Journal: Cell reports

Article Title: The Cyclopeptide Astin C Specifically Inhibits the Innate Immune CDN Sensor STING.

doi: 10.1016/j.celrep.2018.11.097

Figure Lengend Snippet: Figure 4. Astin C May Block the Activation Pocket of STING (A) The in silico virtual simulation analysis for the binding between STING-CTD dimer (green, 4F5D) and astin C (colored) compared with c-di-GMP (yellow). (B) Cell lysates of MEFs (bottom) or HEK293T cells transfected with Flag-tagged hSTING (top) were incubated with biotin or biotinylated astin C (5 mM) in the absence or presence of 10-fold concentration (50 mM) of unlabeled astin C, c-di-GMP, or cGAMP, followed by pull-down analysis with streptavidin-conjugated beads, and immunoblotted with the indicated antibodies. (C and D) MEFs were treated with DMSO or the indicated doses of astin C for 6 hr. Then, the cells were incubated with c-di-GMP (C) or cGAMP (D) by digitonin permeabilization at 37C for 30 min. Six hours after CDN delivery, the induction of Ifnb mRNAs was measured using qPCR assay. (legend continued on next page)

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Critical Commercial Assays Mouse IFN-b ELISA kit PBL assay science Cat#42400-1 Fluoro HepANA Test kit MBL Cat#4220-12CN FastStart Universal SYBR Green Master(ROX) Roche Cat#34226600 Monolith NT.115 Series Capillaries Nanotemper Cat#MO-K005 Monolith Protein labeling kit RED-NHS Nanotemper Cat#MO-L001 CellTiter 96 AQueous One Solution Cell Proliferation Assay (MTS) Promega Cat#G3580 BCA Protein Assay Kit Thermo Scientific Cat#23227 Deposited Data The gene and gene product information of STING GenBank (http://www.ncbi.nlm.nih.gov/Genbank) NM_198282.3, NP_938023.1 The gene and gene product information of SCAP GenBank (http://www.ncbi.nlm.nih.gov/Genbank) NM_012235.2, NP_036367.2 The gene and gene product information of TBK1 GenBank (http://www.ncbi.nlm.nih.gov/Genbank) NM_013254.3, NP_037386.1 The gene and gene product information of IRF3 GenBank (http://www.ncbi.nlm.nih.gov/Genbank) NM_001197122.1, NP_001184051.1 The gene and gene product information of AMFR GenBank (http://www.ncbi.nlm.nih.gov/Genbank) NM_001144.5, NP_001135.3 The gene and gene product information of MAVS GenBank (http://www.ncbi.nlm.nih.gov/Genbank) NM_020746.4, NP_065797.2 The gene and gene product information of INSIG1 GenBank (http://www.ncbi.nlm.nih.gov/Genbank) NM_005542.4, NP_005533.2 The gene and gene product information of cGAS GenBank (http://www.ncbi.nlm.nih.gov/Genbank) NM_138441.2, NP_612450.2 The gene and gene product information of p65 GenBank (http://www.ncbi.nlm.nih.gov/Genbank) NM_021975.3, NP_068810.3 Protein structure of STING C-terminal domain PDB (http://www.rcsb.org/pdb/home/home.do) 4F5D Experimental Models: Cell Lines Mouse: MEFs ATCC Cat#SCRC-1008 Mouse: STING knockout MEFs (Chen et al., 2011) N/A Mouse: Raw264.7 ATCC Cat#TIB-71 Human: HEK293 ATCC Cat# CRL-1573 Human: HEK293T ATCC Cat#ACS-4500 Human: PBMC Allcells Cat#PB003F Human: IMR-90 Cobioer Bioscience Cat#CBP60912 Experimental Models: Organisms/Strains C57BL/6J The Jackson Laboratory Cat#000664 Trex1+/ mice (Hasan et al., 2015) N/A Oligonucleotides See Table S2 for ISD This paper N/A See Table S2 for HSV 60-mer This paper N/A Poly(dA:dT) Sigma Cat#P0883 Poly(I:C) Sigma Cat#P1530 See Table S2 for qPCR primer sequences This paper N/A Recombinant DNA Gene mutants and truncations, see plasmids part in Methods details This paper N/A Software and Algorithms GraphPad Prism 7 GraphPad Software https://www.graphpad.com/ scientific-software/prism/ Origin 8 Origin Lab https://www.originlab.com

Techniques: Blocking Assay, Activation Assay, In Silico, Binding Assay, Transfection, Incubation, Concentration Assay

Journal: Cell reports

Article Title: The Cyclopeptide Astin C Specifically Inhibits the Innate Immune CDN Sensor STING.

doi: 10.1016/j.celrep.2018.11.097

Figure Lengend Snippet: Figure 5. Astin C Suppresses the Recruitment of IRF3 onto the STING Signalosome (A) Flag-tagged IRF3 and HA-tagged STING were cotransfected into HEK293T cells. Fourteen hours after transfection, cells were treated with DMSO, astin C (10 mM), or M11 (10 mM) for 6 hr. Cell lysates were subjected to immunoprecipitation with an anti-FLAG antibody (left) or anti-HA antibody (right) and im- munoblotted with the indicated antibodies. (B) MEFs were pretreated with DMSO, astin C (10 mM), or M11 (10 mM) for 6 hr and then infected with HSV-1 for 6 hr. Cell lysates were immunoprecipitated with an anti-STING antibody and immunoblotted with an anti-IRF3 antibody.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Critical Commercial Assays Mouse IFN-b ELISA kit PBL assay science Cat#42400-1 Fluoro HepANA Test kit MBL Cat#4220-12CN FastStart Universal SYBR Green Master(ROX) Roche Cat#34226600 Monolith NT.115 Series Capillaries Nanotemper Cat#MO-K005 Monolith Protein labeling kit RED-NHS Nanotemper Cat#MO-L001 CellTiter 96 AQueous One Solution Cell Proliferation Assay (MTS) Promega Cat#G3580 BCA Protein Assay Kit Thermo Scientific Cat#23227 Deposited Data The gene and gene product information of STING GenBank (http://www.ncbi.nlm.nih.gov/Genbank) NM_198282.3, NP_938023.1 The gene and gene product information of SCAP GenBank (http://www.ncbi.nlm.nih.gov/Genbank) NM_012235.2, NP_036367.2 The gene and gene product information of TBK1 GenBank (http://www.ncbi.nlm.nih.gov/Genbank) NM_013254.3, NP_037386.1 The gene and gene product information of IRF3 GenBank (http://www.ncbi.nlm.nih.gov/Genbank) NM_001197122.1, NP_001184051.1 The gene and gene product information of AMFR GenBank (http://www.ncbi.nlm.nih.gov/Genbank) NM_001144.5, NP_001135.3 The gene and gene product information of MAVS GenBank (http://www.ncbi.nlm.nih.gov/Genbank) NM_020746.4, NP_065797.2 The gene and gene product information of INSIG1 GenBank (http://www.ncbi.nlm.nih.gov/Genbank) NM_005542.4, NP_005533.2 The gene and gene product information of cGAS GenBank (http://www.ncbi.nlm.nih.gov/Genbank) NM_138441.2, NP_612450.2 The gene and gene product information of p65 GenBank (http://www.ncbi.nlm.nih.gov/Genbank) NM_021975.3, NP_068810.3 Protein structure of STING C-terminal domain PDB (http://www.rcsb.org/pdb/home/home.do) 4F5D Experimental Models: Cell Lines Mouse: MEFs ATCC Cat#SCRC-1008 Mouse: STING knockout MEFs (Chen et al., 2011) N/A Mouse: Raw264.7 ATCC Cat#TIB-71 Human: HEK293 ATCC Cat# CRL-1573 Human: HEK293T ATCC Cat#ACS-4500 Human: PBMC Allcells Cat#PB003F Human: IMR-90 Cobioer Bioscience Cat#CBP60912 Experimental Models: Organisms/Strains C57BL/6J The Jackson Laboratory Cat#000664 Trex1+/ mice (Hasan et al., 2015) N/A Oligonucleotides See Table S2 for ISD This paper N/A See Table S2 for HSV 60-mer This paper N/A Poly(dA:dT) Sigma Cat#P0883 Poly(I:C) Sigma Cat#P1530 See Table S2 for qPCR primer sequences This paper N/A Recombinant DNA Gene mutants and truncations, see plasmids part in Methods details This paper N/A Software and Algorithms GraphPad Prism 7 GraphPad Software https://www.graphpad.com/ scientific-software/prism/ Origin 8 Origin Lab https://www.originlab.com

Techniques: Transfection, Immunoprecipitation, Infection

Journal: PLoS ONE

Article Title: Englerin A Agonizes the TRPC4/C5 Cation Channels to Inhibit Tumor Cell Line Proliferation

doi: 10.1371/journal.pone.0127498

Figure Lengend Snippet: (A ) Effect of TRPC4 siRNA knockdown on viability of A-498 cells in the presence of englerin A. An siRNA targeting luciferase was used as a control (mean+/- S.E.M.) Percent reduction of the TRPC4 mRNA levels are indicated in the legend, KD stands for knockdown. TRPC4 mRNA levels were normalized to peptidyl prolyl isomerase A (PPIA) mRNA levels. ( B ) Effect of TRPC4 siRNA knockdown on viability of A-673 cells in the presence of englerin A (mean +/- S.E.M.) ( C ) Effect of overexpression of TRPC4 by transient transfection on viability of HEK293T cells in the presence of englerin A. TRPC4 expression vector concentrations are indicated by different shapes ( D ) Effect of TRPC4 expression on cell viability in the presence of an englerin A in HEK293T cells engineered to express TRPC4 under control of a Doxycycline (Dox) regulated promoter (mean +/- standard deviation). 100 ng/ml Dox (black circles), 0 ng/ml Dox (open circles). ( E ) Western blot visualizing the levels of TRPC4 in the presence or absence of 100 ng/ml Dox. ( F ) Effect of PKCtheta inhibitor compound 27 on response to englerin A in A-498 cells. ( G ) Effect of PKCtheta inhibitor compound 27 on response to englerin A in A-673 cells.

Article Snippet: They were purchased from ATCC: HEK293T (Cat # CRL-11268), A-498 (Cat # HTB-44), and A-673 (Cat# CRL-1598).

Techniques: Knockdown, Luciferase, Control, Over Expression, Transfection, Expressing, Plasmid Preparation, Standard Deviation, Western Blot

Journal: PLoS ONE

Article Title: Englerin A Agonizes the TRPC4/C5 Cation Channels to Inhibit Tumor Cell Line Proliferation

doi: 10.1371/journal.pone.0127498

Figure Lengend Snippet: ( A ) Expression of TRPC4 and TRPC5 in the cell lines from the CLiP experiment. Each circle represents a single cell line and expression levels were measured by microarray (TRPC4 probe 220818_s_at, TRPC5 probe 220552_at). The circle representing the DMS-79 cell line is indicated. ( B ) The effect of englerin A on cell viability in HEK293T cells transiently transfected with a vector expressing TRPC5 (mean +/- standard deviation).

Article Snippet: They were purchased from ATCC: HEK293T (Cat # CRL-11268), A-498 (Cat # HTB-44), and A-673 (Cat# CRL-1598).

Techniques: Expressing, Microarray, Transfection, Plasmid Preparation, Standard Deviation

Journal: PLoS ONE

Article Title: Englerin A Agonizes the TRPC4/C5 Cation Channels to Inhibit Tumor Cell Line Proliferation

doi: 10.1371/journal.pone.0127498

Figure Lengend Snippet: ( A ) Calcium flux stimulated by englerin A in HEK293T cells overexpressing different TRPC proteins (mean +/- standard deviation): TRPC5 (closed diamonds), TRPC4beta (closed squares), TRPC4 (closed circles), TRPC6 (open squares), mock transfected cells (open circles). ( B ) Membrane depolarization stimulated by englerin A in HEK293T cells overexpressing different TRPC proteins (mean +/- standard deviation), markers as above. ( C ) TRPC4 current evoked by stimulation of 5 μM Englerin A, saline, or 5 μM Englerin A + 10 μM ML204 in 293T cells with Doxycyline-induced TRPC4. Currents were elicited by 200 ms voltage ramps from -100 to +100 mV, applied every 10 s from holding potential of 0 mV. ( D ) Summary of englerin A, englerin-B and ML-204 activity on membrane currents (mean +/- S.E.M.) ( E ) A-673 cell viability in the presence or absence of 50 nM englerin A and/or 50 μM ML204, a TRPC4/C5 channel blocker (mean +/- standard deviation).

Article Snippet: They were purchased from ATCC: HEK293T (Cat # CRL-11268), A-498 (Cat # HTB-44), and A-673 (Cat# CRL-1598).

Techniques: Standard Deviation, Transfection, Membrane, Saline, Activity Assay

Journal: Cell Reports

Article Title: Modular basis for potent SARS-CoV-2 neutralization by a prevalent VH1-2-derived antibody class

doi: 10.1016/j.celrep.2021.108950

Figure Lengend Snippet:

Article Snippet: HEK293T/17(cat# CRL-11268) and Vero E6 cells (cat# CRL-1586) were from ATCC.

Techniques: Bacteria, Virus, Recombinant, Mutagenesis, Plasmid Preparation, Software