agr2 antibody  (Santa Cruz Biotechnology)

Journal: Scientific reports

Article Title: Bispecific antibody simultaneously targeting AGR2 and PD1 mediates cytotoxic T-cell-induced antitumor response in AGR2-dependent manner and inhibits AGR2-induced PDL1 upregulation.

doi: 10.1038/s41598-025-88331-7

Figure Lengend Snippet: Fig. 1. AGR2xPD1 BsAb-mediated T-cell-induced impaired membrane integrity in the AGR2-overexpressing tumor cells. Schematic diagram of AGR2xPD1 BsAb (A), H460 (B), MCF7, MCF7/shAGR2 (C), cells were co-cultured with activated TALL-104 cells and treated with PBS, 18A4HU anti-AGR2 mAb, the combination of 18A4HU and anti-PD1 mAbs, anti-PD1 mAb, and AGR2xPD1 BsAb in respective wells, and incubated for 48 h. Next, TALL-104 cells were washed off with PBS with gentle shaking, and calcein-PI staining solution was added to each well and incubated for 30 min at 37 °C. Then, the staining solution was aspirated, and PBS was added to each well. Pictures were taken under a fluorescence microscope. Scale bar 100 μm. (D), target cell- dependent T-cell mediated cytotoxicity of BsAb (AGR2xPD1) was detected using LDH release assay. H460 cells and PBMCs were used as target cells and effector cells (PBMC: H460, 3:1).

Article Snippet: AGR2 (Santa Cruz), PDL1 (Proteintech), EGFR (ABclonal), p-EGFR (ABclonal), ERK1/2 (CST), pERK (CST), MEK (Proteintech), pMEK (CST), and GAPDH (Proteintech) were the primary antibodies used.

Techniques: Membrane, Cell Culture, Incubation, Gentle, Staining, Fluorescence, Microscopy, Lactate Dehydrogenase Assay

Journal: Scientific reports

Article Title: Bispecific antibody simultaneously targeting AGR2 and PD1 mediates cytotoxic T-cell-induced antitumor response in AGR2-dependent manner and inhibits AGR2-induced PDL1 upregulation.

doi: 10.1038/s41598-025-88331-7

Figure Lengend Snippet: Fig. 2. AGR2-dependent effect of AGR2xPD1 BsAb mediated higher expression of cytolytic proteins in cytotoxic T-cells when co-cultured with AGR2-overexpressing tumor cells, but lower expression of cytolytic proteins when co-culture with AGR2 knockdown cells. Representative images of co-culture of H460 (A-C), MCF7 (D-F), and MCF7/shAGR2 (D-F, last columns) with activated TALL cells, with the expression of GRZB (A, D), perforin (B, E), caspase 3 (C, F). To evaluate the AGR2-dependent effect of AGR2xPD1 BsAb, the co-culture system was made by using the tumor cells and activated (using PMA and ionomycin, incubated for 2 h in 5% CO2 and air at 37 °C) TALL-104 cells. The evaluations were done using fluorescence microscopy (Original magnification 40x). The values are the mean ± SD of the mean fluorescence intensity of 3 randomly selected images. GRZB and perforin expressions in T-cells with H460 (G) and MCF7 (H) were detected by FACS analysis.

Article Snippet: AGR2 (Santa Cruz), PDL1 (Proteintech), EGFR (ABclonal), p-EGFR (ABclonal), ERK1/2 (CST), pERK (CST), MEK (Proteintech), pMEK (CST), and GAPDH (Proteintech) were the primary antibodies used.

Techniques: Expressing, Cell Culture, Co-Culture Assay, Knockdown, Incubation, Fluorescence, Microscopy

Journal: Scientific reports

Article Title: Bispecific antibody simultaneously targeting AGR2 and PD1 mediates cytotoxic T-cell-induced antitumor response in AGR2-dependent manner and inhibits AGR2-induced PDL1 upregulation.

doi: 10.1038/s41598-025-88331-7

Figure Lengend Snippet: Fig. 3. AGR2xPD1 BsAb mediated co-localization of AGR2 and PD1 in the AGR2-rich tumor site. To evaluate the AGR2xPD1 BsAb-mediated co-localization of AGR2 and PD1, AGR2-negative and AGR2-positive tumor mice models were made. One group of mice was injected with AGR2-negative cells B16F10, and the rest of the four groups were injected with AGR2-overexpressing H460 cells to induce tumors. Tumors were treated with the following antibodies twice a week: humanized AGR2xPD1 BsAb (10 mg/kg) to the AGR2-positive and AGR2-negative group of mice, respectively, humanized 18A4HU anti-AGR2 mAb (10 mg/kg), human anti-PD1 mAb (10 mg/kg), human IgG (10 mg/kg) to the AGR2-rich group of mice. After harvesting the tumor, frozen tumors were embedded in OCT and sectioned at 8 μm thickness. (A) Immunofluorescence staining was performed with antibodies against AGR2 (Rabbit) and PD1 (Human). Yellow arrow indicating the co-localizations. (B) The quantification of AGR2 and PD1 was done in IF images. These evaluations were done using fluorescence microscopy (Original magnification 40x). (C) Immunofluorescence staining was performed using anti-CD68 (Rabbit) and anti-AGR2 (Humanized 18A4HU) antibodies. Immunofluorescence analyses showed tumor-infiltrating CD68 + macrophages in the AGR2xPD1 BsAb-treated and IgG-treated tumor sections, scale bar 75 μm. H&E-stained sections showed tumor-infiltrating immune cells represented by the red arrow in the AGR2xPD1 BsAb-treated tumor sections, scale bar 100 μm.

Article Snippet: AGR2 (Santa Cruz), PDL1 (Proteintech), EGFR (ABclonal), p-EGFR (ABclonal), ERK1/2 (CST), pERK (CST), MEK (Proteintech), pMEK (CST), and GAPDH (Proteintech) were the primary antibodies used.

Techniques: Injection, Immunofluorescence, Staining, Fluorescence, Microscopy

Journal: Scientific reports

Article Title: Bispecific antibody simultaneously targeting AGR2 and PD1 mediates cytotoxic T-cell-induced antitumor response in AGR2-dependent manner and inhibits AGR2-induced PDL1 upregulation.

doi: 10.1038/s41598-025-88331-7

Figure Lengend Snippet: Fig. 5. AGR2xPD1 BsAb inhibited AGR2-induced angiogenesis. (A) Representative images of huPBMCs/ H460 cells co-grafting model showing blood vessel density. The number of blood vessels was significantly reduced in the AGR2xPD1 BsAb-treated group compared to the control IgG group. (B) Immunofluorescence staining showed the expression of VEGF in tumor tissues of AGR2xPD1 BsAb and the control IgG-treated group. Significantly lower expression of VEGF was exhibited in the AGR2xPD1 BsAb-treated group.

Article Snippet: AGR2 (Santa Cruz), PDL1 (Proteintech), EGFR (ABclonal), p-EGFR (ABclonal), ERK1/2 (CST), pERK (CST), MEK (Proteintech), pMEK (CST), and GAPDH (Proteintech) were the primary antibodies used.

Techniques: Control, Immunofluorescence, Staining, Expressing

Journal: Scientific reports

Article Title: Bispecific antibody simultaneously targeting AGR2 and PD1 mediates cytotoxic T-cell-induced antitumor response in AGR2-dependent manner and inhibits AGR2-induced PDL1 upregulation.

doi: 10.1038/s41598-025-88331-7

Figure Lengend Snippet: Fig. 8. AGR2xPD1 BsAb mediated T-cell-induced impaired membrane integrity and cytotoxicity. Proposed scheme for redirection of AGR2xPD1 BsAb-mediated cytotoxic T-cells to the AGR2-rich tumor cells and T-cell-induced damaged membrane integrity followed by apoptosis in tumor cells by elevated level of cytolytic proteins, and AGR2-induced PDL1 upregulation inhibited by AGR2xPD1 BsAb.

Article Snippet: AGR2 (Santa Cruz), PDL1 (Proteintech), EGFR (ABclonal), p-EGFR (ABclonal), ERK1/2 (CST), pERK (CST), MEK (Proteintech), pMEK (CST), and GAPDH (Proteintech) were the primary antibodies used.

Techniques: Membrane

Journal: Cell Reports Medicine

Article Title: Disrupting AGR2/IGF1 paracrine and reciprocal signaling for pancreatic cancer therapy

doi: 10.1016/j.xcrm.2024.101927

Figure Lengend Snippet: Secreted AGR2 promotes pancreatic carcinogenesis by activating CAFs (A) Representative immunohistochemical (IHC) images demonstrate AGR2-positive and -negative PDAC tumors (scale bars: 50 μm). (B) Survival curves of patients categorized by AGR2 expression in PDAC tumor samples via IHC ( n = 99). (C) Survival curves of patients stratified according to median AGR2 levels, measured by ELISA, in serum from individuals with PDAC ( n = 172). (D) Western blot analyses of AGR2 expression in human PDAC cell lines (Capan2 and Panc1) following CRISPR-Cas9-mediated AGR2 knockout (performed in triplicate). (E) Images and volumes of control versus AGR2-knockout subcutaneous xenografts derived from Capan2 and Panc1 cell lines in nude mice ( n = 5 per group). (F) Subcutaneous xenografts from AGR2-knockout Capan2 and Panc1 cells (AGR2 KO ), following re-expression of wild-type AGR2 (AGR2 WT ), AGR2 lacking a nuclear localization signal (AGR2 ΔNLS ), and AGR2 lacking a signal peptide (AGR2 ΔSP ) ( n = 4 per group). (G) ELISA analyses of the supernatant from AGR2-knockout Capan2 and Panc1 cells re-expressing AGR2 WT , AGR2 ΔNLS , and AGR2 ΔSP (performed in triplicate). (H) Representative IHC images and quantification of alpha-smooth muscle actin (α-SMA) staining score and collagen score within xenograft tumors across the four groups ( n = 4 per group, scale bars: 50 μm). Statistical significance was determined using a log rank test for (B) and (C) and a one-way ANOVA with multiple comparisons test for (E) through (H). Data are presented as mean ± standard deviation (SD). Significance levels are indicated as ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001.

Article Snippet: Mouse monoclonal anti-AGR2 , Santa Cruz Biotechnology , Cat# sc-101211; RRID: AB_2225121.

Techniques: Immunohistochemical staining, Expressing, Enzyme-linked Immunosorbent Assay, Western Blot, CRISPR, Knock-Out, Control, Derivative Assay, Staining, Standard Deviation

Journal: Cell Reports Medicine

Article Title: Disrupting AGR2/IGF1 paracrine and reciprocal signaling for pancreatic cancer therapy

doi: 10.1016/j.xcrm.2024.101927

Figure Lengend Snippet: Agr2 secretion correlates with desmoplastic reaction in genetic mouse models of PDAC (A) Schematic illustration of the genotypes for KC mice, KC; Agr2 −/− mice, and KC; Agr2 OE mice. (B) Representative hematoxylin and eosin (H&E), Agr2, Krt19, and α-amylase-stained sections, along with α-SMA/BrdU-stained immunofluorescence images of pancreata from KC, KC; Agr2 −/− , and KC; Agr2 OE mice (scale bars: 50 μm; n = 12 mice per group). The images were scored and thereby quantified (right). (C) ELISA analysis of Agr2 levels in the serum of 8-week-old KC, KC; Agr2 −/− and KC; Agr2 OE mice ( n = 12 mice per group). (D) Western blot analysis of α-SMA, α-amylase, and Agr2 expression in the pancreata of KC, KC; Agr2 −/− , and KC; Agr2 OE mice ( n = 4 mice per group). (E) Schematic representation of KC mice injected with AAV-EGFP, AAV- Agr2 WT , and AAV- Agr2 ΔSP particles. (F) Representative images of H&E staining, EGFP immunofluorescence, and IHC for Krt19, α-amylase, and α-SMA in the pancreata of KC mice injected with AAV-EGFP, AAV- Agr2 WT , and AAV- Agr2 ΔSP particles (scale bars: 50 μm; n = 3 mice per group). The images were scored and thereby quantified (right). (G) ELISA analysis of Agr2 levels in the serum of KC mice injected with AAV-EGFP, AAV- Agr2 WT , and AAV- Agr2 ΔSP particles. (H) Western blot analysis of α-SMA, α-amylase, Agr2, and EGFP expression in the pancreata of KC mice injected with AAV-EGFP, AAV- Agr2 WT , and AAV- Agr2 ΔSP particles. (I and J) Schematic representation and survival curves for KC and KC; Agr2 −/− mice over a 1.5-year follow-up period. (K) PDAC incidence in KC (11/34, 32.4%) versus KC; Agr2 −/− mice (5/40, 12.5%). (L) ELISA analysis of Agr2 levels in the serum of KC and KC; Agr2 −/− mice with PDAC ( n = 5 mice per group). (M) Representative H&E-stained sections showing PDAC tumors in KC and KC; Agr2 −/− mice; Sirius red-stained sections showing collagen distribution in tumors; α-SMA/BrdU-stained immunofluorescence images depicting proliferative α-SMA-positive cells in tumors (scale bars: 50 μm; n = 5 mice per group). (N) Western blot analysis of α-SMA and Agr2 expression in tumors from KC mice and KC; Agr2 −/− mice ( n = 5 mice per group). Statistical significance for (B), (C), (G), and (F) was assessed using a one-way ANOVA with multiple comparisons test, (J) with a log rank test, (K) with a chi-squared test, and (L) and (M) with two-tailed, unpaired Student’s t tests. Data are presented as mean ± SD. Significance is denoted as ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001. “ns” indicates no significance.

Article Snippet: Mouse monoclonal anti-AGR2 , Santa Cruz Biotechnology , Cat# sc-101211; RRID: AB_2225121.

Techniques: Staining, Immunofluorescence, Enzyme-linked Immunosorbent Assay, Western Blot, Expressing, Injection, Two Tailed Test

Journal: Cell Reports Medicine

Article Title: Disrupting AGR2/IGF1 paracrine and reciprocal signaling for pancreatic cancer therapy

doi: 10.1016/j.xcrm.2024.101927

Figure Lengend Snippet: IGF1 promotes the secretion of AGR2, which in turn enhances the presentation of the IGF1R on the cell surface (A) The Venn diagram of the upper panel illustrates the count of genes down-regulated in Capan2 and Panc1 cells post AGR2 knockout. The Gene Ontology (GO) analysis of the lower panel identifies enriched biological processes, notably “regulation of IGF receptor signaling pathway” at transcriptional levels, after AGR2 knockout in these cell lines. (B) Western blot and quantitative reverse-transcription PCR (qRT-PCR) analyses assess IGF1R and AGR2 expression in Capan2 and Panc1 cells following AGR2 knockout via the CRISPR-Cas9 system (performed in triplicate). (C) Flow cytometry (FACS) quantifies cell membrane surface expression of IGF1R in Capan2 and Panc1 cells after AGR2 knockout (performed in triplicate). (D) Co-immunoprecipitation assays reveal AGR2’s interaction with pro-IGF1R in Capan2 and Panc1 cells (performed in triplicate). (E) Immunofluorescence imaging displays AGR2 and IGF1R distribution and ER labeling in Panc1 cells (scale bars: 50 μm). (F) Western blot analysis of IGF1R and AGR2 in Panc1 and Capan2 cells with controls (original cell lines), AGR2-knockout (AGR2 KO ) post-expression of AGR2 WT , AGR2 ΔNLS , AGR2 ΔSP , and AGR2 C81A mutation (performed in triplicate). (G) Western blot analysis of IGF1R expression in AGR2 in Panc1 and Capan2 cells with AGR2 knockout (KO) treated with 3-methyladenine (3-MA) (15 mM), bafilomycin A1 (30 nM), chloroquine (20 mM), MLN4929 (1 mM), or MG132 (5 mM) for 12 h (performed in triplicate). (H) Western blot analysis of IGF1R, phosphorylated IGF1R, c-JUN, phosphorylated c-JUN, and AGR2 following 12 h of serum starvation and subsequent IGF1 stimulation (50 ng/mL, performed in triplicate). (I) ELISA measures AGR2 secretion after serum starvation and treatment with PPP (1 μM) and IGF1 (50 ng/mL) over time (performed in triplicate). (J) Identification of potential c-JUN-binding sites within the AGR2 promoter region. (K) Western blot analysis of c-JUN, phosphorylated c-JUN, and AGR2 expression following c-JUN knockdown and IGF1 stimulation over time (performed in triplicate). (L) Western blot shows c-JUN, phosphorylated c-JUN, and AGR2 expression post anisomycin treatment over time (performed in triplicate). (M) Chromatin immunoprecipitation followed by quantitative PCR (ChIP-qPCR) demonstrates c-JUN enrichment at AGR2’s transcription start sites (TSSs) before and after IGF1 treatment (performed in triplicate). (N) Integrative Genomics Viewer (IGV) tracks display c-JUN peaks in AGR2’s promoter region post IGF1 treatment. (O) Dual-luciferase reporter assays in Capan2 and Panc1 cells evaluate AGR2 promoter activity under various lengths and site-specific mutations after IGF1 treatment (performed in triplicate). Statistical analyses: (B) and (C) used a one-way ANOVA with multiple comparisons. (I), (M), (N), and (O) were analyzed using two-tailed, unpaired Student’s t tests. Data are presented as mean ± SD, with significance marked as ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, and “ns” indicates no significance.

Article Snippet: Mouse monoclonal anti-AGR2 , Santa Cruz Biotechnology , Cat# sc-101211; RRID: AB_2225121.

Techniques: Knock-Out, Western Blot, Reverse Transcription, Quantitative RT-PCR, Expressing, CRISPR, Flow Cytometry, Membrane, Immunoprecipitation, Immunofluorescence, Imaging, Labeling, Mutagenesis, Enzyme-linked Immunosorbent Assay, Binding Assay, Knockdown, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Luciferase, Activity Assay, Two Tailed Test

Journal: Cell Reports Medicine

Article Title: Disrupting AGR2/IGF1 paracrine and reciprocal signaling for pancreatic cancer therapy

doi: 10.1016/j.xcrm.2024.101927

Figure Lengend Snippet: Secreted AGR2 promotes IGF1 production from CAFs via the Wnt/β-catenin pathway (A) Western blot analysis evaluates AGR2 and IGF1 levels in three human PDAC-derived CAFs and three PDAC cell lines (Capan2, HPAC, and Panc1) across three independent experiments. (B) qRT-PCR analysis of IGF1 expression and supernatant ELISA analyses of IGF1 secretion in human PDAC-derived CAFs co-cultured with two human PDAC organoids and with or without treatment with Agr2-neutralizing antibody (5 μg/mL) for 48 h ( n = 3 independent experiments). (C) qRT-PCR assesses IGF1 expression in PDAC-derived CAFs co-cultured with AGR2-knockout Capan2 and Panc1 cells, following re-expression of AGR2 WT , AGR2 ΔNLS , and AGR2 ΔSP for 48 h (upper); Western blot analysis investigates IGF1R, phosphorylated IGF1R, c-JUN, phosphorylated c-JUN, and AGR2 levels in AGR2-knockout Capan2 and Panc1 cells after co-culture with PDAC-derived CAFs (lower, n = 3 independent experiments). (D) qRT-PCR explores IGF1 expression in two PDAC-derived CAFs after treatment with rAGR2 (500 ng/mL), rTGF-β1 (4 μg/mL), and rIL-1α (200 ng/mL) for 24 h ( n = 3 independent experiments). (E) Supernatant analysis quantifies collagen levels in two PDAC-derived CAFs following rAGR2 treatment (500 ng/mL) for 24 h ( n = 3 independent experiments). (F) Transwell assays examine cell migration in two PDAC-derived CAFs following rAGR2 treatment (500 ng/mL) for 24 h ( n = 3 independent experiments). (G) Left: scRNA-seq identifies iCAFs and myCAFs within 16 PDAC tissues (GEO: GSE155698), showing iCAFs with elevated IGF1 expression (>mean value). Right: volcano plot displays genes differentially expressed between IGF1 high and IGF1 low CAFs (FDR < 0.01; log 2 FC > 0.5), accompanied by KEGG pathway analysis of the IGF1-CAF signature. (H) Principal component analysis (PCA) of transcriptomic data from CAFs treated with rAGR2, rTGF-β1, and rIL-1α ( n = 3 per group). (I) A heatmap shows genes significantly upregulated in CAFs after treatment with rAGR2, rTGF-β1, and rIL-1α (FDR < 0.01; log2FC > 0.5; left). Bioplant pathway analysis elucidates upregulated gene pathways post rAGR2 treatment in CAFs (right). (J) Identification of potential lymphoid enhancer binding factor 1 (LEF1)-binding sites within the IGF1 promoter region. (K) Western blot analysis shows β-catenin expression in both nuclear and cytoplasmic fractions of PDAC-derived CAFs after AGR2 stimulation (500 ng/mL) for 0.5, 1, 3, and 6 h ( n = 3 independent experiments). (L) Western blot and qRT-PCR analyses evaluate β-catenin and IGF1 levels in PDAC-derived CAFs post β-catenin knockdown or following treatment with ICG-001 (Wnt pathway inhibitor) and rAGR2 (500 ng/mL) for 24 h (M) Luciferase reporter assays in three PDAC-derived CAFs transfected with wild-type and site-specific mutagenized IGF1 promoter sequences based on (J) predictions, post rAGR2 treatment (500 ng/mL) for 24 h ( n = 3 independent experiments). Statistical analysis: one-way ANOVA with multiple comparisons test was used for (B), (C), (D), and (L); two-tailed, unpaired Student’s t tests were employed for (E), (F), and (M). Data are presented as mean ± SD, with ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001 indicating levels of statistical significance.

Article Snippet: Mouse monoclonal anti-AGR2 , Santa Cruz Biotechnology , Cat# sc-101211; RRID: AB_2225121.

Techniques: Western Blot, Derivative Assay, Quantitative RT-PCR, Expressing, Enzyme-linked Immunosorbent Assay, Cell Culture, Knock-Out, Co-Culture Assay, Migration, Binding Assay, Knockdown, Luciferase, Transfection, Two Tailed Test

Journal: Cell Reports Medicine

Article Title: Disrupting AGR2/IGF1 paracrine and reciprocal signaling for pancreatic cancer therapy

doi: 10.1016/j.xcrm.2024.101927

Figure Lengend Snippet: High serum levels of AGR2 and IGF1 are associated with enhanced desmoplastic reactions and immunosuppression in PDAC (A) ELISA analysis of Igf1 in serum from 8-week-old KC mice, KC; Agr2 −/− mice, and KC; Agr2 OE mice reveals a significant difference (left, n = 12 mice per group). Comparison between KC mice and KC; Agr2 −/− mice with PDAC also shows marked differences (right, n = 5 mice per group). (B) Serum levels of AGR2 and IGF1 exhibit a correlation in 145 human patients with PDAC, analyzed using Pearson’s correlation coefficient. (C) IHC images display α-SMA, podoplanin (PDPN), collagen, and IL-6 positivity in tumor areas, comparing AGR2 high ; IGF1 high samples with AGR2 low ; IGF1 low samples, demonstrating a difference in desmoplastic reaction. (D) IHC images illustrate the differential presence of CD3, CD8, CD4, FOXP3, CD68, CD206, and CD20-positive cells in tumors between AGR2 high ; IGF1 high samples and AGR2 low ; IGF1 low samples, indicating variations in immune cell infiltration (scale bars: 50 μm). (E) IHC imaging further reveals the distribution of Cd3, Cd8, Cd4, Foxp3, B220, F4/80, and Cd206-positive cells in tumors from KC mice versus KC; Agr2 −/− mice, emphasizing differences in immunological responses (scale bars: 50 μm). p values in left of (A) was calculated using a one-way ANOVA with a multiple comparisons test, p values in right of (A), (C), (D), and (E) were calculated using two-tailed, unpaired Student’s t tests, and correlation coefficient in (B) was calculated using Pearson’s correlation coefficient. Data are presented as mean ± SD. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001. ns, no significance.

Article Snippet: Mouse monoclonal anti-AGR2 , Santa Cruz Biotechnology , Cat# sc-101211; RRID: AB_2225121.

Techniques: Enzyme-linked Immunosorbent Assay, Comparison, Imaging, Two Tailed Test

Journal: Cell Reports Medicine

Article Title: Disrupting AGR2/IGF1 paracrine and reciprocal signaling for pancreatic cancer therapy

doi: 10.1016/j.xcrm.2024.101927

Figure Lengend Snippet: Combined targeting attenuates desmoplastic reaction and normalizes immunosuppressive microenvironment (A) Western blot analysis reveals Agr2 and Igf1 levels in PSCs isolated from wild-type mice and three mouse PDAC cell lines, highlighting the differential expression patterns. (B) Schematic diagram shows the therapeutic strategy of combining IGF1R inhibitor and AGR2-neutralizing antibody. (C) ELISA and qRT-PCR analyses demonstrate Igf1 levels in PSCs co-cultured with KPC PDAC-derived organoids. The impact of treatments with the IGF1R inhibitor (PPP; 1 μM), Agr2-neutralizing antibody (5 μg/mL) alone, or their combination for 48 h is shown ( n = 3 independent experiments). (D) Western blot results display the expression levels of p-Igf1r, Igf1r, c-Jun, p-c-Jun, Akt, p-Akt, Erk, p-Erk, and Agr2 in mouse PDAC-derived organoids after co-culture with PSC cells and subsequent treatments as mentioned in (C) ( n = 3 independent experiments). (E) Representative images and quantitative analyses show the growth dynamics of PDAC organoids co-cultured with PSC cells under various treatment conditions over 0, 24, 48, and 96 h (scale bars: 50 μm, n = 3 independent experiments). (F) Tumor volume comparisons in KPC mice post caerulein-induced acute pancreatitis and subsequent treatments with Agr2 antibody (4 mg/kg; intraperitoneally [i.p.], three times per week for 2 weeks), PPP (20 mg/kg; i.p., three times per week for 2 weeks), or their combination ( n = 5 for control group, n = 3 for single treatment groups, and n = 5 for combined treatment group). (G) ELISA quantification of Agr2, Igf1, Il-1α, Lif, GM-CSF, and Il-6 in serum samples from the four groups of KPC mice underscores the systemic effects of the treatment modalities on cytokine levels ( n = 5 for control group, n = 3 for single treatment groups, and n = 5 for combined treatment group). (H and I) Representative stained sections and quantitative statistics of H&E, Pdpn, α-SMA, collagen, Cd3, Cd4, Foxp3, B220, and Cd206-positive cells within PDAC tumors (scale bars: 50 μm, n = 5 mice per group). (J) Representative IHC highlights CD8-positive cells in lymph nodes adjacent to the tumors (scale bars: 50 μm). p values in (C), (F), and (G) were calculated using a one-way ANOVA with a multiple comparisons test, and p values in (H) and (I) were calculated using two-tailed, unpaired Student’s t tests. Data are presented as mean ± SD. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001.

Article Snippet: Mouse monoclonal anti-AGR2 , Santa Cruz Biotechnology , Cat# sc-101211; RRID: AB_2225121.

Techniques: Western Blot, Isolation, Expressing, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Cell Culture, Derivative Assay, Co-Culture Assay, Control, Staining, Two Tailed Test

Journal: Cell Reports Medicine

Article Title: Disrupting AGR2/IGF1 paracrine and reciprocal signaling for pancreatic cancer therapy

doi: 10.1016/j.xcrm.2024.101927

Figure Lengend Snippet:

Article Snippet: Mouse monoclonal anti-AGR2 , Santa Cruz Biotechnology , Cat# sc-101211; RRID: AB_2225121.

Techniques: Virus, Recombinant, Control, Enzyme-linked Immunosorbent Assay, Isolation, Membrane, Protein Extraction, Chromatin Immunoprecipitation, Bicinchoninic Acid Protein Assay, Sircol Collagen Assay, Luciferase, RNA Sequencing Assay, Sequencing, Expressing, Real-time Polymerase Chain Reaction, shRNA, Plasmid Preparation, Software, Flow Cytometry

Journal: The EMBO Journal

Article Title: Activation of goblet-cell stress sensor IRE1β is controlled by the mucin chaperone AGR2

doi: 10.1038/s44318-023-00015-y

Figure Lengend Snippet: ( A ) Verification of transgene expression and successful immunoprecipitation of FLAG-tagged IRE1 in the samples analyzed by MS in ( B , C ). Lysates were probed for IRE1-FLAG expression using anti-FLAG, and actin was used as a loading control. ( B ) Proteins with a log 2 FC enrichment of >2 and log 10 Adj P value of >2 for IRE1α-FLAG and IRE1β-FLAG immunoprecipitation (IP) compared to control cells. The Venn diagram shows the number of proteins that were detected uniquely associated with one of the two IRE1 paralogues or that were commonly identified with both IRE1 paralogues. ( C ) Volcano plot depicting the cutoff criteria and significantly enriched proteins in each IP. X axis shows the log 2 fold change of the measured peptide intensities of a given protein in the control condition over the IRE1 IP condition. Y axis shows the FDR corrected P value obtained by two-sample t test in Perseus. ( D ) Confirmation of specific interaction between AGR2 and IRE1β, but not IRE1α. IRE1 proteins were tagged with an Avi-tag that is specifically biotinylated upon BirA co-expression. The biotinylated Avi-tag was precipitated using streptavidin beads. For control conditions, BirA was omitted. Blots were probed for co-precipitation of AGR2 and streptavidin to detect Avi-tag-biotinylated IRE1. Tubulin was used as a loading control. Non-specific signal is indicated with an asterisk. Representative of two independent experiments. ( E ) Confirmation of the AGR2-IRE1β interaction in murine tissue. Colons were isolated and digested, and IP was performed using anti-AGR2. Agr2-deficient mice were used as a negative control to assess whether IRE1β binds specifically to the antibody/bead complex. IP samples were probed for IRE1β co-precipitation via immunoblot. Tubulin was used as a loading control. Representative of two independent experiments. .

Article Snippet: Anti-AGR2 antibody (clone 6C5, Santa Cruz Biotechnology) was first bound to Dynabeads Protein G (Invitrogen) according to the manufacturer’s instructions.

Techniques: Expressing, Immunoprecipitation, Control, Isolation, Negative Control, Western Blot

Journal: The EMBO Journal

Article Title: Activation of goblet-cell stress sensor IRE1β is controlled by the mucin chaperone AGR2

doi: 10.1038/s44318-023-00015-y

Figure Lengend Snippet: ( A ) AGR2 (purple) and ERN2 (blue insert, same data as shown in Fig. ) transcript expression in cell lines assayed by RT-qPCR. N = 3 culture dishes were sampled and AGR2 and ERN2 expression is shown relative to the expression detected in LS174T parental cells. Error bars show SEM. Bottom picture shows protein expression by western blot. Protein lysates were probed for AGR2 and tubulin was used as a loading control. ( B ) HSPA5 (purple) and ERN1 (blue insert, same data as shown in Fig. ) transcript expression in cell lines assayed by RT-qPCR. N = 3 culture dishes were sampled and HSPA5 and ERN2 expression is shown relative to the expression detected in LS174T parental cells. Error bars show SEM. Bottom picture shows protein expression by western blot. Protein lysates were probed for BiP and tubulin was used as a loading control. ( C ) IRE1β-FLAG transgene expression over time by western blot in cell lysates derived from Calu-1 ERN1-/-IRE1βFLAG-DOX co-expressing ER-targeted BirA as a control protein (left, orange), or AGR2 (right, purple). Cells received 1 μg/ml doxycycline to induce expression of IRE1β. Protein lysates were prepared at the indicated times and probed for IRE1β-FLAG expression, XBP1S and tubulin as a loading control. ( D ) Quantification of IRE1β-FLAG and XPP1S protein levels normalized to tubulin in three replicate experiments represented in ( C ). Error bars represent SEM. ( E ) Gating strategy to assess cell death. Doublets were gated out and dead cells were gated on via Annexin V and Live/Dead positive staining. All cells staining positive for a single, or both cell death markers were considered dead (red gate).

Article Snippet: Anti-AGR2 antibody (clone 6C5, Santa Cruz Biotechnology) was first bound to Dynabeads Protein G (Invitrogen) according to the manufacturer’s instructions.

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Control, Derivative Assay, Staining

Journal: The EMBO Journal

Article Title: Activation of goblet-cell stress sensor IRE1β is controlled by the mucin chaperone AGR2

doi: 10.1038/s44318-023-00015-y

Figure Lengend Snippet: Calu-1 ERN1 -/-IRE1βFLAG-DOX cells were transduced with a constitutive AGR2 transgene (“Calu-1 AGR2 “). Cells denoted as “Calu-1” are the original Calu-1 ERN1 -/-IRE1βFLAG-DOX cells. ( A ) RT-qPCR analysis of XBP1 S/T transcript levels after 24 h of transgene induction using 1 μg/ml doxycycline. Bottom picture shows XBP1 splicing in the same samples assayed by conventional PCR. Representative of four independent experiments with three replicate wells per condition. Error bars show SEM. ( B ) RT-qPCR analysis of BLOC1S1 transcript levels after 24 h of transgene induction using 1 μg/ml doxycycline. Representative of five independent experiments with three replicates per condition. Error bars show SEM. ( C ) Photographs showing the phenotype of cultures overexpressing IRE1β-FLAG with and without exogenous expression of AGR2. Left panels show untreated cultures, middle panels show cultures that received 1 μg/ml doxycycline for 72 h, and cultures in the right panels received 1 μg/ml doxycycline and 1 μM 4μ8C. Scale bar represents 100 µm. Representative of three independent experiments. ( D ) Quantification of cell death in cultures overexpressing IRE1β-FLAG with and without exogenously added AGR2 after 48 h. All cells in the culture dish were stained with Annexin V and Live/Dead stain and analyzed by flow cytometry. All single and double positive cells were considered as dead cells. Representative of two independent experiments with three replicate wells per condition. Error bars show SEM. ( E ) Analysis of IRE1β-FLAG and AGR2 expression in the cell lines used for ( C ). Only cells that remained attached in the dish were collected and lysates were probed for IRE1β expression using anti-FLAG and AGR2 expression using anti-AGR2. Tubulin was used as a loading control. ( F ) RT-qPCR validation of AGR2 knockdown efficiency in LS174T ERN1-/-IRE1βFLAG-DOX cells. NTC is a non-targeting control pool of siRNA’s, #1 and #3 are siRNA’s targeting AGR2. Representative of three independent experiments with n = 3 replicate wells per condition. Error bars show SEM. ( G ) Log 2 fold changes in XBP1 splicing after AGR2 partial knockdown and/or treatment with 4μ8C or DMSO (vehicle). Splicing is shown as a log 2 fold change of XBP1S mRNA over the NTC/vehicle-treated cells. Representative of three independent experiments with n = 3 replicate wells per condition. Error bars show SEM. ( H ) Western blot confirmation of ( F , G ). Proteins were extracted after 72 hours and probed for XBP1S, AGR2 and tubulin expression. Representative of two independent replicates. ( I ) Quantification of XPP1S protein levels normalized to tubulin in n = 2 experiments represented in ( H ). .

Article Snippet: Anti-AGR2 antibody (clone 6C5, Santa Cruz Biotechnology) was first bound to Dynabeads Protein G (Invitrogen) according to the manufacturer’s instructions.

Techniques: Transduction, Quantitative RT-PCR, Expressing, Staining, Flow Cytometry, Control, Biomarker Discovery, Knockdown, Western Blot

Journal: The EMBO Journal

Article Title: Activation of goblet-cell stress sensor IRE1β is controlled by the mucin chaperone AGR2

doi: 10.1038/s44318-023-00015-y

Figure Lengend Snippet: ( A ) Schematic overview of gel filtration experiments in ( B , C ). ( B ) msfGFP fluorescence measured during elution of HEK293T lysates overexpressing IRE1β in the absence of AGR2 (black dotted trace) or in the presence of AGR2 (orange and purple traces indicating different ratios of transfected AGR2:IRE1β plasmid). Top scale represents the approximate elution profile and expected MW of protein standards. Bottom drawings indicate expected oligomerization status based on protein standards and the previously obtained elution profile (Grey et al, ). Each trace shows a single chromatography run. ( C ) IRE1β-FLAG expression in fractions collected after gel filtration of protein lysates from Calu-1 ERN1-/-IRE1βFLAG-DOX cells, in the absence or presence of additional AGR2 expression. Line graph shows quantification of band intensities from the gel of three independent replicates. Error bar shows SEM. ( D ) Schematic representation of competition IP experiments in ( E , F ). IRE1β is expressed with an Avi-tag or FLAG-tag in equimolar amounts. After biotinylation of the Avi-tag by BirA, both the Avi-tag and FLAG-tag will be detected after streptavidin IP if dimers have been formed. If addition of another protein (e.g., AGR2) would block this process, a loss of signal is expected. ( E ) Competition IP showing loss of dimer formation upon co-expression of AGR2. Samples were immunoblotted with anti-AGR2, anti-FLAG and Streptavidin. Tubulin was used as a loading control in input samples. Representative of three independent experiments. Non-specific signal is indicated with an asterisk. ( F ) Competition IP demonstrating concentration-dependent loss of dimer formation upon increasing AGR2 co-expression. Samples were immunoblotted with anti-AGR2, anti-FLAG and Streptavidin. Tubulin was used as a loading control in input samples. Representative of two independent experiments. .

Article Snippet: Anti-AGR2 antibody (clone 6C5, Santa Cruz Biotechnology) was first bound to Dynabeads Protein G (Invitrogen) according to the manufacturer’s instructions.

Techniques: Filtration, Fluorescence, Transfection, Plasmid Preparation, Chromatography, Expressing, FLAG-tag, Blocking Assay, Control, Concentration Assay

Journal: The EMBO Journal

Article Title: Activation of goblet-cell stress sensor IRE1β is controlled by the mucin chaperone AGR2

doi: 10.1038/s44318-023-00015-y

Figure Lengend Snippet: ( A ) The structure of AGR2 (pdb: 2LNS) visualized in PyMol with the relevant mutations indicated. Purple and grey cartoons depict two AGR2 molecules and their dimer structure (Patel et al, ), specific residues are represented as ball-and-sticks. ( B ) Schematic overview of competition IP using AGR2 mutants. ( C ) Competition IP showing loss of dimer inhibition using C81S and H117Y AGR2 mutants. Samples were immunoblotted with anti-AGR2, anti-FLAG-IRE1β and Streptavidin. Tubulin was used as a loading control in input samples. Representative of three independent experiments. ( D ) Quantification of IP band intensities normalized over corresponding input sample on western blots from ( C ), with each data point representing one experiment. Error bars show SEM. ( E ) Photographs showing Calu-1 ERN1-/-IRE1βFLAG-DOX cultures, transduced with a constitutive AGR2 transgene (wild-type or the indicated mutants). IRE1β-FLAG overexpression was induced with 1 μg/ml doxycycline for 72 h. Scale bar represents 200 µm. Representative of two independent experiments. ( F ) Quantification of cell death in cell lines from ( E ) after 72 h of transgene expression. All cells in the culture dish were stained with Annexin V and Live/Dead stain and analyzed by flow cytometry. All single and double positive cells were considered as dead cells. Representative of two independent experiments with n = 2 culture dishes. ( G ) RT-qPCR analysis of XBP1 S/T transcript levels after 24 h of transgene induction using 1 μg/ml doxycycline. Representative of two independent experiments with three replicates per condition. Error bars show SEM. ( H ) RT-qPCR analysis of BLOC1S1 transcript levels after 24 h of transgene induction using 1 μg/ml doxycycline. Representative of two independent experiments with three replicates per condition. Error bars show SEM. .

Article Snippet: Anti-AGR2 antibody (clone 6C5, Santa Cruz Biotechnology) was first bound to Dynabeads Protein G (Invitrogen) according to the manufacturer’s instructions.

Techniques: Inhibition, Control, Western Blot, Transduction, Over Expression, Expressing, Staining, Flow Cytometry, Quantitative RT-PCR

Journal: The EMBO Journal

Article Title: Activation of goblet-cell stress sensor IRE1β is controlled by the mucin chaperone AGR2

doi: 10.1038/s44318-023-00015-y

Figure Lengend Snippet: AGR2 expression in cultures analyzed in Fig. D, . Tubulin was used as a loading control.

Article Snippet: Anti-AGR2 antibody (clone 6C5, Santa Cruz Biotechnology) was first bound to Dynabeads Protein G (Invitrogen) according to the manufacturer’s instructions.

Techniques: Expressing, Control

Journal: The EMBO Journal

Article Title: Activation of goblet-cell stress sensor IRE1β is controlled by the mucin chaperone AGR2

doi: 10.1038/s44318-023-00015-y

Figure Lengend Snippet: ( A ) BLAST alignment of the regions containing cysteines in human IRE1α and IRE1β. Green square indicates the sole conserved cysteine in IRE1α and IRE1β luminal domain, orange squares show cysteines present in only one of the paralogues. ( B ) Highest scoring AlphaFold2-Multimer model (pTM score = 0.662), modeled using IRE1β residues 35-377 (Uniprot Q76MJ5 ) and AGR2 residues 41-175 (Uniprot O95994 ). The IRE1β luminal domain is shown in orange and AGR2 in grey. Labels indicate the highlighted green residues. ( C ) Predicted aligned error (PAE) plot for the model shown in B.

Article Snippet: Anti-AGR2 antibody (clone 6C5, Santa Cruz Biotechnology) was first bound to Dynabeads Protein G (Invitrogen) according to the manufacturer’s instructions.

Techniques:

Journal: The EMBO Journal

Article Title: Activation of goblet-cell stress sensor IRE1β is controlled by the mucin chaperone AGR2

doi: 10.1038/s44318-023-00015-y

Figure Lengend Snippet: In steady-state conditions, AGR2 is bound to most IRE1β molecules and under these conditions, IRE1β is mainly present in the inactive, monomeric forms. As a result, overall IRE1β activity will be low. In conditions where an activating trigger is present (possibly unfolded MUC2 polypeptides, though this remains to be demonstrated), AGR2 is released from IRE1β in favor of binding other AGR2 chaperone substrates. As a result, IRE1β is released, activated and overall IRE1β activity will be high. In case of AGR2 C81S and AGR2 H117Y , interaction with IRE1β is disrupted leading to spontaneous IRE1β dimerization triggering its activity.

Article Snippet: Anti-AGR2 antibody (clone 6C5, Santa Cruz Biotechnology) was first bound to Dynabeads Protein G (Invitrogen) according to the manufacturer’s instructions.

Techniques: Activity Assay, Binding Assay