Journal: bioRxiv

Article Title: Analysis of Serologic Cross-Reactivity Between Common Human Coronaviruses and SARS-CoV-2 Using Coronavirus Antigen Microarray

doi: 10.1101/2020.03.24.006544

Figure Lengend Snippet: Content of coronavirus antigen microarray.

Article Snippet: Influenza , H1N1 , A/Beijing/22808/2009 , HA1+HA2 , ADD64203.1 , HEK293 , Sino Biological , N-(AA1-529)-His-C , 40035-V08H.

Techniques: Microarray

Journal: bioRxiv

Article Title: Analysis of Serologic Cross-Reactivity Between Common Human Coronaviruses and SARS-CoV-2 Using Coronavirus Antigen Microarray

doi: 10.1101/2020.03.24.006544

Figure Lengend Snippet: Non-coronavirus respiratory virus antigens on microarray.

Article Snippet: Influenza , H1N1 , A/Beijing/22808/2009 , HA1+HA2 , ADD64203.1 , HEK293 , Sino Biological , N-(AA1-529)-His-C , 40035-V08H.

Techniques: Microarray, Expressing, Construct

Journal: Cell Death & Disease

Article Title: Subverted regulation of Nox1 NADPH oxidase-dependent oxidant generation by protein disulfide isomerase A1 in colon carcinoma cells with overactivated KRas

doi: 10.1038/s41419-019-1402-y

Figure Lengend Snippet: a KRas expression and activity in Caco2, HKE3, and HCT116 cells. Active KRas was pulled down with GST-RBD beads from lysates of serum-starved cells treated with EGF 25 ng/mL for 10 min. Aliquots of the lysates were blotted for total KRas and GAPDH as loading control. b RNAseq analysis of P4HB (PDIA1) gene in Caco2, HCT116, and HCT15 cells, showing mean expression in FPKM (Fragments Per Kilobase Million), P4HB fold-change expression in HCT15 vs. Caco2 (q < 0.000001) and HCT116 vs. Caco2 (q < 0.000001). c PDIA1 basal protein expression by western analysis, normalized for GAPDH ( n = 3) and quantified using Odyssey software. d Intracellular PDIA1 titration, using Human P4HB ELISA Pair Set (SinoBiological). Test t ** p < 0.01, ( n = 4); e–g ROS production after 72 h of PDIA1 silencing, measured by HPLC analysis of DHE oxidation products. DHE oxidation produces, among many others, two major products: 2-hidroxyethidium (EOH), which is representative of superoxide species and ethidium, representative of other oxidant species. Ctrl negative, si-RNA control; si-PDI, si-RNA against PDIA1 protein. Representative immunoblots of PDI silencing for each cell type Test t * p < 0.05; ** p < 0.01. For Caco2 cells, n = 3; HKE3 cells, n = 4; HCT116 cells, n = 5

Article Snippet: Active KRas was pulled down with GST-RBD beads from lysates of serum-starved cells treated with EGF 25 ng/mL for 10 min. Aliquots of the lysates were blotted for total KRas and GAPDH as loading control. b RNAseq analysis of P4HB (PDIA1) gene in Caco2, HCT116, and HCT15 cells, showing mean expression in FPKM (Fragments Per Kilobase Million), P4HB fold-change expression in HCT15 vs. Caco2 (q < 0.000001) and HCT116 vs. Caco2 (q < 0.000001). c PDIA1 basal protein expression by western analysis, normalized for GAPDH ( n = 3) and quantified using Odyssey software. d Intracellular PDIA1 titration, using Human P4HB ELISA Pair Set (SinoBiological).

Techniques: Expressing, Activity Assay, Western Blot, Software, Titration, Enzyme-linked Immunosorbent Assay

Journal: Cell Death & Disease

Article Title: Subverted regulation of Nox1 NADPH oxidase-dependent oxidant generation by protein disulfide isomerase A1 in colon carcinoma cells with overactivated KRas

doi: 10.1038/s41419-019-1402-y

Figure Lengend Snippet: a Nox1, NoxA1, p67phox, NoxO1, and p47phox basal protein expression by western analysis normalized for GADPH. Nox1 immunoblot densities were quantified using Odyssey software. * P < 0.05, ANOVA plus Tukey's multiple comparisons test ( n = 3). b RhoGDIα, RhoA, and Rac1 basal protein expression by western analysis normalized for GAPDH. Rac1 immunoblot densities were quantified using Odyssey software. ** P < 0.01; *** P < 0.005, ANOVA plus Tukey's multiple comparisons test ( n = 3). c Nox1 complex scheme. d Rac1 basal activity, using Rac1 G-LISA activation assay (Cytoskeleton,inc). Test t * p < 0.05, ( n = 2). e Rac1 protein expression by western analysis after 72 h of PDIA1 silencing. Rac1 Immunoblot densities were quantified using Odyssey software ** P < 0.01; ns: not significant, Student's t -test ( n = 3). f Western analysis of NoxO1 and RhoGDIα protein expression before and after PDIA1 silencing, normalized for GAPDH. RhoGDIα immunoblot densities were quantified using Odyssey software, **** P < 0.001; ns: not significant, Student's t -test ( n = 3). g Superoxide production after 72h of PDIA1 silencing in HCT116 cells treated or not with 10 µM of NoxA1ds Nox1’s peptide inhibitor. EOH 2-hidroxyethidiium relative superoxide production, si-PDI, si-RNA against PDIA1 protein; Scrmb NoxA1ds, NoxA1ds negative control peptide; NoxA1ds, Nox1 peptide inhibitor. * P < 0.05; ** P < 0.01, ANOVA plus Tukey's multiple comparisons test ( n = 3)

Article Snippet: Active KRas was pulled down with GST-RBD beads from lysates of serum-starved cells treated with EGF 25 ng/mL for 10 min. Aliquots of the lysates were blotted for total KRas and GAPDH as loading control. b RNAseq analysis of P4HB (PDIA1) gene in Caco2, HCT116, and HCT15 cells, showing mean expression in FPKM (Fragments Per Kilobase Million), P4HB fold-change expression in HCT15 vs. Caco2 (q < 0.000001) and HCT116 vs. Caco2 (q < 0.000001). c PDIA1 basal protein expression by western analysis, normalized for GAPDH ( n = 3) and quantified using Odyssey software. d Intracellular PDIA1 titration, using Human P4HB ELISA Pair Set (SinoBiological).

Techniques: Expressing, Western Blot, Software, Activity Assay, Activation Assay, Negative Control

Journal: Cell Death & Disease

Article Title: Subverted regulation of Nox1 NADPH oxidase-dependent oxidant generation by protein disulfide isomerase A1 in colon carcinoma cells with overactivated KRas

doi: 10.1038/s41419-019-1402-y

Figure Lengend Snippet: a Superoxide production after 72 h of PDIA1 silencing in HKE3 transfected with Rac1 G12V , treated or not with 10 µM of NoxA1ds (Nox1 peptide inhibitor), measured by DHE oxidation detected by HPLC. ** p < 0.01, ANOVA plus Tukey's multiple comparison test ( n = 3). b Superoxide production after 72 h of PDIA1 silencing in HCT116 cells treated or not with 50 µM of W56 Rac1 peptide inhibitor. * p < 0.05, ANOVA plus Tukey's multiple comparison test ( n = 4). c PDIA1 co-immunoprecipitation, IP: PDIA1 immunoprecipitation in Caco2, HKE3, and HCT116 cells, IgG: Immunoglobulin control, Input: 1% of total protein lysate, IB: immunoblot against PDIA1, KRas, Rac1, GAPDH, and tubulin ( n = 2). EOH: 2-hidroxyethidiium relative superoxide production

Article Snippet: Active KRas was pulled down with GST-RBD beads from lysates of serum-starved cells treated with EGF 25 ng/mL for 10 min. Aliquots of the lysates were blotted for total KRas and GAPDH as loading control. b RNAseq analysis of P4HB (PDIA1) gene in Caco2, HCT116, and HCT15 cells, showing mean expression in FPKM (Fragments Per Kilobase Million), P4HB fold-change expression in HCT15 vs. Caco2 (q < 0.000001) and HCT116 vs. Caco2 (q < 0.000001). c PDIA1 basal protein expression by western analysis, normalized for GAPDH ( n = 3) and quantified using Odyssey software. d Intracellular PDIA1 titration, using Human P4HB ELISA Pair Set (SinoBiological).

Techniques: Transfection, Immunoprecipitation, Western Blot

Journal: Cell Death & Disease

Article Title: Subverted regulation of Nox1 NADPH oxidase-dependent oxidant generation by protein disulfide isomerase A1 in colon carcinoma cells with overactivated KRas

doi: 10.1038/s41419-019-1402-y

Figure Lengend Snippet: 3D cell proliferation assay: a Representative phase-contrast images of spheroid proliferation with or without PDIA1 silencing; pictures were taken at T0 and T48 h after spheroid formation on methylcellulose media. Scale bar, 500 µm. b Proliferation analysis: spheroid surface area was measured at T0 and T48 h, using ImageJ software. Spheroid growth was calculated as T48 h spheroid area/T0 spheroid area ; between 17 and 27 spheroids were analyzed for each condition. Data are mean fold ± SD, ** p < 0.01; **** p < 0.0001 vs. HKE3 scrmb, ANOVA plus Tukey's multiple comparison test. c Effect of PDIA1 silencing on cell invasion: representative phase-contrast images of spheroid invasion in 2D fibronectin matrix (10 µM); pictures were taken at T0 and T48 h after spheroids were laid down on matrix. Scale bar, 500 µm. d Spheroid 2D invasion analysis: total spheroid expansion was measured at T0 and T48 h using ImageJ software. Spheroid expansion was calculated as (T48 h total evasion area—T0 initial spheroid area)/T0 initial spheroid area ; between 12 and 17 spheroids were analyzed for each condition. Data are mean ± SD, **** p < 0.0001 vs. HKE3 scrmb, ANOVA plus Tukey's multiple comparison test

Article Snippet: Active KRas was pulled down with GST-RBD beads from lysates of serum-starved cells treated with EGF 25 ng/mL for 10 min. Aliquots of the lysates were blotted for total KRas and GAPDH as loading control. b RNAseq analysis of P4HB (PDIA1) gene in Caco2, HCT116, and HCT15 cells, showing mean expression in FPKM (Fragments Per Kilobase Million), P4HB fold-change expression in HCT15 vs. Caco2 (q < 0.000001) and HCT116 vs. Caco2 (q < 0.000001). c PDIA1 basal protein expression by western analysis, normalized for GAPDH ( n = 3) and quantified using Odyssey software. d Intracellular PDIA1 titration, using Human P4HB ELISA Pair Set (SinoBiological).

Techniques: Proliferation Assay, Software

Journal: Cell Death & Disease

Article Title: Subverted regulation of Nox1 NADPH oxidase-dependent oxidant generation by protein disulfide isomerase A1 in colon carcinoma cells with overactivated KRas

doi: 10.1038/s41419-019-1402-y

Figure Lengend Snippet: PathScan array analysis. All array’s spots were quantified and analyzed using ImageJ software. PDIA1 silencing was checked by immunoblot analysis. Ctrl, si-RNA negative control; si-PDI, si-RNA against PDIA1; ERK1/2, Thr202/Tyr204 phosphorylation; Stat1, Tyr701 phosphorylation; Stat3, Tyr705 phosphorylation; Akt, Thr308 phosphorylation; Akt, Ser473 phosphorylation; AMPKa, Thr172 phosphorylation, s6 ribosomal protein, Ser235/236 phosphorylation; mTOR, Ser2448 phosphorylation; HSP27, Ser78 phosphorylation; Bad, Ser112 phosphorylation; p70 S6 kinase, Thr389 phosphorylation; PRAS40, Thr246 phosphorylation; p53, Ser15 phosphorylation; p38, Thr180/Tyr182 phosphorylation; SAPK/JNK, Thr183/Tyr185 phosphorylation; PARP, Asp214 cleavage; Caspase-3, Asp175 Cleavage; GSK-3β, Ser9 phosphorylation. b GSK-3β, Ser9 phosphorylation plot. c Stat3, Tyr705 phosphorylation. d Basal E-cadherin (E-cad) protein expression by immunoblot normalized for GAPDH ( n = 3). e Immunoblot for E-cadherin before and after PDIA1 silencing in Caco2 cells ( n = 2). f Immunoblot for E-cadherin before and after PDIA1 silencing in HKE3 cells.; ns: not significant, Student's t test ( n = 3). g Immunoblot for E-cadherin before and after PDIA1 silencing in HCT116 cells * P < 0.05, Student's t test ( n = 3). Immunoblot densities were quantified using Odyssey software

Article Snippet: Active KRas was pulled down with GST-RBD beads from lysates of serum-starved cells treated with EGF 25 ng/mL for 10 min. Aliquots of the lysates were blotted for total KRas and GAPDH as loading control. b RNAseq analysis of P4HB (PDIA1) gene in Caco2, HCT116, and HCT15 cells, showing mean expression in FPKM (Fragments Per Kilobase Million), P4HB fold-change expression in HCT15 vs. Caco2 (q < 0.000001) and HCT116 vs. Caco2 (q < 0.000001). c PDIA1 basal protein expression by western analysis, normalized for GAPDH ( n = 3) and quantified using Odyssey software. d Intracellular PDIA1 titration, using Human P4HB ELISA Pair Set (SinoBiological).

Techniques: Software, Western Blot, Negative Control, Expressing

Journal: Cell Death & Disease

Article Title: Subverted regulation of Nox1 NADPH oxidase-dependent oxidant generation by protein disulfide isomerase A1 in colon carcinoma cells with overactivated KRas

doi: 10.1038/s41419-019-1402-y

Figure Lengend Snippet: a Interaction map fashioned with String 10.5 program ( http://string-db.org ), Network nodes represent proteins of Nox1, PDIA1 (P4HB), RhoGDIα (ARHGDIA), RAC1, BRAF, KRAS, GSK3β (GSK3B), STAT3, and E-cadherin (CDH1). Different colored lines displays predicted functional links. Interactions experimentally determined appear in pink, interactions curated from databases in blue, co-expression was represented in black and text data mining in green. The analysis showed: nine nodes with an average node degree of 3.56; number of edges: 16; expected number of edges: 4; average local clustering coefficient: 0.333; PPI enrichment p -value: 2.02e −05 . b Top 10 ranked enriched proteins KEGG pathway analysis. Bar graphs show the –log of FDR (False discovery rate). High values correlated to higher probabilities. c Cellular component Gene ontology (GO) analysis

Article Snippet: Active KRas was pulled down with GST-RBD beads from lysates of serum-starved cells treated with EGF 25 ng/mL for 10 min. Aliquots of the lysates were blotted for total KRas and GAPDH as loading control. b RNAseq analysis of P4HB (PDIA1) gene in Caco2, HCT116, and HCT15 cells, showing mean expression in FPKM (Fragments Per Kilobase Million), P4HB fold-change expression in HCT15 vs. Caco2 (q < 0.000001) and HCT116 vs. Caco2 (q < 0.000001). c PDIA1 basal protein expression by western analysis, normalized for GAPDH ( n = 3) and quantified using Odyssey software. d Intracellular PDIA1 titration, using Human P4HB ELISA Pair Set (SinoBiological).

Techniques: Functional Assay, Expressing

Journal: Cell Death & Disease

Article Title: Subverted regulation of Nox1 NADPH oxidase-dependent oxidant generation by protein disulfide isomerase A1 in colon carcinoma cells with overactivated KRas

doi: 10.1038/s41419-019-1402-y

Figure Lengend Snippet: Model of PDIA1-associated regulation of Nox1 in cells with normal or overactivated KRas

Article Snippet: Active KRas was pulled down with GST-RBD beads from lysates of serum-starved cells treated with EGF 25 ng/mL for 10 min. Aliquots of the lysates were blotted for total KRas and GAPDH as loading control. b RNAseq analysis of P4HB (PDIA1) gene in Caco2, HCT116, and HCT15 cells, showing mean expression in FPKM (Fragments Per Kilobase Million), P4HB fold-change expression in HCT15 vs. Caco2 (q < 0.000001) and HCT116 vs. Caco2 (q < 0.000001). c PDIA1 basal protein expression by western analysis, normalized for GAPDH ( n = 3) and quantified using Odyssey software. d Intracellular PDIA1 titration, using Human P4HB ELISA Pair Set (SinoBiological).

Techniques:

Journal: PLOS Neglected Tropical Diseases

Article Title: Antibodies against medically relevant arthropod-borne viruses in the ubiquitous African rodent Mastomys natalensis

doi: 10.1371/journal.pntd.0012233

Figure Lengend Snippet: Recombinant arbovirus proteins used for the bead coupling and the inoculation of captive Mastomys natalensis .

Article Snippet: Flaviviridae , West Nile virus (WNV) , Nonstructural protein 1 (40346-V07H) , Sinobiological (Eschborn, Germany).

Techniques: Recombinant, Virus

Journal: PLOS Neglected Tropical Diseases

Article Title: Antibodies against medically relevant arthropod-borne viruses in the ubiquitous African rodent Mastomys natalensis

doi: 10.1371/journal.pntd.0012233

Figure Lengend Snippet: Total seroprevalence of each arbovirus and virus family in the wild-caught M . natalensis sample set.

Article Snippet: Flaviviridae , West Nile virus (WNV) , Nonstructural protein 1 (40346-V07H) , Sinobiological (Eschborn, Germany).

Techniques: Virus