplemented with 100 μMAcLEHD-afc (Santa Cruz; sc-311277) was incubated in the presence of CA-074-Me, E64d (Sigma; E8640) and the Caspase-9/11 inhibitor Z-LEHD-FMK (Abcam; ab142026) or a DMSO solvent control as the indicated concentrations on the figure legend.

Techniques: Expressing, Infection, Ligand Binding Assay, Activity Assay, Purification, Positive Control, Standard Deviation

Journal: Nature microbiology

Article Title: Spatiotemporal proteomics uncovers cathepsin-dependent macrophage cell death during Salmonella infection

doi: 10.1038/s41564-020-0736-7

Figure Lengend Snippet: CA-074-Me does not affect Caspase-11 expression or processing. Wildtype BMDMs were infected with wildtype S Tm for 20 hours in the presence of CA-074-Me (25 μM) or DMSO control. Lysates were probed for Caspase-11 by immunoblot and using GAPDH as a loading control. Experiment was performed once.

Article Snippet: In brief, 10 U/100 μL of recombinant Caspase-11 (mouse, Enzo life sciences; BML-SE155-5000) in caspase activity buffer (200 mM NaCl, 50 mM HEPES pH 8.0, 50 mM KCl, 10 mM DTT) supplemented with 100 μMAcLEHD-afc (Santa Cruz; sc-311277) was incubated in the presence of CA-074-Me, E64d (Sigma; E8640) and the Caspase-9/11 inhibitor Z-LEHD-FMK (Abcam; ab142026) or a DMSO solvent control as the indicated concentrations on the figure legend.

Techniques: Expressing, Infection, Western Blot