envelope proteins Search Results


ab180207  (Danaher Inc)
99
Danaher Inc ab180207
Primary and secondary antibodies used for Western blotting, immunohistochemistry and confocal microscopy
Ab180207, supplied by Danaher Inc, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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e protein e trunc  (ProSci Incorporated)
91
ProSci Incorporated e protein e trunc
Primary and secondary antibodies used for Western blotting, immunohistochemistry and confocal microscopy
E Protein E Trunc, supplied by ProSci Incorporated, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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anti rabbit antibodies  (Proteintech)
95
Proteintech anti rabbit antibodies
Primary and secondary antibodies used for Western blotting, immunohistochemistry and confocal microscopy
Anti Rabbit Antibodies, supplied by Proteintech, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/envelope+proteins/10__3390_slash_molecules24061090-194-54-56?v=Proteintech
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α tubulin  (Santa Cruz Biotechnology)
93
Santa Cruz Biotechnology α tubulin
Hypoxic preconditioning induced gene regulation. ( a ) The HIF1- α mRNA level in hESC neurospheres was upregulated after 3–24 h under the 0.1% oxygen condition, revealed by RT-PCR measurements. The last column shows that the HIF1- α level remained high after finishing a 12-h hypoxia plus 12-h back to 21% oxygen (reoxygenation). No change in the expression of the proapoptotic gene BNIP3 was observed. β -Actin was used as a loading control. ( b ) Western blot analysis shows the upregulation of the protein levels of HIF1- α and pAkt induced by the hypoxia treatment. The Akt activation (phosphorylation of S473) was inhibited by the PI3K inhibitor Wortmannin that also partially inhibited hypoxic stabilization of HIF1- α . ( c ) Western blot analysis showed significant upregulation of several survival genes, including HIF1- α , HIF2- α , pAkt, EPO and Bcl-2 after the 12-h 0.1% oxygen treatment. <t>α</t> <t>-Tubulin</t> was used as a loading control. ( d ) Gene regulation after the termination of hypoxic treatment. Different time points after hypoxia, cells were returned to 21% oxygen. The protective gene bcl-2 decreased and proapoptotic gene Bax increased after reoxygenation. However, a significant upregulation of EPO, mature VEGF and Bcl-2, as well as a downregulation of Bax, were evident after 72-h reoxygenation. * P <0.05 between normoxic controls and 12-h hypoxic culture; ** P <0.05 between normoxic controls and 72-h reoxygenation
α Tubulin, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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sars cov 2 e  (Novus Biologicals)
94
Novus Biologicals sars cov 2 e
Hypoxic preconditioning induced gene regulation. ( a ) The HIF1- α mRNA level in hESC neurospheres was upregulated after 3–24 h under the 0.1% oxygen condition, revealed by RT-PCR measurements. The last column shows that the HIF1- α level remained high after finishing a 12-h hypoxia plus 12-h back to 21% oxygen (reoxygenation). No change in the expression of the proapoptotic gene BNIP3 was observed. β -Actin was used as a loading control. ( b ) Western blot analysis shows the upregulation of the protein levels of HIF1- α and pAkt induced by the hypoxia treatment. The Akt activation (phosphorylation of S473) was inhibited by the PI3K inhibitor Wortmannin that also partially inhibited hypoxic stabilization of HIF1- α . ( c ) Western blot analysis showed significant upregulation of several survival genes, including HIF1- α , HIF2- α , pAkt, EPO and Bcl-2 after the 12-h 0.1% oxygen treatment. <t>α</t> <t>-Tubulin</t> was used as a loading control. ( d ) Gene regulation after the termination of hypoxic treatment. Different time points after hypoxia, cells were returned to 21% oxygen. The protective gene bcl-2 decreased and proapoptotic gene Bax increased after reoxygenation. However, a significant upregulation of EPO, mature VEGF and Bcl-2, as well as a downregulation of Bax, were evident after 72-h reoxygenation. * P <0.05 between normoxic controls and 12-h hypoxic culture; ** P <0.05 between normoxic controls and 72-h reoxygenation
Sars Cov 2 E, supplied by Novus Biologicals, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/envelope+proteins/pm39075394-98-11-13?v=Novus+Biologicals
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anti e antibodies  (ProSci Incorporated)
90
ProSci Incorporated anti e antibodies
Hypoxic preconditioning induced gene regulation. ( a ) The HIF1- α mRNA level in hESC neurospheres was upregulated after 3–24 h under the 0.1% oxygen condition, revealed by RT-PCR measurements. The last column shows that the HIF1- α level remained high after finishing a 12-h hypoxia plus 12-h back to 21% oxygen (reoxygenation). No change in the expression of the proapoptotic gene BNIP3 was observed. β -Actin was used as a loading control. ( b ) Western blot analysis shows the upregulation of the protein levels of HIF1- α and pAkt induced by the hypoxia treatment. The Akt activation (phosphorylation of S473) was inhibited by the PI3K inhibitor Wortmannin that also partially inhibited hypoxic stabilization of HIF1- α . ( c ) Western blot analysis showed significant upregulation of several survival genes, including HIF1- α , HIF2- α , pAkt, EPO and Bcl-2 after the 12-h 0.1% oxygen treatment. <t>α</t> <t>-Tubulin</t> was used as a loading control. ( d ) Gene regulation after the termination of hypoxic treatment. Different time points after hypoxia, cells were returned to 21% oxygen. The protective gene bcl-2 decreased and proapoptotic gene Bax increased after reoxygenation. However, a significant upregulation of EPO, mature VEGF and Bcl-2, as well as a downregulation of Bax, were evident after 72-h reoxygenation. * P <0.05 between normoxic controls and 12-h hypoxic culture; ** P <0.05 between normoxic controls and 72-h reoxygenation
Anti E Antibodies, supplied by ProSci Incorporated, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/envelope+proteins/pmc08256659__mmc1-173-43-45?v=ProSci+Incorporated
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catalog number 100 401 p12  (Rockland Immunochemicals)
90
Rockland Immunochemicals catalog number 100 401 p12
Hypoxic preconditioning induced gene regulation. ( a ) The HIF1- α mRNA level in hESC neurospheres was upregulated after 3–24 h under the 0.1% oxygen condition, revealed by RT-PCR measurements. The last column shows that the HIF1- α level remained high after finishing a 12-h hypoxia plus 12-h back to 21% oxygen (reoxygenation). No change in the expression of the proapoptotic gene BNIP3 was observed. β -Actin was used as a loading control. ( b ) Western blot analysis shows the upregulation of the protein levels of HIF1- α and pAkt induced by the hypoxia treatment. The Akt activation (phosphorylation of S473) was inhibited by the PI3K inhibitor Wortmannin that also partially inhibited hypoxic stabilization of HIF1- α . ( c ) Western blot analysis showed significant upregulation of several survival genes, including HIF1- α , HIF2- α , pAkt, EPO and Bcl-2 after the 12-h 0.1% oxygen treatment. <t>α</t> <t>-Tubulin</t> was used as a loading control. ( d ) Gene regulation after the termination of hypoxic treatment. Different time points after hypoxia, cells were returned to 21% oxygen. The protective gene bcl-2 decreased and proapoptotic gene Bax increased after reoxygenation. However, a significant upregulation of EPO, mature VEGF and Bcl-2, as well as a downregulation of Bax, were evident after 72-h reoxygenation. * P <0.05 between normoxic controls and 12-h hypoxic culture; ** P <0.05 between normoxic controls and 72-h reoxygenation
Catalog Number 100 401 P12, supplied by Rockland Immunochemicals, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/envelope+proteins/pm35947647-178-8-5?v=Rockland+Immunochemicals
Average 90 stars, based on 1 article reviews
catalog number 100 401 p12 - by Bioz Stars, 2026-08
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pan flavivirus anti env 4g2 primary antibody  (Novus Biologicals)
93
Novus Biologicals pan flavivirus anti env 4g2 primary antibody
Hypoxic preconditioning induced gene regulation. ( a ) The HIF1- α mRNA level in hESC neurospheres was upregulated after 3–24 h under the 0.1% oxygen condition, revealed by RT-PCR measurements. The last column shows that the HIF1- α level remained high after finishing a 12-h hypoxia plus 12-h back to 21% oxygen (reoxygenation). No change in the expression of the proapoptotic gene BNIP3 was observed. β -Actin was used as a loading control. ( b ) Western blot analysis shows the upregulation of the protein levels of HIF1- α and pAkt induced by the hypoxia treatment. The Akt activation (phosphorylation of S473) was inhibited by the PI3K inhibitor Wortmannin that also partially inhibited hypoxic stabilization of HIF1- α . ( c ) Western blot analysis showed significant upregulation of several survival genes, including HIF1- α , HIF2- α , pAkt, EPO and Bcl-2 after the 12-h 0.1% oxygen treatment. <t>α</t> <t>-Tubulin</t> was used as a loading control. ( d ) Gene regulation after the termination of hypoxic treatment. Different time points after hypoxia, cells were returned to 21% oxygen. The protective gene bcl-2 decreased and proapoptotic gene Bax increased after reoxygenation. However, a significant upregulation of EPO, mature VEGF and Bcl-2, as well as a downregulation of Bax, were evident after 72-h reoxygenation. * P <0.05 between normoxic controls and 12-h hypoxic culture; ** P <0.05 between normoxic controls and 72-h reoxygenation
Pan Flavivirus Anti Env 4g2 Primary Antibody, supplied by Novus Biologicals, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/envelope+proteins/pmc12698002-289-20-25?v=Novus+Biologicals
Average 93 stars, based on 1 article reviews
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egr2  (Danaher Inc)
99
Danaher Inc egr2
(A) Proliferation of siRNA transfected patient-derived colon cancer cells in non-adherent cell culture compared to control cells (mean ± SD; data from three independent experiments). *p-value < 0.05; ***p-value < 0.001 (t test). (B) Fold expression of ALDH1A1, <t>EGR2,</t> EGR3, HDGFRP3 OLFML2, PCP4, PEG10, PRKACB and THBS1 RT-PCR gene expression data (±95% confidence intervals) in siRNA transfected 278-ML-P cells (n=3 independent cell preparations) over the comparator population (control siRNA transfected 278-ML-P cells) (see also Table S2 and S4). (C) Frequency of siRNA EGR2 spheroid formation in non-adherent cell culture compared to control transfected cells (mean ± SD; data from three independent experiments). ns = not significant; *p-value < 0.05; **p-value < 0.01 (t test). (D) Representative images of a 278-ML-P control spheroid (LHS) and a siRNA EGR2 spheroid (RHS) in non-adherent cell culture (Bars = 100 µm). (E) Table shows results of limiting dilution transplantation of control virus transduced and shRNA EGR2 transduced 195-CB-P cells. The number of established tumors as a fraction of the number of animals transplanted is given. P-values for pairwise tests of differences in CSC frequencies between control virus versus shRNA EGR2 1, shRNA EGR2 2 and shRNA EGR2 3 195-CB-P cells are 6.9 ×10 −9 , 4.9 × 10 −6 and 6.92 × 10 −8 , respectively. (F) Growth curves for xenografts derived from control virus transduced cells and shRNA EGR2 transduced cells. (G) Fold expression of EGR2 , proliferation, differentiation, stem cell genes, Wnt signaling and EGR2 NCSC target genes RT-PCR gene expression data (±95% confidence intervals) in four separate 195-CB-P shRNA EGR2 tumors over the comparator population (four control virus transduced 195-CB-P xenografts). Significant differences are as follows: ∗p < 0.05, ∗∗p < 0.01. (see also Table S3).
Egr2, supplied by Danaher Inc, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/envelope+proteins/bio_rxiv__2021__02__02__428317-99-20-22?v=Danaher+Inc
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rabbit anti sars cov 2 envelope protein polyclonal antibody pab  (Cell Signaling Technology Inc)
93
Cell Signaling Technology Inc rabbit anti sars cov 2 envelope protein polyclonal antibody pab
(A) Proliferation of siRNA transfected patient-derived colon cancer cells in non-adherent cell culture compared to control cells (mean ± SD; data from three independent experiments). *p-value < 0.05; ***p-value < 0.001 (t test). (B) Fold expression of ALDH1A1, <t>EGR2,</t> EGR3, HDGFRP3 OLFML2, PCP4, PEG10, PRKACB and THBS1 RT-PCR gene expression data (±95% confidence intervals) in siRNA transfected 278-ML-P cells (n=3 independent cell preparations) over the comparator population (control siRNA transfected 278-ML-P cells) (see also Table S2 and S4). (C) Frequency of siRNA EGR2 spheroid formation in non-adherent cell culture compared to control transfected cells (mean ± SD; data from three independent experiments). ns = not significant; *p-value < 0.05; **p-value < 0.01 (t test). (D) Representative images of a 278-ML-P control spheroid (LHS) and a siRNA EGR2 spheroid (RHS) in non-adherent cell culture (Bars = 100 µm). (E) Table shows results of limiting dilution transplantation of control virus transduced and shRNA EGR2 transduced 195-CB-P cells. The number of established tumors as a fraction of the number of animals transplanted is given. P-values for pairwise tests of differences in CSC frequencies between control virus versus shRNA EGR2 1, shRNA EGR2 2 and shRNA EGR2 3 195-CB-P cells are 6.9 ×10 −9 , 4.9 × 10 −6 and 6.92 × 10 −8 , respectively. (F) Growth curves for xenografts derived from control virus transduced cells and shRNA EGR2 transduced cells. (G) Fold expression of EGR2 , proliferation, differentiation, stem cell genes, Wnt signaling and EGR2 NCSC target genes RT-PCR gene expression data (±95% confidence intervals) in four separate 195-CB-P shRNA EGR2 tumors over the comparator population (four control virus transduced 195-CB-P xenografts). Significant differences are as follows: ∗p < 0.05, ∗∗p < 0.01. (see also Table S3).
Rabbit Anti Sars Cov 2 Envelope Protein Polyclonal Antibody Pab, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/envelope+proteins/bio_rxiv__2024__09__05__611372-196-9-58?v=Cell+Signaling+Technology+Inc
Average 93 stars, based on 1 article reviews
rabbit anti sars cov 2 envelope protein polyclonal antibody pab - by Bioz Stars, 2026-08
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sars cov 2 envelope protein  (ProSci Incorporated)
86
ProSci Incorporated sars cov 2 envelope protein
(A) Proliferation of siRNA transfected patient-derived colon cancer cells in non-adherent cell culture compared to control cells (mean ± SD; data from three independent experiments). *p-value < 0.05; ***p-value < 0.001 (t test). (B) Fold expression of ALDH1A1, <t>EGR2,</t> EGR3, HDGFRP3 OLFML2, PCP4, PEG10, PRKACB and THBS1 RT-PCR gene expression data (±95% confidence intervals) in siRNA transfected 278-ML-P cells (n=3 independent cell preparations) over the comparator population (control siRNA transfected 278-ML-P cells) (see also Table S2 and S4). (C) Frequency of siRNA EGR2 spheroid formation in non-adherent cell culture compared to control transfected cells (mean ± SD; data from three independent experiments). ns = not significant; *p-value < 0.05; **p-value < 0.01 (t test). (D) Representative images of a 278-ML-P control spheroid (LHS) and a siRNA EGR2 spheroid (RHS) in non-adherent cell culture (Bars = 100 µm). (E) Table shows results of limiting dilution transplantation of control virus transduced and shRNA EGR2 transduced 195-CB-P cells. The number of established tumors as a fraction of the number of animals transplanted is given. P-values for pairwise tests of differences in CSC frequencies between control virus versus shRNA EGR2 1, shRNA EGR2 2 and shRNA EGR2 3 195-CB-P cells are 6.9 ×10 −9 , 4.9 × 10 −6 and 6.92 × 10 −8 , respectively. (F) Growth curves for xenografts derived from control virus transduced cells and shRNA EGR2 transduced cells. (G) Fold expression of EGR2 , proliferation, differentiation, stem cell genes, Wnt signaling and EGR2 NCSC target genes RT-PCR gene expression data (±95% confidence intervals) in four separate 195-CB-P shRNA EGR2 tumors over the comparator population (four control virus transduced 195-CB-P xenografts). Significant differences are as follows: ∗p < 0.05, ∗∗p < 0.01. (see also Table S3).
Sars Cov 2 Envelope Protein, supplied by ProSci Incorporated, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/envelope+proteins/pmc07447529-46-12-18?v=ProSci+Incorporated
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recombinant proteins zika virus full length envelope e protein aviva systems biology opma04848 zika virus edi edii peptides  (Aviva Systems)
93
Aviva Systems recombinant proteins zika virus full length envelope e protein aviva systems biology opma04848 zika virus edi edii peptides
Figure 1. <t>ZIKV</t> TCI vaccine design and antigen expression (A) Schematic diagram of plasmid design. The gene sequence encoding for the NS3 protein was split into three parts (denoted 1, 2, and 3). The 30 nucleotide bases before and after any cleaved sequence were placed in front of each region to preserve any epitopes that may have been disrupted. The ORF gene en- coding for a mouse/human monomer of ubiquitin (Ub) was placed immediately upstream of the rearranged NS3 sequence. A glycine at the 76th residue was modified to encode an alanine to enhance the stability of the Ub/NS3 complex. (B) 293T cells were transfected with the rearranged Ub/NS3 plasmid overnight. The cells were allowed to stably express plasmid for 36 h. After this period, MG132 was added overnight. The cell lysate was analyzed via western blot for the expression of NS3 and beta-actin. A representative blot from three ex- periments is shown. (C) Total band density of western blot was quantified using ImageJ and analyzed using GraphPad (Prism). The data are represented as mean ± SEM (n = 2 for each experiment, three experiments total). *p < 0.05; **p < 0.01.
Recombinant Proteins Zika Virus Full Length Envelope E Protein Aviva Systems Biology Opma04848 Zika Virus Edi Edii Peptides, supplied by Aviva Systems, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Image Search Results


Primary and secondary antibodies used for Western blotting, immunohistochemistry and confocal microscopy

Journal: Molecular Neurobiology

Article Title: High Expression of Nicotinamide N -Methyltransferase in Patients with Sporadic Alzheimer’s Disease

doi: 10.1007/s12035-020-02259-9

Figure Lengend Snippet: Primary and secondary antibodies used for Western blotting, immunohistochemistry and confocal microscopy

Article Snippet: Tubulin , 1:1000 , - , ab180207 (Abcam) , 1:5000 (A4416, Sigma) , -.

Techniques: Western Blot, Immunohistochemistry, Microscopy, Confocal Microscopy

Hypoxic preconditioning induced gene regulation. ( a ) The HIF1- α mRNA level in hESC neurospheres was upregulated after 3–24 h under the 0.1% oxygen condition, revealed by RT-PCR measurements. The last column shows that the HIF1- α level remained high after finishing a 12-h hypoxia plus 12-h back to 21% oxygen (reoxygenation). No change in the expression of the proapoptotic gene BNIP3 was observed. β -Actin was used as a loading control. ( b ) Western blot analysis shows the upregulation of the protein levels of HIF1- α and pAkt induced by the hypoxia treatment. The Akt activation (phosphorylation of S473) was inhibited by the PI3K inhibitor Wortmannin that also partially inhibited hypoxic stabilization of HIF1- α . ( c ) Western blot analysis showed significant upregulation of several survival genes, including HIF1- α , HIF2- α , pAkt, EPO and Bcl-2 after the 12-h 0.1% oxygen treatment. α -Tubulin was used as a loading control. ( d ) Gene regulation after the termination of hypoxic treatment. Different time points after hypoxia, cells were returned to 21% oxygen. The protective gene bcl-2 decreased and proapoptotic gene Bax increased after reoxygenation. However, a significant upregulation of EPO, mature VEGF and Bcl-2, as well as a downregulation of Bax, were evident after 72-h reoxygenation. * P <0.05 between normoxic controls and 12-h hypoxic culture; ** P <0.05 between normoxic controls and 72-h reoxygenation

Journal: Cell Death & Disease

Article Title: Human embryonic stem cell neural differentiation and enhanced cell survival promoted by hypoxic preconditioning

doi: 10.1038/cddis.2009.22

Figure Lengend Snippet: Hypoxic preconditioning induced gene regulation. ( a ) The HIF1- α mRNA level in hESC neurospheres was upregulated after 3–24 h under the 0.1% oxygen condition, revealed by RT-PCR measurements. The last column shows that the HIF1- α level remained high after finishing a 12-h hypoxia plus 12-h back to 21% oxygen (reoxygenation). No change in the expression of the proapoptotic gene BNIP3 was observed. β -Actin was used as a loading control. ( b ) Western blot analysis shows the upregulation of the protein levels of HIF1- α and pAkt induced by the hypoxia treatment. The Akt activation (phosphorylation of S473) was inhibited by the PI3K inhibitor Wortmannin that also partially inhibited hypoxic stabilization of HIF1- α . ( c ) Western blot analysis showed significant upregulation of several survival genes, including HIF1- α , HIF2- α , pAkt, EPO and Bcl-2 after the 12-h 0.1% oxygen treatment. α -Tubulin was used as a loading control. ( d ) Gene regulation after the termination of hypoxic treatment. Different time points after hypoxia, cells were returned to 21% oxygen. The protective gene bcl-2 decreased and proapoptotic gene Bax increased after reoxygenation. However, a significant upregulation of EPO, mature VEGF and Bcl-2, as well as a downregulation of Bax, were evident after 72-h reoxygenation. * P <0.05 between normoxic controls and 12-h hypoxic culture; ** P <0.05 between normoxic controls and 72-h reoxygenation

Article Snippet: Membranes were blocked for 1 h with 5% BSA containing phosphatase inhibitors (50 mM NaF, 200 μ M Na 3 VO 4 ) before overnight incubation with primary antibodies: VEGF (sc-507; 1 : 1000), Flk-1 (sc-6251; 1 : 1000), EPOR (sc-697; 1 : 1000), Akt (sc-8312; 1 : 1000), Bcl-2 (sc-7382; 1 : 500), Bcl-Xl (sc-8392; 1 : 500), BDNF (sc-546; 1 : 1000), α -tubulin (sc-5286; 1 : 5000), EPAS-1 (sc-28706; 1 : 2000), EPO (sc-7956; 1 : 2000; Santa Cruz Biotechnology, Santa Cruz, CA, USA), phospho-Akt (S473) (#9271; 1 : 1000), NF-ÊB (#3035; 1 : 2000; Cell Signaling Technology, Danvers, MA, USA), GLUT-1 (AB1340; 1 : 7500), 17 kDa Caspase 3 (AB3623; 1 : 2000), FGF-2 (AB1458; 1 : 500; Chemicon/Millipore), HIF1- α (NB100-479; 1 : 2000; Novus Biologicals, Littleton, CO, USA), Bax (no. 54179; 1 : 500; Anaspec, Fremont, CA, USA).

Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Control, Western Blot, Activation Assay, Phospho-proteomics

(A) Proliferation of siRNA transfected patient-derived colon cancer cells in non-adherent cell culture compared to control cells (mean ± SD; data from three independent experiments). *p-value < 0.05; ***p-value < 0.001 (t test). (B) Fold expression of ALDH1A1, EGR2, EGR3, HDGFRP3 OLFML2, PCP4, PEG10, PRKACB and THBS1 RT-PCR gene expression data (±95% confidence intervals) in siRNA transfected 278-ML-P cells (n=3 independent cell preparations) over the comparator population (control siRNA transfected 278-ML-P cells) (see also Table S2 and S4). (C) Frequency of siRNA EGR2 spheroid formation in non-adherent cell culture compared to control transfected cells (mean ± SD; data from three independent experiments). ns = not significant; *p-value < 0.05; **p-value < 0.01 (t test). (D) Representative images of a 278-ML-P control spheroid (LHS) and a siRNA EGR2 spheroid (RHS) in non-adherent cell culture (Bars = 100 µm). (E) Table shows results of limiting dilution transplantation of control virus transduced and shRNA EGR2 transduced 195-CB-P cells. The number of established tumors as a fraction of the number of animals transplanted is given. P-values for pairwise tests of differences in CSC frequencies between control virus versus shRNA EGR2 1, shRNA EGR2 2 and shRNA EGR2 3 195-CB-P cells are 6.9 ×10 −9 , 4.9 × 10 −6 and 6.92 × 10 −8 , respectively. (F) Growth curves for xenografts derived from control virus transduced cells and shRNA EGR2 transduced cells. (G) Fold expression of EGR2 , proliferation, differentiation, stem cell genes, Wnt signaling and EGR2 NCSC target genes RT-PCR gene expression data (±95% confidence intervals) in four separate 195-CB-P shRNA EGR2 tumors over the comparator population (four control virus transduced 195-CB-P xenografts). Significant differences are as follows: ∗p < 0.05, ∗∗p < 0.01. (see also Table S3).

Journal: bioRxiv

Article Title: Identification of a Nervous System Gene Expression Signature in Colon Cancer Stem Cells Reveals a Role for Neural Crest Regulators EGR2 and SOX2 in Tumorigenesis

doi: 10.1101/2021.02.02.428317

Figure Lengend Snippet: (A) Proliferation of siRNA transfected patient-derived colon cancer cells in non-adherent cell culture compared to control cells (mean ± SD; data from three independent experiments). *p-value < 0.05; ***p-value < 0.001 (t test). (B) Fold expression of ALDH1A1, EGR2, EGR3, HDGFRP3 OLFML2, PCP4, PEG10, PRKACB and THBS1 RT-PCR gene expression data (±95% confidence intervals) in siRNA transfected 278-ML-P cells (n=3 independent cell preparations) over the comparator population (control siRNA transfected 278-ML-P cells) (see also Table S2 and S4). (C) Frequency of siRNA EGR2 spheroid formation in non-adherent cell culture compared to control transfected cells (mean ± SD; data from three independent experiments). ns = not significant; *p-value < 0.05; **p-value < 0.01 (t test). (D) Representative images of a 278-ML-P control spheroid (LHS) and a siRNA EGR2 spheroid (RHS) in non-adherent cell culture (Bars = 100 µm). (E) Table shows results of limiting dilution transplantation of control virus transduced and shRNA EGR2 transduced 195-CB-P cells. The number of established tumors as a fraction of the number of animals transplanted is given. P-values for pairwise tests of differences in CSC frequencies between control virus versus shRNA EGR2 1, shRNA EGR2 2 and shRNA EGR2 3 195-CB-P cells are 6.9 ×10 −9 , 4.9 × 10 −6 and 6.92 × 10 −8 , respectively. (F) Growth curves for xenografts derived from control virus transduced cells and shRNA EGR2 transduced cells. (G) Fold expression of EGR2 , proliferation, differentiation, stem cell genes, Wnt signaling and EGR2 NCSC target genes RT-PCR gene expression data (±95% confidence intervals) in four separate 195-CB-P shRNA EGR2 tumors over the comparator population (four control virus transduced 195-CB-P xenografts). Significant differences are as follows: ∗p < 0.05, ∗∗p < 0.01. (see also Table S3).

Article Snippet: Immunohistochemistry was carried out via standard techniques with non-phospho (Active) β-Catenin (#8814, rabbit monoclonal, Cell Signaling Technology; diluted 1:200) and EGR2 (ab43020, Abcam, rabbit IgG, polyclonal, diluted 1:1000) antibodies.

Techniques: Transfection, Derivative Assay, Cell Culture, Control, Expressing, Reverse Transcription Polymerase Chain Reaction, Gene Expression, Transplantation Assay, Virus, shRNA

(A) Immunofluorescence staining of PDO for EGR2 (green) and F-ACTIN (red). Nuclei are stained blue with DAPI (Bars = 20 µm). Immunostaining of PDX tissue (B) and tissue microarrays of normal intestinal mucosa and colorectal cancer patient tissue (C) for EGR2. (Bars = 200 µm). Related to .

Journal: bioRxiv

Article Title: Identification of a Nervous System Gene Expression Signature in Colon Cancer Stem Cells Reveals a Role for Neural Crest Regulators EGR2 and SOX2 in Tumorigenesis

doi: 10.1101/2021.02.02.428317

Figure Lengend Snippet: (A) Immunofluorescence staining of PDO for EGR2 (green) and F-ACTIN (red). Nuclei are stained blue with DAPI (Bars = 20 µm). Immunostaining of PDX tissue (B) and tissue microarrays of normal intestinal mucosa and colorectal cancer patient tissue (C) for EGR2. (Bars = 200 µm). Related to .

Article Snippet: Immunohistochemistry was carried out via standard techniques with non-phospho (Active) β-Catenin (#8814, rabbit monoclonal, Cell Signaling Technology; diluted 1:200) and EGR2 (ab43020, Abcam, rabbit IgG, polyclonal, diluted 1:1000) antibodies.

Techniques: Immunofluorescence, Staining, Immunostaining

(A) Expression of EGR2, ATOH1, HOXA2, HOXA4, HOXA5, HOXA7, HOXB2, HOXB3, HOXD10 and SOX2 in colorectal cancer patients across different tumor stages (T1 v T4, p-value = 0.027, 0.53, 0.026, 0.000075, 0.001, 0.009, 0.075, 0.0016, 0.043 and 0.1, respectively). Of these, HOXA4, HOXA5, HOXA7 , and HOXB3 are significant at FDR < 5%. RNAseq and clinical data of 533 patients (n=378 colon adenocarcinoma, n=155 rectal adenocarcinoma) was extracted from cBioPortal. (B) Kaplan-Meier survival curves for EGR2, ATOH1, HOXA2, HOXA4, HOXA5, HOXA7, HOXB2, HOXB3, HOXD10 and SOX2 in colorectal cancer patients comparing lower third percentile to upper third percentile (logrank p-values = 0.00017, 0.0013, 0.0028, 0.0006, 0.0043, 0.0022, 0.00025, 0.019, 0.11 and 0.21, respectively. Of these, higher EGR2, HOXA2, HOXA4, HOXA5 and HOXA7 are significant at FDR < 5%. Results based upon data generated by the Kaplan-Meier Plotter ( www.kmplot.com/analysis ) .

Journal: bioRxiv

Article Title: Identification of a Nervous System Gene Expression Signature in Colon Cancer Stem Cells Reveals a Role for Neural Crest Regulators EGR2 and SOX2 in Tumorigenesis

doi: 10.1101/2021.02.02.428317

Figure Lengend Snippet: (A) Expression of EGR2, ATOH1, HOXA2, HOXA4, HOXA5, HOXA7, HOXB2, HOXB3, HOXD10 and SOX2 in colorectal cancer patients across different tumor stages (T1 v T4, p-value = 0.027, 0.53, 0.026, 0.000075, 0.001, 0.009, 0.075, 0.0016, 0.043 and 0.1, respectively). Of these, HOXA4, HOXA5, HOXA7 , and HOXB3 are significant at FDR < 5%. RNAseq and clinical data of 533 patients (n=378 colon adenocarcinoma, n=155 rectal adenocarcinoma) was extracted from cBioPortal. (B) Kaplan-Meier survival curves for EGR2, ATOH1, HOXA2, HOXA4, HOXA5, HOXA7, HOXB2, HOXB3, HOXD10 and SOX2 in colorectal cancer patients comparing lower third percentile to upper third percentile (logrank p-values = 0.00017, 0.0013, 0.0028, 0.0006, 0.0043, 0.0022, 0.00025, 0.019, 0.11 and 0.21, respectively. Of these, higher EGR2, HOXA2, HOXA4, HOXA5 and HOXA7 are significant at FDR < 5%. Results based upon data generated by the Kaplan-Meier Plotter ( www.kmplot.com/analysis ) .

Article Snippet: Immunohistochemistry was carried out via standard techniques with non-phospho (Active) β-Catenin (#8814, rabbit monoclonal, Cell Signaling Technology; diluted 1:200) and EGR2 (ab43020, Abcam, rabbit IgG, polyclonal, diluted 1:1000) antibodies.

Techniques: Expressing, Generated

Figure 1. ZIKV TCI vaccine design and antigen expression (A) Schematic diagram of plasmid design. The gene sequence encoding for the NS3 protein was split into three parts (denoted 1, 2, and 3). The 30 nucleotide bases before and after any cleaved sequence were placed in front of each region to preserve any epitopes that may have been disrupted. The ORF gene en- coding for a mouse/human monomer of ubiquitin (Ub) was placed immediately upstream of the rearranged NS3 sequence. A glycine at the 76th residue was modified to encode an alanine to enhance the stability of the Ub/NS3 complex. (B) 293T cells were transfected with the rearranged Ub/NS3 plasmid overnight. The cells were allowed to stably express plasmid for 36 h. After this period, MG132 was added overnight. The cell lysate was analyzed via western blot for the expression of NS3 and beta-actin. A representative blot from three ex- periments is shown. (C) Total band density of western blot was quantified using ImageJ and analyzed using GraphPad (Prism). The data are represented as mean ± SEM (n = 2 for each experiment, three experiments total). *p < 0.05; **p < 0.01.

Journal: Cell reports

Article Title: A vaccine inducing solely cytotoxic T lymphocytes fully prevents Zika virus infection and fetal damage.

doi: 10.1016/j.celrep.2021.109107

Figure Lengend Snippet: Figure 1. ZIKV TCI vaccine design and antigen expression (A) Schematic diagram of plasmid design. The gene sequence encoding for the NS3 protein was split into three parts (denoted 1, 2, and 3). The 30 nucleotide bases before and after any cleaved sequence were placed in front of each region to preserve any epitopes that may have been disrupted. The ORF gene en- coding for a mouse/human monomer of ubiquitin (Ub) was placed immediately upstream of the rearranged NS3 sequence. A glycine at the 76th residue was modified to encode an alanine to enhance the stability of the Ub/NS3 complex. (B) 293T cells were transfected with the rearranged Ub/NS3 plasmid overnight. The cells were allowed to stably express plasmid for 36 h. After this period, MG132 was added overnight. The cell lysate was analyzed via western blot for the expression of NS3 and beta-actin. A representative blot from three ex- periments is shown. (C) Total band density of western blot was quantified using ImageJ and analyzed using GraphPad (Prism). The data are represented as mean ± SEM (n = 2 for each experiment, three experiments total). *p < 0.05; **p < 0.01.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Anti-mouse CD4 (IgG2b) antibody Bio X Cell BP0003-1; RRID:AB_1107636 Anti-mouse CD8a (IgG2b) antibody Bio X Cell BP0061; RRID:AB_1125541 Rat IgG2b isotype control antibody Bio X Cell BP0090; RRID:AB_1107780 Anti-mouse-CD8a (IgG2a) antibody Bio X Cell BE0004-1; RRID:AB_1107671 Rat IgG2a isotype control antibody Bio X Cell BE0089; RRID:AB_1107769 Anti-mouse IFNAR1 (MAR1-5A3) antibody Leinco I-401; RRID:AB_2491621 ZIKV EDIII-specific human antibody, ZV-64 Absolute Antibody Ab00811-10.0 Rabbit anti-vimentin antibody Abcam Ab92547; RRID:AB_10562134 Rabbit anti-active caspase-3 antibody Abcam Ab2302; RRID:AB_302962 FITC-anti-human antibody Sigma 9512-1mL; RRID:AB_259808 Anti-rabbit Alexa Fluor 647 antibody Abcam Ab150075; RRID:AB_2752244 FITC-anti-mouse antibody Biolegend 406001; RRID:AB_315029 Anti-flavivirus 4G2 antibody BEI Resources NR-50327 PerCP-Cy5.5-anti-mouse CD8 antibody BD Biosciences 551162; RRID:AB_394081 FITC-anti-mouse IL-2 antibody BD Biosciences 554427; RRID:AB_395385 FITC-anti-mouse CD4 antibody BD Biosciences 553729; RRID:AB_395013 PE-anti-mouse IFN-g antibody BD Biosciences 554412; RRID:AB_395376 Brilliant Violet 421-anti-mouse TNF-a antibody Biolegend 506328; RRID:AB_2562902 HRP-conjugated anti-mouse IgG-Fab antibody Sigma a9917-1mL; RRID:AB_258476 Anti-ZIKV NS3 antibody Genetex GTX133309; RRID:AB_2756864 Bacterial and virus strains Zika virus (human strain R103451 (2015/ Honduras)) BEI Resources NR-50355 Zika virus (human strain PAN2016 (2016/ Panama)) BEI Resources NR-50210 Chemicals, peptides, and recombinant proteins Zika virus full-length envelope (E) protein Aviva Systems Biology OPMA04848 Zika virus EDI/EDII peptides (see below peptide 1-5) BEI Resources NR-50553 Peptide 1: IGVSNRDFVEGMSGG BEI Resources N/A Peptide 2: TWVDVVLEHGGCVTV BEI Resources N/A Peptide 3: MAQDKPTVDIELVTT BEI Resources N/A Peptide 4: VDRGWGNGCGLFGKG BEI Resources N/A Peptide 5: VVLGSQEGAVHTALA BEI Resources N/A Zika virus NS3 overlapping peptides (see below peptide 6-15) GenScript N/A Peptide 6: AETDEDHAHWLEARM GenScript N/A Peptide 7: HAHWLEARMLLDNIY GenScript N/A Peptide 8: ARMLLDNIYLQDGLI GenScript N/A Peptide 9: NIYLQDGLIASLYRP GenScript N/A Peptide 10: GLIASLYRPEADKVA GenScript N/A Peptide 11: YRPEADKVAAIEGEF GenScript N/A (Continued on next page) e1 Cell Reports 35, 109107, May 11, 2021

Techniques: Expressing, Plasmid Preparation, Sequencing, Ubiquitin Proteomics, Residue, Transfection, Stable Transfection, Western Blot

Figure 2. ZIKV TCI-DNA vaccine protected adult male and female Ifnar1/ mice against ZIKV challenge with complete survival and reduced viral titers, including reproductive organs Equal numbers of male and female Ifnar1/ mice were immunized with ZIKV E ectodomain, EDI/II peptides, TCI-DNA, or PBS control, and sera were collected at 10 days post-second immunization. (A and B) At 13 days post-second immunization, the mice were challenged (i.p.) with ZIKV (strain R103451, 103 PFU/mouse) and recorded for weight (A) and survival (B) daily for 14 days (n = 6). (C) In a separate experiment, the immunized Ifnar1/ mice were challenged with ZIKV (strain PAN2016, 103 PFU/mouse), and 6 days later, sera and tissues (including brain, kidney, lung, heart, liver, spleen, muscle, and testis) were collected for detection of viral titers using plaque-forming assay. The detection limit was 25 PFU/ml (for sera), 12.5 PFU/g (for brain), 20 PFU/g (for kidney, spleen, and muscle), and 25 PFU/g (for lung, heart, liver, and testis). The data are represented as mean ± SEM (n = 3 for testis and n = 6 for other groups). *p < 0.05; **p < 0.01; ***p < 0.001.

Journal: Cell reports

Article Title: A vaccine inducing solely cytotoxic T lymphocytes fully prevents Zika virus infection and fetal damage.

doi: 10.1016/j.celrep.2021.109107

Figure Lengend Snippet: Figure 2. ZIKV TCI-DNA vaccine protected adult male and female Ifnar1/ mice against ZIKV challenge with complete survival and reduced viral titers, including reproductive organs Equal numbers of male and female Ifnar1/ mice were immunized with ZIKV E ectodomain, EDI/II peptides, TCI-DNA, or PBS control, and sera were collected at 10 days post-second immunization. (A and B) At 13 days post-second immunization, the mice were challenged (i.p.) with ZIKV (strain R103451, 103 PFU/mouse) and recorded for weight (A) and survival (B) daily for 14 days (n = 6). (C) In a separate experiment, the immunized Ifnar1/ mice were challenged with ZIKV (strain PAN2016, 103 PFU/mouse), and 6 days later, sera and tissues (including brain, kidney, lung, heart, liver, spleen, muscle, and testis) were collected for detection of viral titers using plaque-forming assay. The detection limit was 25 PFU/ml (for sera), 12.5 PFU/g (for brain), 20 PFU/g (for kidney, spleen, and muscle), and 25 PFU/g (for lung, heart, liver, and testis). The data are represented as mean ± SEM (n = 3 for testis and n = 6 for other groups). *p < 0.05; **p < 0.01; ***p < 0.001.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Anti-mouse CD4 (IgG2b) antibody Bio X Cell BP0003-1; RRID:AB_1107636 Anti-mouse CD8a (IgG2b) antibody Bio X Cell BP0061; RRID:AB_1125541 Rat IgG2b isotype control antibody Bio X Cell BP0090; RRID:AB_1107780 Anti-mouse-CD8a (IgG2a) antibody Bio X Cell BE0004-1; RRID:AB_1107671 Rat IgG2a isotype control antibody Bio X Cell BE0089; RRID:AB_1107769 Anti-mouse IFNAR1 (MAR1-5A3) antibody Leinco I-401; RRID:AB_2491621 ZIKV EDIII-specific human antibody, ZV-64 Absolute Antibody Ab00811-10.0 Rabbit anti-vimentin antibody Abcam Ab92547; RRID:AB_10562134 Rabbit anti-active caspase-3 antibody Abcam Ab2302; RRID:AB_302962 FITC-anti-human antibody Sigma 9512-1mL; RRID:AB_259808 Anti-rabbit Alexa Fluor 647 antibody Abcam Ab150075; RRID:AB_2752244 FITC-anti-mouse antibody Biolegend 406001; RRID:AB_315029 Anti-flavivirus 4G2 antibody BEI Resources NR-50327 PerCP-Cy5.5-anti-mouse CD8 antibody BD Biosciences 551162; RRID:AB_394081 FITC-anti-mouse IL-2 antibody BD Biosciences 554427; RRID:AB_395385 FITC-anti-mouse CD4 antibody BD Biosciences 553729; RRID:AB_395013 PE-anti-mouse IFN-g antibody BD Biosciences 554412; RRID:AB_395376 Brilliant Violet 421-anti-mouse TNF-a antibody Biolegend 506328; RRID:AB_2562902 HRP-conjugated anti-mouse IgG-Fab antibody Sigma a9917-1mL; RRID:AB_258476 Anti-ZIKV NS3 antibody Genetex GTX133309; RRID:AB_2756864 Bacterial and virus strains Zika virus (human strain R103451 (2015/ Honduras)) BEI Resources NR-50355 Zika virus (human strain PAN2016 (2016/ Panama)) BEI Resources NR-50210 Chemicals, peptides, and recombinant proteins Zika virus full-length envelope (E) protein Aviva Systems Biology OPMA04848 Zika virus EDI/EDII peptides (see below peptide 1-5) BEI Resources NR-50553 Peptide 1: IGVSNRDFVEGMSGG BEI Resources N/A Peptide 2: TWVDVVLEHGGCVTV BEI Resources N/A Peptide 3: MAQDKPTVDIELVTT BEI Resources N/A Peptide 4: VDRGWGNGCGLFGKG BEI Resources N/A Peptide 5: VVLGSQEGAVHTALA BEI Resources N/A Zika virus NS3 overlapping peptides (see below peptide 6-15) GenScript N/A Peptide 6: AETDEDHAHWLEARM GenScript N/A Peptide 7: HAHWLEARMLLDNIY GenScript N/A Peptide 8: ARMLLDNIYLQDGLI GenScript N/A Peptide 9: NIYLQDGLIASLYRP GenScript N/A Peptide 10: GLIASLYRPEADKVA GenScript N/A Peptide 11: YRPEADKVAAIEGEF GenScript N/A (Continued on next page) e1 Cell Reports 35, 109107, May 11, 2021

Techniques: Control

Figure 3. ZIKV TCI-DNA vaccine protected female pregnant Ifnar1/ mice and their fetuses against ZIKV challenge Female Ifnar1/ mice were immunized with ZIKV E ectodomain, EDI/II peptides, TCI-DNA, or PBS control and mated with male Ifnar1/ mice for pregnancy at day 10 post-second immunization. The pregnant mice (E10–E12) were challenged with ZIKV (strain R103451, 104 PFU/mouse), and 6 days later, uteri and fetuses were collected to evaluate morphological changes as well as sera, body fluid, and tissues (including placenta) to measure viral titers using plaque-forming assay. Placenta was also observed for apoptosis and vascular damage and examined for inflammatory cytokines and chemokines as described below. (A and B) Representative image of morphology of uteri (E16–E18) and fetuses from pregnant mice challenged with ZIKV at E10-E12 (A), among which total numbers and dead fetuses from each group are shown (B). (C–E) Viral titers in placenta, fetal brain (C), as well as sera and amniotic fluid (D) and tissues (including brain, kidney, liver, spleen, heart, and muscle) (E), 6 days post-challenge. The detection limit was 12.5 PFU/g (for placenta and fetal brain), 20 PFU/g (for heart), 25 PFU/ml (for sera and amniotic fluid), and 25 PFU/g (for brain, kidney, liver, spleen, and muscle). The data are represented as mean ± SEM (n = 6). *p < 0.05; **p < 0.01; ***p < 0.001.

Journal: Cell reports

Article Title: A vaccine inducing solely cytotoxic T lymphocytes fully prevents Zika virus infection and fetal damage.

doi: 10.1016/j.celrep.2021.109107

Figure Lengend Snippet: Figure 3. ZIKV TCI-DNA vaccine protected female pregnant Ifnar1/ mice and their fetuses against ZIKV challenge Female Ifnar1/ mice were immunized with ZIKV E ectodomain, EDI/II peptides, TCI-DNA, or PBS control and mated with male Ifnar1/ mice for pregnancy at day 10 post-second immunization. The pregnant mice (E10–E12) were challenged with ZIKV (strain R103451, 104 PFU/mouse), and 6 days later, uteri and fetuses were collected to evaluate morphological changes as well as sera, body fluid, and tissues (including placenta) to measure viral titers using plaque-forming assay. Placenta was also observed for apoptosis and vascular damage and examined for inflammatory cytokines and chemokines as described below. (A and B) Representative image of morphology of uteri (E16–E18) and fetuses from pregnant mice challenged with ZIKV at E10-E12 (A), among which total numbers and dead fetuses from each group are shown (B). (C–E) Viral titers in placenta, fetal brain (C), as well as sera and amniotic fluid (D) and tissues (including brain, kidney, liver, spleen, heart, and muscle) (E), 6 days post-challenge. The detection limit was 12.5 PFU/g (for placenta and fetal brain), 20 PFU/g (for heart), 25 PFU/ml (for sera and amniotic fluid), and 25 PFU/g (for brain, kidney, liver, spleen, and muscle). The data are represented as mean ± SEM (n = 6). *p < 0.05; **p < 0.01; ***p < 0.001.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Anti-mouse CD4 (IgG2b) antibody Bio X Cell BP0003-1; RRID:AB_1107636 Anti-mouse CD8a (IgG2b) antibody Bio X Cell BP0061; RRID:AB_1125541 Rat IgG2b isotype control antibody Bio X Cell BP0090; RRID:AB_1107780 Anti-mouse-CD8a (IgG2a) antibody Bio X Cell BE0004-1; RRID:AB_1107671 Rat IgG2a isotype control antibody Bio X Cell BE0089; RRID:AB_1107769 Anti-mouse IFNAR1 (MAR1-5A3) antibody Leinco I-401; RRID:AB_2491621 ZIKV EDIII-specific human antibody, ZV-64 Absolute Antibody Ab00811-10.0 Rabbit anti-vimentin antibody Abcam Ab92547; RRID:AB_10562134 Rabbit anti-active caspase-3 antibody Abcam Ab2302; RRID:AB_302962 FITC-anti-human antibody Sigma 9512-1mL; RRID:AB_259808 Anti-rabbit Alexa Fluor 647 antibody Abcam Ab150075; RRID:AB_2752244 FITC-anti-mouse antibody Biolegend 406001; RRID:AB_315029 Anti-flavivirus 4G2 antibody BEI Resources NR-50327 PerCP-Cy5.5-anti-mouse CD8 antibody BD Biosciences 551162; RRID:AB_394081 FITC-anti-mouse IL-2 antibody BD Biosciences 554427; RRID:AB_395385 FITC-anti-mouse CD4 antibody BD Biosciences 553729; RRID:AB_395013 PE-anti-mouse IFN-g antibody BD Biosciences 554412; RRID:AB_395376 Brilliant Violet 421-anti-mouse TNF-a antibody Biolegend 506328; RRID:AB_2562902 HRP-conjugated anti-mouse IgG-Fab antibody Sigma a9917-1mL; RRID:AB_258476 Anti-ZIKV NS3 antibody Genetex GTX133309; RRID:AB_2756864 Bacterial and virus strains Zika virus (human strain R103451 (2015/ Honduras)) BEI Resources NR-50355 Zika virus (human strain PAN2016 (2016/ Panama)) BEI Resources NR-50210 Chemicals, peptides, and recombinant proteins Zika virus full-length envelope (E) protein Aviva Systems Biology OPMA04848 Zika virus EDI/EDII peptides (see below peptide 1-5) BEI Resources NR-50553 Peptide 1: IGVSNRDFVEGMSGG BEI Resources N/A Peptide 2: TWVDVVLEHGGCVTV BEI Resources N/A Peptide 3: MAQDKPTVDIELVTT BEI Resources N/A Peptide 4: VDRGWGNGCGLFGKG BEI Resources N/A Peptide 5: VVLGSQEGAVHTALA BEI Resources N/A Zika virus NS3 overlapping peptides (see below peptide 6-15) GenScript N/A Peptide 6: AETDEDHAHWLEARM GenScript N/A Peptide 7: HAHWLEARMLLDNIY GenScript N/A Peptide 8: ARMLLDNIYLQDGLI GenScript N/A Peptide 9: NIYLQDGLIASLYRP GenScript N/A Peptide 10: GLIASLYRPEADKVA GenScript N/A Peptide 11: YRPEADKVAAIEGEF GenScript N/A (Continued on next page) e1 Cell Reports 35, 109107, May 11, 2021

Techniques: Control

Figure 4. ZIKV TCI-DNA vaccine prevented ZIKV-caused apoptosis in placenta of female pregnant Ifnar1/ mice Placenta collected from above ZIKV-challenged pregnant (E10–E12) Ifnar1/ mice was stained for activated form of caspase-3 (an apoptotic marker) by immunofluorescence assay. (A) Representative images of immunofluorescence staining of activated caspase-3 in placenta. ZIKV (green), activated caspase-3 (red), and nuclei (blue) were stained with anti-ZIKV antibody, anti-active caspase-3 antibody, and DAPI, respectively. The images were magnified at 633, with a scale bar of 10 mm. (B and C) Quantification of ZIKV+ (B) and activated caspase-3+ (C) staining in (A) by ImageJ software. The data are presented as mean ± SEM of fluorescence intensity for ZIKV+ or caspase-3+ staining in each field (n = 6; n indicates numbers of images from different placentas). *p < 0.05; **p < 0.01; ***p < 0.001.

Journal: Cell reports

Article Title: A vaccine inducing solely cytotoxic T lymphocytes fully prevents Zika virus infection and fetal damage.

doi: 10.1016/j.celrep.2021.109107

Figure Lengend Snippet: Figure 4. ZIKV TCI-DNA vaccine prevented ZIKV-caused apoptosis in placenta of female pregnant Ifnar1/ mice Placenta collected from above ZIKV-challenged pregnant (E10–E12) Ifnar1/ mice was stained for activated form of caspase-3 (an apoptotic marker) by immunofluorescence assay. (A) Representative images of immunofluorescence staining of activated caspase-3 in placenta. ZIKV (green), activated caspase-3 (red), and nuclei (blue) were stained with anti-ZIKV antibody, anti-active caspase-3 antibody, and DAPI, respectively. The images were magnified at 633, with a scale bar of 10 mm. (B and C) Quantification of ZIKV+ (B) and activated caspase-3+ (C) staining in (A) by ImageJ software. The data are presented as mean ± SEM of fluorescence intensity for ZIKV+ or caspase-3+ staining in each field (n = 6; n indicates numbers of images from different placentas). *p < 0.05; **p < 0.01; ***p < 0.001.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Anti-mouse CD4 (IgG2b) antibody Bio X Cell BP0003-1; RRID:AB_1107636 Anti-mouse CD8a (IgG2b) antibody Bio X Cell BP0061; RRID:AB_1125541 Rat IgG2b isotype control antibody Bio X Cell BP0090; RRID:AB_1107780 Anti-mouse-CD8a (IgG2a) antibody Bio X Cell BE0004-1; RRID:AB_1107671 Rat IgG2a isotype control antibody Bio X Cell BE0089; RRID:AB_1107769 Anti-mouse IFNAR1 (MAR1-5A3) antibody Leinco I-401; RRID:AB_2491621 ZIKV EDIII-specific human antibody, ZV-64 Absolute Antibody Ab00811-10.0 Rabbit anti-vimentin antibody Abcam Ab92547; RRID:AB_10562134 Rabbit anti-active caspase-3 antibody Abcam Ab2302; RRID:AB_302962 FITC-anti-human antibody Sigma 9512-1mL; RRID:AB_259808 Anti-rabbit Alexa Fluor 647 antibody Abcam Ab150075; RRID:AB_2752244 FITC-anti-mouse antibody Biolegend 406001; RRID:AB_315029 Anti-flavivirus 4G2 antibody BEI Resources NR-50327 PerCP-Cy5.5-anti-mouse CD8 antibody BD Biosciences 551162; RRID:AB_394081 FITC-anti-mouse IL-2 antibody BD Biosciences 554427; RRID:AB_395385 FITC-anti-mouse CD4 antibody BD Biosciences 553729; RRID:AB_395013 PE-anti-mouse IFN-g antibody BD Biosciences 554412; RRID:AB_395376 Brilliant Violet 421-anti-mouse TNF-a antibody Biolegend 506328; RRID:AB_2562902 HRP-conjugated anti-mouse IgG-Fab antibody Sigma a9917-1mL; RRID:AB_258476 Anti-ZIKV NS3 antibody Genetex GTX133309; RRID:AB_2756864 Bacterial and virus strains Zika virus (human strain R103451 (2015/ Honduras)) BEI Resources NR-50355 Zika virus (human strain PAN2016 (2016/ Panama)) BEI Resources NR-50210 Chemicals, peptides, and recombinant proteins Zika virus full-length envelope (E) protein Aviva Systems Biology OPMA04848 Zika virus EDI/EDII peptides (see below peptide 1-5) BEI Resources NR-50553 Peptide 1: IGVSNRDFVEGMSGG BEI Resources N/A Peptide 2: TWVDVVLEHGGCVTV BEI Resources N/A Peptide 3: MAQDKPTVDIELVTT BEI Resources N/A Peptide 4: VDRGWGNGCGLFGKG BEI Resources N/A Peptide 5: VVLGSQEGAVHTALA BEI Resources N/A Zika virus NS3 overlapping peptides (see below peptide 6-15) GenScript N/A Peptide 6: AETDEDHAHWLEARM GenScript N/A Peptide 7: HAHWLEARMLLDNIY GenScript N/A Peptide 8: ARMLLDNIYLQDGLI GenScript N/A Peptide 9: NIYLQDGLIASLYRP GenScript N/A Peptide 10: GLIASLYRPEADKVA GenScript N/A Peptide 11: YRPEADKVAAIEGEF GenScript N/A (Continued on next page) e1 Cell Reports 35, 109107, May 11, 2021

Techniques: Staining, Marker, Software

Figure 5. ZIKV TCI-DNA vaccine prevented ZIKV-caused inflammation in placenta of female pregnant Ifnar1/ mice Placentas collected from above challenged pregnant (E10–E12) Ifnar1/ mice were examined for inflammatory cytokines (A) and chemokines (B) by Mouse Inflammatory Cytokines Multi-Analyte ELISArray Kit and Mouse Common Chemokines Multi-Analyte ELISArray Kit, respectively. The data are presented as mean ± SEM (n = 6). *p < 0.05; **p < 0.01; ***p < 0.001.

Journal: Cell reports

Article Title: A vaccine inducing solely cytotoxic T lymphocytes fully prevents Zika virus infection and fetal damage.

doi: 10.1016/j.celrep.2021.109107

Figure Lengend Snippet: Figure 5. ZIKV TCI-DNA vaccine prevented ZIKV-caused inflammation in placenta of female pregnant Ifnar1/ mice Placentas collected from above challenged pregnant (E10–E12) Ifnar1/ mice were examined for inflammatory cytokines (A) and chemokines (B) by Mouse Inflammatory Cytokines Multi-Analyte ELISArray Kit and Mouse Common Chemokines Multi-Analyte ELISArray Kit, respectively. The data are presented as mean ± SEM (n = 6). *p < 0.05; **p < 0.01; ***p < 0.001.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Anti-mouse CD4 (IgG2b) antibody Bio X Cell BP0003-1; RRID:AB_1107636 Anti-mouse CD8a (IgG2b) antibody Bio X Cell BP0061; RRID:AB_1125541 Rat IgG2b isotype control antibody Bio X Cell BP0090; RRID:AB_1107780 Anti-mouse-CD8a (IgG2a) antibody Bio X Cell BE0004-1; RRID:AB_1107671 Rat IgG2a isotype control antibody Bio X Cell BE0089; RRID:AB_1107769 Anti-mouse IFNAR1 (MAR1-5A3) antibody Leinco I-401; RRID:AB_2491621 ZIKV EDIII-specific human antibody, ZV-64 Absolute Antibody Ab00811-10.0 Rabbit anti-vimentin antibody Abcam Ab92547; RRID:AB_10562134 Rabbit anti-active caspase-3 antibody Abcam Ab2302; RRID:AB_302962 FITC-anti-human antibody Sigma 9512-1mL; RRID:AB_259808 Anti-rabbit Alexa Fluor 647 antibody Abcam Ab150075; RRID:AB_2752244 FITC-anti-mouse antibody Biolegend 406001; RRID:AB_315029 Anti-flavivirus 4G2 antibody BEI Resources NR-50327 PerCP-Cy5.5-anti-mouse CD8 antibody BD Biosciences 551162; RRID:AB_394081 FITC-anti-mouse IL-2 antibody BD Biosciences 554427; RRID:AB_395385 FITC-anti-mouse CD4 antibody BD Biosciences 553729; RRID:AB_395013 PE-anti-mouse IFN-g antibody BD Biosciences 554412; RRID:AB_395376 Brilliant Violet 421-anti-mouse TNF-a antibody Biolegend 506328; RRID:AB_2562902 HRP-conjugated anti-mouse IgG-Fab antibody Sigma a9917-1mL; RRID:AB_258476 Anti-ZIKV NS3 antibody Genetex GTX133309; RRID:AB_2756864 Bacterial and virus strains Zika virus (human strain R103451 (2015/ Honduras)) BEI Resources NR-50355 Zika virus (human strain PAN2016 (2016/ Panama)) BEI Resources NR-50210 Chemicals, peptides, and recombinant proteins Zika virus full-length envelope (E) protein Aviva Systems Biology OPMA04848 Zika virus EDI/EDII peptides (see below peptide 1-5) BEI Resources NR-50553 Peptide 1: IGVSNRDFVEGMSGG BEI Resources N/A Peptide 2: TWVDVVLEHGGCVTV BEI Resources N/A Peptide 3: MAQDKPTVDIELVTT BEI Resources N/A Peptide 4: VDRGWGNGCGLFGKG BEI Resources N/A Peptide 5: VVLGSQEGAVHTALA BEI Resources N/A Zika virus NS3 overlapping peptides (see below peptide 6-15) GenScript N/A Peptide 6: AETDEDHAHWLEARM GenScript N/A Peptide 7: HAHWLEARMLLDNIY GenScript N/A Peptide 8: ARMLLDNIYLQDGLI GenScript N/A Peptide 9: NIYLQDGLIASLYRP GenScript N/A Peptide 10: GLIASLYRPEADKVA GenScript N/A Peptide 11: YRPEADKVAAIEGEF GenScript N/A (Continued on next page) e1 Cell Reports 35, 109107, May 11, 2021

Techniques:

Figure 6. ZIKV TCI-DNA vaccine induced low to no ZIKV-, E-, and NS3-specific antibodies without ADE effect Mouse sera collected at 10 days post-second immunization were detected for ZIKV E-, NS3-, and ZIKV-specific IgG antibody, neutralizing antibodies, and ADE of ZIKV infection. ZIKV strain R103451 was used for the neutralization and ADE tests. (A and B) ELISA was used for detection of IgG antibody specific to ZIKV E ectodomain, NS3 peptides, and ZIKV (R103451 strain) in sera of BALB/c (A) or Ifnar1/

Journal: Cell reports

Article Title: A vaccine inducing solely cytotoxic T lymphocytes fully prevents Zika virus infection and fetal damage.

doi: 10.1016/j.celrep.2021.109107

Figure Lengend Snippet: Figure 6. ZIKV TCI-DNA vaccine induced low to no ZIKV-, E-, and NS3-specific antibodies without ADE effect Mouse sera collected at 10 days post-second immunization were detected for ZIKV E-, NS3-, and ZIKV-specific IgG antibody, neutralizing antibodies, and ADE of ZIKV infection. ZIKV strain R103451 was used for the neutralization and ADE tests. (A and B) ELISA was used for detection of IgG antibody specific to ZIKV E ectodomain, NS3 peptides, and ZIKV (R103451 strain) in sera of BALB/c (A) or Ifnar1/

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Anti-mouse CD4 (IgG2b) antibody Bio X Cell BP0003-1; RRID:AB_1107636 Anti-mouse CD8a (IgG2b) antibody Bio X Cell BP0061; RRID:AB_1125541 Rat IgG2b isotype control antibody Bio X Cell BP0090; RRID:AB_1107780 Anti-mouse-CD8a (IgG2a) antibody Bio X Cell BE0004-1; RRID:AB_1107671 Rat IgG2a isotype control antibody Bio X Cell BE0089; RRID:AB_1107769 Anti-mouse IFNAR1 (MAR1-5A3) antibody Leinco I-401; RRID:AB_2491621 ZIKV EDIII-specific human antibody, ZV-64 Absolute Antibody Ab00811-10.0 Rabbit anti-vimentin antibody Abcam Ab92547; RRID:AB_10562134 Rabbit anti-active caspase-3 antibody Abcam Ab2302; RRID:AB_302962 FITC-anti-human antibody Sigma 9512-1mL; RRID:AB_259808 Anti-rabbit Alexa Fluor 647 antibody Abcam Ab150075; RRID:AB_2752244 FITC-anti-mouse antibody Biolegend 406001; RRID:AB_315029 Anti-flavivirus 4G2 antibody BEI Resources NR-50327 PerCP-Cy5.5-anti-mouse CD8 antibody BD Biosciences 551162; RRID:AB_394081 FITC-anti-mouse IL-2 antibody BD Biosciences 554427; RRID:AB_395385 FITC-anti-mouse CD4 antibody BD Biosciences 553729; RRID:AB_395013 PE-anti-mouse IFN-g antibody BD Biosciences 554412; RRID:AB_395376 Brilliant Violet 421-anti-mouse TNF-a antibody Biolegend 506328; RRID:AB_2562902 HRP-conjugated anti-mouse IgG-Fab antibody Sigma a9917-1mL; RRID:AB_258476 Anti-ZIKV NS3 antibody Genetex GTX133309; RRID:AB_2756864 Bacterial and virus strains Zika virus (human strain R103451 (2015/ Honduras)) BEI Resources NR-50355 Zika virus (human strain PAN2016 (2016/ Panama)) BEI Resources NR-50210 Chemicals, peptides, and recombinant proteins Zika virus full-length envelope (E) protein Aviva Systems Biology OPMA04848 Zika virus EDI/EDII peptides (see below peptide 1-5) BEI Resources NR-50553 Peptide 1: IGVSNRDFVEGMSGG BEI Resources N/A Peptide 2: TWVDVVLEHGGCVTV BEI Resources N/A Peptide 3: MAQDKPTVDIELVTT BEI Resources N/A Peptide 4: VDRGWGNGCGLFGKG BEI Resources N/A Peptide 5: VVLGSQEGAVHTALA BEI Resources N/A Zika virus NS3 overlapping peptides (see below peptide 6-15) GenScript N/A Peptide 6: AETDEDHAHWLEARM GenScript N/A Peptide 7: HAHWLEARMLLDNIY GenScript N/A Peptide 8: ARMLLDNIYLQDGLI GenScript N/A Peptide 9: NIYLQDGLIASLYRP GenScript N/A Peptide 10: GLIASLYRPEADKVA GenScript N/A Peptide 11: YRPEADKVAAIEGEF GenScript N/A (Continued on next page) e1 Cell Reports 35, 109107, May 11, 2021

Techniques: Infection, Neutralization, Enzyme-linked Immunosorbent Assay

Figure 7. CD8+ T cell immune responses induced by ZIKV TCI-DNA vaccine played a key role in protecting pregnant mothers and their fe- tuses against ZIKV infection Female BALB/c mice were immunized with ZIKV TCI-DNA or PBS control for two doses and mated with male BALB/c mice for pregnancy at 10 days post-second immunization. The pregnant (E10–E12) mice were then injected (i.p.) with anti-CD8a (for depleting CD8+ T cells) or IgG2a isotype control (i.e., Iso con; without depleting CD8+ T cells) antibody (200 mg/mouse) for three times (2, 1, and 3 days p.i.). One day before challenge, the mice were also injected with anti-IFNAR1 blocking antibody (for depleting type I IFN; 2 mg/mouse) and then infected with ZIKV (strain R103451, 106 PFU/mouse). (A–C) Six days post-challenge, the mice were euthanized, recorded for morphology of uteri and fetuses (A), and counted for dead and total fetuses (B), and viral titers were determined by plaque-forming assay in placenta, amniotic fluid, and fetal brain, as well as sera collected at 3 and 6 days post-challenge (C). The detection limit was 20 PFU/g (for placenta), 40 PFU/g (for fetal brain), and 50 PFU/ml (for sera and amniotic fluid). Six days post-challenge, splenocytes were isolated from the mice injected with isotype control antibody (i.e., Iso con) and analyzed for ZIKV-specific CD8+ T cell responses by flow cytometry analysis. (D) Quantification of the frequencies of CD8+ T cells (left), as well as IL2+, IFN-g+, and tumor necrosis factor (TNF)-a secretion in CD8+ T cells (right) in splenocytes. The data are represented as mean ± SEM (n = 6). *p < 0.05; **p < 0.01; ***p < 0.001.

Journal: Cell reports

Article Title: A vaccine inducing solely cytotoxic T lymphocytes fully prevents Zika virus infection and fetal damage.

doi: 10.1016/j.celrep.2021.109107

Figure Lengend Snippet: Figure 7. CD8+ T cell immune responses induced by ZIKV TCI-DNA vaccine played a key role in protecting pregnant mothers and their fe- tuses against ZIKV infection Female BALB/c mice were immunized with ZIKV TCI-DNA or PBS control for two doses and mated with male BALB/c mice for pregnancy at 10 days post-second immunization. The pregnant (E10–E12) mice were then injected (i.p.) with anti-CD8a (for depleting CD8+ T cells) or IgG2a isotype control (i.e., Iso con; without depleting CD8+ T cells) antibody (200 mg/mouse) for three times (2, 1, and 3 days p.i.). One day before challenge, the mice were also injected with anti-IFNAR1 blocking antibody (for depleting type I IFN; 2 mg/mouse) and then infected with ZIKV (strain R103451, 106 PFU/mouse). (A–C) Six days post-challenge, the mice were euthanized, recorded for morphology of uteri and fetuses (A), and counted for dead and total fetuses (B), and viral titers were determined by plaque-forming assay in placenta, amniotic fluid, and fetal brain, as well as sera collected at 3 and 6 days post-challenge (C). The detection limit was 20 PFU/g (for placenta), 40 PFU/g (for fetal brain), and 50 PFU/ml (for sera and amniotic fluid). Six days post-challenge, splenocytes were isolated from the mice injected with isotype control antibody (i.e., Iso con) and analyzed for ZIKV-specific CD8+ T cell responses by flow cytometry analysis. (D) Quantification of the frequencies of CD8+ T cells (left), as well as IL2+, IFN-g+, and tumor necrosis factor (TNF)-a secretion in CD8+ T cells (right) in splenocytes. The data are represented as mean ± SEM (n = 6). *p < 0.05; **p < 0.01; ***p < 0.001.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Anti-mouse CD4 (IgG2b) antibody Bio X Cell BP0003-1; RRID:AB_1107636 Anti-mouse CD8a (IgG2b) antibody Bio X Cell BP0061; RRID:AB_1125541 Rat IgG2b isotype control antibody Bio X Cell BP0090; RRID:AB_1107780 Anti-mouse-CD8a (IgG2a) antibody Bio X Cell BE0004-1; RRID:AB_1107671 Rat IgG2a isotype control antibody Bio X Cell BE0089; RRID:AB_1107769 Anti-mouse IFNAR1 (MAR1-5A3) antibody Leinco I-401; RRID:AB_2491621 ZIKV EDIII-specific human antibody, ZV-64 Absolute Antibody Ab00811-10.0 Rabbit anti-vimentin antibody Abcam Ab92547; RRID:AB_10562134 Rabbit anti-active caspase-3 antibody Abcam Ab2302; RRID:AB_302962 FITC-anti-human antibody Sigma 9512-1mL; RRID:AB_259808 Anti-rabbit Alexa Fluor 647 antibody Abcam Ab150075; RRID:AB_2752244 FITC-anti-mouse antibody Biolegend 406001; RRID:AB_315029 Anti-flavivirus 4G2 antibody BEI Resources NR-50327 PerCP-Cy5.5-anti-mouse CD8 antibody BD Biosciences 551162; RRID:AB_394081 FITC-anti-mouse IL-2 antibody BD Biosciences 554427; RRID:AB_395385 FITC-anti-mouse CD4 antibody BD Biosciences 553729; RRID:AB_395013 PE-anti-mouse IFN-g antibody BD Biosciences 554412; RRID:AB_395376 Brilliant Violet 421-anti-mouse TNF-a antibody Biolegend 506328; RRID:AB_2562902 HRP-conjugated anti-mouse IgG-Fab antibody Sigma a9917-1mL; RRID:AB_258476 Anti-ZIKV NS3 antibody Genetex GTX133309; RRID:AB_2756864 Bacterial and virus strains Zika virus (human strain R103451 (2015/ Honduras)) BEI Resources NR-50355 Zika virus (human strain PAN2016 (2016/ Panama)) BEI Resources NR-50210 Chemicals, peptides, and recombinant proteins Zika virus full-length envelope (E) protein Aviva Systems Biology OPMA04848 Zika virus EDI/EDII peptides (see below peptide 1-5) BEI Resources NR-50553 Peptide 1: IGVSNRDFVEGMSGG BEI Resources N/A Peptide 2: TWVDVVLEHGGCVTV BEI Resources N/A Peptide 3: MAQDKPTVDIELVTT BEI Resources N/A Peptide 4: VDRGWGNGCGLFGKG BEI Resources N/A Peptide 5: VVLGSQEGAVHTALA BEI Resources N/A Zika virus NS3 overlapping peptides (see below peptide 6-15) GenScript N/A Peptide 6: AETDEDHAHWLEARM GenScript N/A Peptide 7: HAHWLEARMLLDNIY GenScript N/A Peptide 8: ARMLLDNIYLQDGLI GenScript N/A Peptide 9: NIYLQDGLIASLYRP GenScript N/A Peptide 10: GLIASLYRPEADKVA GenScript N/A Peptide 11: YRPEADKVAAIEGEF GenScript N/A (Continued on next page) e1 Cell Reports 35, 109107, May 11, 2021

Techniques: Infection, Control, Injection, Blocking Assay, Isolation, Cytometry