Journal: International Journal of Molecular Sciences

Article Title: High-Density Real-Time PCR-Based in Vivo Toxicogenomic Screen to Predict Organ-Specific Toxicity

doi: 10.3390/ijms12096116

Figure Lengend Snippet: List of marker genes, their references and Taqman probes used in this study.

Article Snippet: 28 , Reg3a , regenerating islet-derived 3a , NM_011259.1 , Mm00441121_m1 , heart, liver , [ ] .

Techniques: Marker, Binding Assay, Ubiquitin Proteomics, Inhibition, Control

Journal: Communications Biology

Article Title: REG3A/REG3B promotes acinar to ductal metaplasia through binding to EXTL3 and activating the RAS-RAF-MEK-ERK signaling pathway

doi: 10.1038/s42003-021-02193-z

Figure Lengend Snippet: a H&E staining shows histological evidence of transformation from normal acini to ADM and to PDAC. Magnification, ×10; Scale bar: 2 mm. b Enlarged view of ADM area in a . Magnification, ×100; Scale bar: 200 μm. Black arrows indicate typical ADM circular structures. c Single antibody immunohistochemical staining shows intense REG3A expression the ADM zone. Magnification, ×10; Scale bar: 2 mm. d Enlarged view of ADM area in c . Black arrows indicate typical ADM stained strongly with REG3A. Magnification, ×100; Scale bar: 200 μm.

Article Snippet: Recombinant human REG3A and mouse REG3B protein were purchased from R&D Systems (5940-RG and 5110-RG).

Techniques: Staining, Transformation Assay, Immunohistochemical staining, Expressing

Journal: Communications Biology

Article Title: REG3A/REG3B promotes acinar to ductal metaplasia through binding to EXTL3 and activating the RAS-RAF-MEK-ERK signaling pathway

doi: 10.1038/s42003-021-02193-z

Figure Lengend Snippet: a Bright field images showing an increase in ADM events (depicted by black arrows) in cultured mouse primary acinar cells after 5 days of REG3B treatment and in cultured human primary acinar cells after 5 days of REG3A treatment (Magnification, ×200). TGFα-treated mouse primary acinar cells served as a positive control. b Bar graph showing increase in ADM quantity in 3D culture of mouse and human primary acinar cells during the 5-day REG3B or REG3A or TGFα treatment ( n = 3, 15 fields each group, one-way ANOVA and student’s t -test). c Bright field images in the upper row showing ADM events in cultured mouse primary acinar cells after 5 days of REG3B treatment and in cultured human primary acinar cells after 5 days of REG3A treatment (magnification, ×630). Lower four rows are corresponding confocal immunofluorescence images showing a decrease in AMYLASE protein expression (green) and an increase in CK19 (red) protein in REG3B-induced, REG3A-induced, or TGFα-induced ADM (magnification, ×630. Scale bars: 20 μm). d RT-qPCR analysis showed a decrease in acinar-specific mRNA (Ptf1a, Cpa, and Mist1) and an increase in duct-specific mRNA (Ck19 and Nestin) in mouse and human primary acinar cells after 48 h of REG3B or REG3A treatment. ( n = 3 per group, student’s t -test). Values are represented as mean ± standard deviation. * P < 0.05, ** P < 0.01, *** P < 0.001. Non-significant (n.s.) if P > 0.05.

Article Snippet: Recombinant human REG3A and mouse REG3B protein were purchased from R&D Systems (5940-RG and 5110-RG).

Techniques: Cell Culture, Positive Control, Immunofluorescence, Expressing, Quantitative RT-PCR, Standard Deviation, IF-P

Journal: Communications Biology

Article Title: REG3A/REG3B promotes acinar to ductal metaplasia through binding to EXTL3 and activating the RAS-RAF-MEK-ERK signaling pathway

doi: 10.1038/s42003-021-02193-z

Figure Lengend Snippet: a Western blot showing increased expression of p-ERK, p-MEK, p-BRAF, KRAS, and active RAS in the pancreatic tissue of REG3B-treated WT mice with caerulein-induced pancreatitis (WT cae+REG3B) and a moderate increase in TG mice with caerulein-induced pancreatitis (TG cae) ( n = 3, one-way ANOVA). b Western blots demonstrating increased expression of p-ERK, p-MEK, p-BRAF, KRAS, and active RAS in cultured human primary acinar cells and mouse acinar cell line 266-6 after REG3A or REG3B treatment respectively, for 48 h ( n = 3, student’s t -test). c Western blot showing the reduced expression of phosphorylated ERK, MEK, BRAF, and total KRAS in the 266-6 cell line after Reg3b gene knockdown by siRNA ( n = 3, student t -test). d In the upper panel, LY3009120 inhibited ERK, MEK, BRAF phosphorylation in a dose-dependent manner in 266-6 cell line. In the lower panel, LY3009120 (5 μM) blocked REG3B-induced MEK and ERK phosphorylation ( n = 3, one-way ANOVA). e Upper panel, Trametinib efficiently attenuated ERK phosphorylation in a dose-dependent manner in the AR42J cell line ( n = 3, one-way ANOVA). Lower panel, Trametinib (100 nM) blocked REG3B-induced ERK phosphorylation. f , g Bright field images ( g ) with quantification ( f ) show that Trametinib (100 nM) and LY3009120 (5 μM) treatment hindered REG3B-induced ADM in 3D cultures of mouse primary acinar cells (200×, n = 3, one-way ANOVA). h Confocal microscopy shows that Trametinib (100 nM) and LY3009120 (5 μM) treatment reduced CK19 protein expression (red) and increased AMYLASE protein expression (green) in 3D culture of mouse primary acinar cells (Scale bar: 20 μm). Data are represented as means ± SD, n = 3. * P < 0.05, ** P < 0.01, *** P < 0.001. Non-significant (n.s.) if P > 0.05.

Article Snippet: Recombinant human REG3A and mouse REG3B protein were purchased from R&D Systems (5940-RG and 5110-RG).

Techniques: Western Blot, Expressing, Cell Culture, Knockdown, Phospho-proteomics, Confocal Microscopy, IF-P

Journal: Communications Biology

Article Title: REG3A/REG3B promotes acinar to ductal metaplasia through binding to EXTL3 and activating the RAS-RAF-MEK-ERK signaling pathway

doi: 10.1038/s42003-021-02193-z

Figure Lengend Snippet: a Co-localization of EXTL3 (green) with human REG3A (red) or rodent REG3B (red) in ADM zones derived from human primary acinar cells in 3D culture, and AR42J and 266-6 cell lines in 2D culture by immunofluorescence microscopy. (magnification: ×630, Scale bars: 10 μm). b Co-immunoprecipitation of REG3B and EXTL3 in mouse primary acinar cells, rat AR42J and mouse 266-6 acinar cell lines. Lysate-bead/antibody conjugate mixture was eluted with sample buffer without DTT for 10 min at 50 °C (elution 1). Sample buffer with DTT (100 mM) was added to the pelleted beads from elution 1 and boiled for 5 min (elution 2). c Confocal immunofluorescence microscopy showed that EXTL3 monoclonal antibody treatment effectively blocks REG3B-induced ADM as indicated by increased expression of the acinar marker AMYLASE and decreased expression of the ductal marker CK19. (magnification: ×630, Scale bar: 20 μm). d – f Extl3 siRNA or neutralizing antibody treatment reduced the protein expression of KRAS and phosphorylated ERK, MEK, and BRAF in the presence or absence of REG3B for 48 h. d Western blotting analysis of the knockdown of Extl3 by siRNA (20 nM) in 266-6 cell line, e knockdown of Extl3 by siRNA (20 nM) in the context of the 266-6 cell line treated with REG3B for 48 h, f Western blotting analysis of EXTL3 neutralizing antibody treatment (2 μg/ml) in 266-6 and AR42J cell lines and mouse primary acinar cells treated with or without REG3B for 48 h. Human primary acinar cells were treated for 30 min. TGFα treatment serves as a positive control for ADM induction.

Article Snippet: Recombinant human REG3A and mouse REG3B protein were purchased from R&D Systems (5940-RG and 5110-RG).

Techniques: Derivative Assay, Immunofluorescence, Microscopy, Immunoprecipitation, Expressing, Marker, Western Blot, Knockdown, Positive Control

Journal: Communications Biology

Article Title: REG3A/REG3B promotes acinar to ductal metaplasia through binding to EXTL3 and activating the RAS-RAF-MEK-ERK signaling pathway

doi: 10.1038/s42003-021-02193-z

Figure Lengend Snippet: REG3B/REG3A binds to its receptor, EXTL3 receptor on the acinar cell membrane, and promotes ADM by activating the downstream RAS-RAF-MEK-ERK signaling pathway, in the absence of oncogenic Kras mutation. Targeting REG3B/REG3A, neutralizing its receptor EXTL3, or inhibiting downstream signaling molecules, such as B-RAF (LY3009120) or MEK1/2 (Trametinib), could interrupt the ADM process and potentially prevent early PDAC carcinogenesis.

Article Snippet: Recombinant human REG3A and mouse REG3B protein were purchased from R&D Systems (5940-RG and 5110-RG).

Techniques: Membrane, Mutagenesis

Journal: Medical Microbiology and Immunology

Article Title: Inflammation, the kynurenines, and mucosal injury during human experimental enterotoxigenic Escherichia coli infection

doi: 10.1007/s00430-024-00786-z

Figure Lengend Snippet: Overview plasma markers analyzed in the present study

Article Snippet: Plasma concentrations of regenerating islet-derived protein 3 alpha (Reg3A) were estimated using the Human Reg3A DuoSet ELISA kit (Catalog # DY5940-05; R&D Systems, Minneapolis, MN, USA) (Table ).

Techniques: Concentration Assay, Activation Assay

Journal: Scientific Reports

Article Title: Combined use of GM2AP and TCP1- eta urinary levels predicts recovery from intrinsic acute kidney injury

doi: 10.1038/s41598-020-68398-0

Figure Lengend Snippet: Biomarker levels in Control (n = 18) and AKI (n = 85) patients. * p < 0.05; ** p < 0.01; *** p < 0.001 versus “Control” group. AU, arbitrary units; Cr u , urinary creatinine; FABP1: fatty acid binding protein 1; GM2AP, GM2-activator protein; KIM-1, kidney injury molecule 1; NAG, N-acetyl-β- d -glucosaminidase; NGAL, neutrophil gelatinase-associated lipocalin; REG3A, regenerating islet-derived 3 alpha; SD, Standard deviation; TCP1- eta , T-complex protein 1 eta .

Article Snippet: Proteins were transferred to an Immobilon-P Transfer Membrane (Millipore, Madrid, Spain) and incubated with the following primary antibodies: (1) Anti KIM-1 (R&D Systems, Minneapolis, MN, USA); (2) TCP1-eta antibody (Novus Biologicals, Littleton, CO, USA); (3) Reg3A (R&D Systems, Minneapolis, MN, USA); (4) GM2AP [our polyclonal antibody, described in ; (5) gelsolin (Santa Cruz Biotechnology, Dallas, TX, USA); and (6) FABP1 (SAB Signalway Antibody, College Park, MD, USA).

Techniques: Biomarker Discovery, Control, Binding Assay, Derivative Assay, Standard Deviation

Journal: Scientific Reports

Article Title: Combined use of GM2AP and TCP1- eta urinary levels predicts recovery from intrinsic acute kidney injury

doi: 10.1038/s41598-020-68398-0

Figure Lengend Snippet: Biomarker levels in AKI patients

Article Snippet: Proteins were transferred to an Immobilon-P Transfer Membrane (Millipore, Madrid, Spain) and incubated with the following primary antibodies: (1) Anti KIM-1 (R&D Systems, Minneapolis, MN, USA); (2) TCP1-eta antibody (Novus Biologicals, Littleton, CO, USA); (3) Reg3A (R&D Systems, Minneapolis, MN, USA); (4) GM2AP [our polyclonal antibody, described in ; (5) gelsolin (Santa Cruz Biotechnology, Dallas, TX, USA); and (6) FABP1 (SAB Signalway Antibody, College Park, MD, USA).

Techniques: Biomarker Discovery, Clinical Proteomics, Comparison

Journal: Scientific Reports

Article Title: Combined use of GM2AP and TCP1- eta urinary levels predicts recovery from intrinsic acute kidney injury

doi: 10.1038/s41598-020-68398-0

Figure Lengend Snippet: ROC curves of urinary biomarkers for intrinsic AKI patients sub stratified according to the double Cr u /Cr p and FENa criteria (i.e. patients catalogued positive for intrinsic AKI complied with both criteria, n = 55). AUC: area under the curve; CI: confidence interval; Cr p : plasma creatinine; Cr u : urinary creatinine; FABP1: fatty acid binding protein 1; FENa, fractional excretion of sodium; GM2AP, GM2-activator protein; KIM-1, kidney injury molecule 1; NAG, N-acetyl-β- d -glucosaminidase; NGAL, neutrophil gelatinase-associated lipocalin; REG3A, regenerating islet-derived 3 alpha; RFI, renal failure index; SD, Standard deviation; TCP1- eta , T-complex protein 1 eta .

Article Snippet: Proteins were transferred to an Immobilon-P Transfer Membrane (Millipore, Madrid, Spain) and incubated with the following primary antibodies: (1) Anti KIM-1 (R&D Systems, Minneapolis, MN, USA); (2) TCP1-eta antibody (Novus Biologicals, Littleton, CO, USA); (3) Reg3A (R&D Systems, Minneapolis, MN, USA); (4) GM2AP [our polyclonal antibody, described in ; (5) gelsolin (Santa Cruz Biotechnology, Dallas, TX, USA); and (6) FABP1 (SAB Signalway Antibody, College Park, MD, USA).

Techniques: Clinical Proteomics, Binding Assay, Derivative Assay, Standard Deviation

Journal: Scientific Reports

Article Title: Combined use of GM2AP and TCP1- eta urinary levels predicts recovery from intrinsic acute kidney injury

doi: 10.1038/s41598-020-68398-0

Figure Lengend Snippet: ROC curves of urinary biomarkers for intrinsic AKI patients sub stratified according to the double Cr u /Cr p and RFI criteria (i.e. patients catalogued positive for intrinsic AKI complied with both criteria, n = 55). AUC: area under the curve; CI: confidence interval; Cr p : plasma creatinine; Cr u : urinary creatinine; FABP1: fatty acid binding protein 1; GM2AP, GM2-activator protein; KIM-1, kidney injury molecule 1; NAG, N-acetyl-β- d -glucosaminidase; NGAL, neutrophil gelatinase-associated lipocalin; REG3A, regenerating islet-derived 3 alpha; RFI, renal failure index; SD, Standard deviation; TCP1- eta , T-complex protein 1 eta .

Article Snippet: Proteins were transferred to an Immobilon-P Transfer Membrane (Millipore, Madrid, Spain) and incubated with the following primary antibodies: (1) Anti KIM-1 (R&D Systems, Minneapolis, MN, USA); (2) TCP1-eta antibody (Novus Biologicals, Littleton, CO, USA); (3) Reg3A (R&D Systems, Minneapolis, MN, USA); (4) GM2AP [our polyclonal antibody, described in ; (5) gelsolin (Santa Cruz Biotechnology, Dallas, TX, USA); and (6) FABP1 (SAB Signalway Antibody, College Park, MD, USA).

Techniques: Clinical Proteomics, Binding Assay, Derivative Assay, Standard Deviation

Journal: Scientific Reports

Article Title: Combined use of GM2AP and TCP1- eta urinary levels predicts recovery from intrinsic acute kidney injury

doi: 10.1038/s41598-020-68398-0

Figure Lengend Snippet: ROC curves of urinary biomarkers for intrinsic AKI patients sub stratified according to the double FENa and RFI criteria (i.e. patients catalogued positive for intrinsic AKI complied with both criteria, n = 65). AUC: area under the curve; CI: confidence interval; FABP1: fatty acid binding protein 1; FENa, fractional excretion of sodium; GM2AP, GM2-activator protein; KIM-1, kidney injury molecule 1; NAG, N-acetyl-β- d -glucosaminidase; NGAL, neutrophil gelatinase-associated lipocalin; REG3A, regenerating islet-derived 3 alpha; RFI, renal failure index; SD, Standard deviation; TCP1- eta , T-complex protein 1 eta .

Article Snippet: Proteins were transferred to an Immobilon-P Transfer Membrane (Millipore, Madrid, Spain) and incubated with the following primary antibodies: (1) Anti KIM-1 (R&D Systems, Minneapolis, MN, USA); (2) TCP1-eta antibody (Novus Biologicals, Littleton, CO, USA); (3) Reg3A (R&D Systems, Minneapolis, MN, USA); (4) GM2AP [our polyclonal antibody, described in ; (5) gelsolin (Santa Cruz Biotechnology, Dallas, TX, USA); and (6) FABP1 (SAB Signalway Antibody, College Park, MD, USA).

Techniques: Binding Assay, Derivative Assay, Standard Deviation

Journal: Scientific Reports

Article Title: Combined use of GM2AP and TCP1- eta urinary levels predicts recovery from intrinsic acute kidney injury

doi: 10.1038/s41598-020-68398-0

Figure Lengend Snippet: Urinary biomarker levels in pre-renal and renal-type AKI patients based on the Cr u /Cr p criterion.

Article Snippet: Proteins were transferred to an Immobilon-P Transfer Membrane (Millipore, Madrid, Spain) and incubated with the following primary antibodies: (1) Anti KIM-1 (R&D Systems, Minneapolis, MN, USA); (2) TCP1-eta antibody (Novus Biologicals, Littleton, CO, USA); (3) Reg3A (R&D Systems, Minneapolis, MN, USA); (4) GM2AP [our polyclonal antibody, described in ; (5) gelsolin (Santa Cruz Biotechnology, Dallas, TX, USA); and (6) FABP1 (SAB Signalway Antibody, College Park, MD, USA).

Techniques: Biomarker Discovery

Journal: Scientific Reports

Article Title: Combined use of GM2AP and TCP1- eta urinary levels predicts recovery from intrinsic acute kidney injury

doi: 10.1038/s41598-020-68398-0

Figure Lengend Snippet: Urinary biomarker levels in pre-renal and renal-type AKI patients based on the FENa criterion.

Article Snippet: Proteins were transferred to an Immobilon-P Transfer Membrane (Millipore, Madrid, Spain) and incubated with the following primary antibodies: (1) Anti KIM-1 (R&D Systems, Minneapolis, MN, USA); (2) TCP1-eta antibody (Novus Biologicals, Littleton, CO, USA); (3) Reg3A (R&D Systems, Minneapolis, MN, USA); (4) GM2AP [our polyclonal antibody, described in ; (5) gelsolin (Santa Cruz Biotechnology, Dallas, TX, USA); and (6) FABP1 (SAB Signalway Antibody, College Park, MD, USA).

Techniques: Biomarker Discovery

Journal: Scientific Reports

Article Title: Combined use of GM2AP and TCP1- eta urinary levels predicts recovery from intrinsic acute kidney injury

doi: 10.1038/s41598-020-68398-0

Figure Lengend Snippet: Urinary biomarker levels in pre-renal and renal-type AKI patients based on the RFI criterion.

Article Snippet: Proteins were transferred to an Immobilon-P Transfer Membrane (Millipore, Madrid, Spain) and incubated with the following primary antibodies: (1) Anti KIM-1 (R&D Systems, Minneapolis, MN, USA); (2) TCP1-eta antibody (Novus Biologicals, Littleton, CO, USA); (3) Reg3A (R&D Systems, Minneapolis, MN, USA); (4) GM2AP [our polyclonal antibody, described in ; (5) gelsolin (Santa Cruz Biotechnology, Dallas, TX, USA); and (6) FABP1 (SAB Signalway Antibody, College Park, MD, USA).

Techniques: Biomarker Discovery

Journal: Journal of Extracellular Vesicles

Article Title: WJMSC‐derived small extracellular vesicle enhance T cell suppression through PD‐L1

doi: 10.1002/jev2.12067

Figure Lengend Snippet: Decreased plasma ST2 and REG3A levels in aGvHD patients with WJMSC infusion. (a,b) ELISA of ST2 (a) and REG3A (b) in the plasma samples from aGvHD patients with the WJMSC infusion. Left, combined analysis; Middle, low‐dose (1.2 × 10 6 /kg WJMSCs); Right, high‐dose (10 × 10 6 /kg WJMSCs). Two cell infusion time points were indicated by red arrows (a,b). Data are mean ± s.e.m. and analysed by unpaired one‐tailed Student's t‐test (a,b). * P < 0.05, ** P < 0.01, *** P < 0.005

Article Snippet: For detection of IFN‐γ, ST2 and REG3 alpha (REG3A), plates were prepared using Duoset human IFN‐γ, ST2 and REG3A ELISA kit according to the manufacturers’ instructions (R&D systems, USA).

Techniques: Clinical Proteomics, Enzyme-linked Immunosorbent Assay, One-tailed Test