Journal: Theranostics

Article Title: LRG1 inhibition promotes acute pancreatitis recovery by inducing cholecystokinin Type 1 receptor expression via Akt.

doi: 10.7150/thno.110116

Figure Lengend Snippet: Figure 1. Loss of LRG1 does not affect the structure and function of the normal mouse pancreas. (A) Immunofluorescence staining of LRG1 (red), CD31 or NG2 (green), and DAPI (blue) in the normal mouse pancreas. (B) Pancreas weight to body weight ratio of wild-type and Lrg1-/- mice. (C) H&E staining showing vasculature of wild-type

Article Snippet: CCK1R antagonist and LRG1 neutralizing antibody treatment in-vivo L364, 718 (#HY-106301, MedChemExpress, USA), a competitive non-peptide antagonist of CCK1R was administered intraperitoneally in Lrg1−/− mice at a dose of 0.1mg/kg, 30 minutes before each caerulein injection.

Techniques: Immunofluorescence, Staining

Journal: Theranostics

Article Title: LRG1 inhibition promotes acute pancreatitis recovery by inducing cholecystokinin Type 1 receptor expression via Akt.

doi: 10.7150/thno.110116

Figure Lengend Snippet: Figure 2. LRG1 is highly induced in humans and mice during AP. (A) ELISA analysis of LRG1 levels in the serum of healthy controls (n = 10) and AP patients (n = 18). (B) Correlation analysis with regression line (95% confidence intervals) of serum LRG1 and CRP in AP patients. (C) Schematic diagram of caerulein-induced AP in mice. (D)

Article Snippet: CCK1R antagonist and LRG1 neutralizing antibody treatment in-vivo L364, 718 (#HY-106301, MedChemExpress, USA), a competitive non-peptide antagonist of CCK1R was administered intraperitoneally in Lrg1−/− mice at a dose of 0.1mg/kg, 30 minutes before each caerulein injection.

Techniques: Enzyme-linked Immunosorbent Assay

Journal: Theranostics

Article Title: LRG1 inhibition promotes acute pancreatitis recovery by inducing cholecystokinin Type 1 receptor expression via Akt.

doi: 10.7150/thno.110116

Figure Lengend Snippet: Figure 3. LRG1 is regulated by IL-6 in pancreatic acinar cells during AP. (A) ELISA analysis of IL-6 levels in the serum of healthy controls (n = 10) and AP patients (n = 18). (B) Correlation analysis with regression line (95% confidence intervals) of serum LRG1 and IL-6 in AP patients. (C) ELISA analysis of serum IL-6 levels in mice subjected to caerulein-induced AP. (D) qRT-PCR analysis of Il6 in mouse pancreas at various time points during AP progression. (E) qRT-PCR analysis of Il6 in isolated acinar cells 24 hours following the first caerulein injection. (F) Western blot (left) and densitometry analysis (right) of phosphorylated and total STAT3, and LRG1 in wild-type acinar cells subjected to DMSO or STATTIC treatment with or without addition of IL-6. (G) Schematic indicating organization of the mouse LRG1 promoter containing two putative STAT3 transcription factor binding sites. (H) DNA agarose gel (left) and quantitative analysis (right) of chromatin immunoprecipitation assay for STAT3 and LRG1 promoter association in the presence or absence of IL-6 in primary acinar cells. All images are representative. Data are presented as mean ± s.e.m. Significance was determined by one-way ANOVA followed by Holm-Sidak’s multiple comparison test or unpaired, two-tailed Student’s t-test of n ≥ 3 mice or independent experiments unless stated otherwise; *: p < 0.05, **: p < 0.01.

Article Snippet: CCK1R antagonist and LRG1 neutralizing antibody treatment in-vivo L364, 718 (#HY-106301, MedChemExpress, USA), a competitive non-peptide antagonist of CCK1R was administered intraperitoneally in Lrg1−/− mice at a dose of 0.1mg/kg, 30 minutes before each caerulein injection.

Techniques: Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Isolation, Injection, Western Blot, Binding Assay, Agarose Gel Electrophoresis, Chromatin Immunoprecipitation, Comparison, Two Tailed Test

Journal: Theranostics

Article Title: LRG1 inhibition promotes acute pancreatitis recovery by inducing cholecystokinin Type 1 receptor expression via Akt.

doi: 10.7150/thno.110116

Figure Lengend Snippet: Figure 4. LRG1 deficiency exacerbates caerulein-induced AP. (A) H&E images demonstrating the overall pancreatic damage in saline or caerulein-treated wild-type and Lrg1-/- mice (Inter- and intralobular damage (asterisk), infiltrated inflammatory cells (arrowhead), and edema (arrow)). Scale bar: 50μm, scale bar for boxed regions: 25μm. Histopathological grading of (B) overall pancreatic damage (C) lobular damage, (D) inflammatory cell infiltration, and (E) edema in H&E-stained wild-type and Lrg1-/- pancreatic tissues. (F) Immunofluorescent staining against MPO (red) and DAPI (blue) (left) and quantification (right) of MPO+ inflammatory cells (arrow) in wild-type and Lrg1-/- pancreas. Scale bar: 20μm. qRT-PCR analysis of the mRNA levels of inflammatory cytokines, (G) Tnfa, (H) Il6, and (I) Cxcl1 in wild-type and Lrg1-deficient pancreas. (J) Analysis of serum amylase activity in wild-type and Lrg1-/- mice. qRT-PCR analysis of mRNA levels of pancreatic (K) Amy2 and (L) Krt19. All analyses were performed 24 hours after the induction of AP. Images are representative. Data are presented as the mean ± s.e.m. Significance was determined by one-way ANOVA followed by Holm-Sidak’s multiple comparison test or unpaired, two-tailed Student’s t-test of n ≥ 6 mice; *: p < 0.05, **: p < 0.01, ***: p < 0.001.

Article Snippet: CCK1R antagonist and LRG1 neutralizing antibody treatment in-vivo L364, 718 (#HY-106301, MedChemExpress, USA), a competitive non-peptide antagonist of CCK1R was administered intraperitoneally in Lrg1−/− mice at a dose of 0.1mg/kg, 30 minutes before each caerulein injection.

Techniques: Saline, Staining, Quantitative RT-PCR, Activity Assay, Comparison, Two Tailed Test

Journal: Theranostics

Article Title: LRG1 inhibition promotes acute pancreatitis recovery by inducing cholecystokinin Type 1 receptor expression via Akt.

doi: 10.7150/thno.110116

Figure Lengend Snippet: Figure 5. Non-myeloid cell-derived LRG1 protects against AP-induced damage. (A) Schematic diagram of bone marrow transplantation with wild-type mice reconstituted with wild-type BMCs (Wild-type Wild-type), Lrg1-/- mice reconstituted with wild-type BMCs (Wild-type Lrg1-/-), wild-type mice reconstituted with Lrg1-/- BMCs (Lrg1-/- Wild-type) and Lrg1-/- mice reconstituted with Lrg1-/- BMCs (Lrg1-/- Lrg1-/-). (B) H&E staining and (C) histopathological scoring of overall pancreatic damage. Scale bar: 100μm, scale bar of boxed region: 50μm. (D) Immunofluorescent staining against MPO (red) and DAPI (blue) (left) and quantification (right) of infiltrated MPO+ inflammatory cells (arrow) in the pancreas. Scale bar: 25μm. qRT-PCR analysis of mRNA levels of (E) Cxcl1, (F) Ccl2, (G) Tnfa, (H) Il6 and (I) Amy2 in the pancreas. (J) Western blot (top) and densitometry analysis (bottom) for cleaved caspase 3 levels in primary wild-type or Lrg1-/- acinar cells subjected to saline or caerulein treatment. qRT-PCR analysis of mRNA levels of (K) Tnfa, (L) Il6, and (M) Cxcl1 in primary acinar cells isolated from wild-type or Lrg1-/- mice subjected to saline or caerulein treatment. All analyses were performed 24 hours after the induction of AP. Images are representative. Data are presented as the mean ± s.e.m. Significance was determined by one-way ANOVA followed by Holm-Sidak’s multiple comparisons test of n ≥ 4 mice; *: p < 0.05, **: p < 0.01, ***: p < 0.001.

Article Snippet: CCK1R antagonist and LRG1 neutralizing antibody treatment in-vivo L364, 718 (#HY-106301, MedChemExpress, USA), a competitive non-peptide antagonist of CCK1R was administered intraperitoneally in Lrg1−/− mice at a dose of 0.1mg/kg, 30 minutes before each caerulein injection.

Techniques: Derivative Assay, Transplantation Assay, Staining, Quantitative RT-PCR, Western Blot, Saline, Isolation

Journal: Theranostics

Article Title: LRG1 inhibition promotes acute pancreatitis recovery by inducing cholecystokinin Type 1 receptor expression via Akt.

doi: 10.7150/thno.110116

Figure Lengend Snippet: Figure 6. Lrg1-deletion benefits AP recovery in mice in AP. (A) H&E staining and (B) histopathological grading demonstrating overall pancreatic damage. Scale bar: 50μm, Scale bar of boxed regions: 25μm. qRT-PCR analysis of mRNA levels of (C) Tnfa, (D) Il6, and (E) Il1. (F) Serum amylase activity in saline or caerulein-treated wild-type and Lrg1-deficient mice. qRT-PCR analysis of (G) Amy2 mRNA levels in the pancreas. qRT-PCR analysis of mRNA levels of anti-inflammatory cytokines (H) Il10 (I) Nfkbia and proliferative markers (J) Cyclin B, Ccnb, (K) Cyclin D, Ccnd, and (L) Cyclin E, Ccne in the pancreas. (M) Immunofluorescent staining against Ki67 (red), AMY (green), and DAPI (blue) (left) and quantification (right) of Ki67+ proliferating acinar cells (arrow) in the pancreas. Scale bar: 20μm. All images are representative. Figures (A)-(G) were performed in the pancreas of saline or caerulein-treated wild-type and Lrg1-deficient mice at 3- and 7-day post the induction of AP. Figures (H)-(I) and (J)-(M) were performed in the pancreas of saline- or caerulein-injected wild-type and Lrg1-/- mice 24 hours and 3 days post the induction of AP respectively. Data are presented as the mean ± s.e.m. Significance was determined by one-way ANOVA followed by Holm-Sidak’s multiple comparisons test or unpaired, two-tailed Student’s t-test of n ≥ 4 mice; *: p < 0.05, **: p < 0.01, ***: p < 0.001, n.s.: not significant, p > 0.05.

Article Snippet: CCK1R antagonist and LRG1 neutralizing antibody treatment in-vivo L364, 718 (#HY-106301, MedChemExpress, USA), a competitive non-peptide antagonist of CCK1R was administered intraperitoneally in Lrg1−/− mice at a dose of 0.1mg/kg, 30 minutes before each caerulein injection.

Techniques: Staining, Quantitative RT-PCR, Activity Assay, Saline, Injection, Two Tailed Test

Journal: Theranostics

Article Title: LRG1 inhibition promotes acute pancreatitis recovery by inducing cholecystokinin Type 1 receptor expression via Akt.

doi: 10.7150/thno.110116

Figure Lengend Snippet: Figure 7. LRG1 regulates acinar cell function through AKT-mediated CCK1R expression. (A) qRT-PCR analysis of cholecystokinin A receptor (Cck1r) mRNA levels and (B) Western blot (top) and densitometry analysis (bottom) of CCK1R protein levels in the pancreas of adult wild-type and Lrg1-/- mice. (C) Western blot (left) and densitometry analysis (right) of phosphorylated and total levels of ALK5, AKT, ERK, and SMAD2 in the pancreatic of wild-type and Lrg1-deficient mice. (D) Western blot (left) and densitometry analysis (right) of phosphorylated and total levels of ALK5 and AKT protein in primary acinar cells isolated from wild-type or Lrg1-/- mice in the presence or absence of rhLRG1. (E) qRT-PCR analysis Cck1r mRNA levels and (F) Western blot (left) and densitometry analysis (right) of CCK1R protein levels in primary acinar cells isolated from wild-type or Lrg1-/- mice in the presence or absence of rhLRG1. (G) Western blot (left) and densitometry analysis (right) of pALK5, ALK5, pAKT, and AKT levels in primary acinar cells isolated from Lrg1-/- mice subjected to the treatment with ALK5 (SB 431542) or AKT(MK2206) specific inhibitor. (H) qRT-PCR analysis of Cck1r mRNA levels and (I) Western blot (left) and densitometry analysis (right) of CCK1R protein levels in primary acinar cells isolated from Lrg1-/- mice subjected to the treatment with ALK5 or AKT inhibitor. All images are representative. Data are presented as the mean ± s.e.m. Significance was determined by one-way ANOVA followed by Holm-Sidak’s multiple comparisons test or unpaired, two-tailed Student’s t-test of n ≥ 4 mice or independent experiments; *: p < 0.05, **: p < 0.01, ***: p < 0.001.

Article Snippet: CCK1R antagonist and LRG1 neutralizing antibody treatment in-vivo L364, 718 (#HY-106301, MedChemExpress, USA), a competitive non-peptide antagonist of CCK1R was administered intraperitoneally in Lrg1−/− mice at a dose of 0.1mg/kg, 30 minutes before each caerulein injection.

Techniques: Cell Function Assay, Expressing, Quantitative RT-PCR, Western Blot, Isolation, Two Tailed Test

Journal: Theranostics

Article Title: LRG1 inhibition promotes acute pancreatitis recovery by inducing cholecystokinin Type 1 receptor expression via Akt.

doi: 10.7150/thno.110116

Figure Lengend Snippet: Figure 8. LRG1 regulates AP pathology in a CCK1R-dependent manner. (A) Schematic diagram of CCK1R antagonist L364,718 administration strategy. (B) H&E staining and (C) histopathological scoring of overall pancreatic damage in vehicle or L364,718-treated Lrg1-/- mice 24 hours after the induction of AP. Scale bar: 100μm, scale bar of boxed regions: 50μm. (D) Immunofluorescent staining against MPO (red) and DAPI (blue) (left) and quantification (right) of infiltrated MPO+ inflammatory cells (arrow) of the pancreas tissues of caerulein-treated Lrg1-/- mice following the treatment with either vehicle control or L364,718. Scale bar: 25μm. qRT-PCR analysis of pancreatic mRNA levels of (E) Il6 and (F) Cxcl1 (G) Ccl2 in vehicle or L364,718 treated Lrg1-deficient mice following the induction of AP. (H) Serum amylase activity of the vehicle or L364,718 treated Lrg1-/- mice 24 hours following the AP induction. qRT-PCR analysis of pancreatic mRNA levels of (I) Amy2 and (J) Krt19. All images are representative. Data are presented as the mean ± s.e.m. Significance was determined by unpaired, two-tailed Student’s t-test of n ≥ 5 mice; *: p < 0.05, **: p < 0.01, ***: p < 0.001.

Article Snippet: CCK1R antagonist and LRG1 neutralizing antibody treatment in-vivo L364, 718 (#HY-106301, MedChemExpress, USA), a competitive non-peptide antagonist of CCK1R was administered intraperitoneally in Lrg1−/− mice at a dose of 0.1mg/kg, 30 minutes before each caerulein injection.

Techniques: Staining, Control, Quantitative RT-PCR, Activity Assay, Two Tailed Test

Journal: Theranostics

Article Title: LRG1 inhibition promotes acute pancreatitis recovery by inducing cholecystokinin Type 1 receptor expression via Akt.

doi: 10.7150/thno.110116

Figure Lengend Snippet: Figure 9. LRG1 inhibition promotes pancreatic recovery in AP. (A) Schematic diagram of LRG1 antibody treatment strategy. (B) Western blot (left) and densitometry analysis (right) of pancreatic phosphorylated and total ALK5 and AKT levels in AP mice treated with IgG or LRG1 antibody. (C) qRT-PCR analysis of pancreatic Cck1r mRNA levels in AP mice treated with IgG or LRG1 antibody. (D) Western blot (top) and densitometry analysis (bottom) of pancreatic CCK1R protein levels in AP mice treated with IgG or LRG1 antibody. (E) H&E staining demonstrating inflammatory cell infiltration (arrowhead) and (F) histopathological grading of the pancreas of AP mice subjected to IgG or LRG1 antibody treatment. Scale bar: 100μm, Scale bar of boxed regions: 50μm. qRT-PCR analysis of pancreatic (G) Amy2, (H) Il10, (I) Nfkbia, and (J) Ccnb (K) Ccne levels in IgG or LRG1 antibody-treated mice at Day 2 following AP induction. (L) Immunofluorescent staining against Ki67 (red), AMY (green), and DAPI (blue) (top) and quantification (bottom) of Ki67+ acinar cells (arrow) in IgG and LRG1 antibody-treated mice at Day 3 post-AP induction. Scale bar: 20μm. Data are presented as the mean ± s.e.m. All images are representative and all experiments are conducted 2 days post-AP induction unless specified. Significance was determined by unpaired, two-tailed Student’s t-test of n ≥ 5 mice; *: p < 0.05, **: p < 0.01.

Article Snippet: CCK1R antagonist and LRG1 neutralizing antibody treatment in-vivo L364, 718 (#HY-106301, MedChemExpress, USA), a competitive non-peptide antagonist of CCK1R was administered intraperitoneally in Lrg1−/− mice at a dose of 0.1mg/kg, 30 minutes before each caerulein injection.

Techniques: Inhibition, Western Blot, Quantitative RT-PCR, Staining, Two Tailed Test

Journal: Theranostics

Article Title: LRG1 inhibition promotes acute pancreatitis recovery by inducing cholecystokinin Type 1 receptor expression via Akt.

doi: 10.7150/thno.110116

Figure Lengend Snippet: Figure 10. Mechanistic summary of the role of LRG1 in pancreatic injury and regeneration. In wild-type acinar cells, the binding of caerulein to CCK1R on the surface of acinar cells triggers a cascade of signaling events, including the activation of PKC, STAT3, and JNK pathways. This leads to acinar cell apoptosis and the production of pro-inflammatory cytokines such as TNF-α and IL-6, which amplify the inflammatory response initiated by the initial acinar cell injury. Concurrently, IL-6 signals through its receptor, IL-6R, to activate the transcription factor STAT3, which subsequently induces LRG1 expression in acinar cells. LRG1, in turn, antagonizes the TGFβRII/ALK5/AKT-mediated expression of CCK1R, a trophic factor for acinar cells, thereby limiting pancreatic regeneration. In Lrg1-/- acinar cells, the inhibitory effect of LRG1 on the TGFβRII/ALK5/AKT pathway is absent, resulting in elevated CCK1R expression compared to wild-type acinar cells. Consequently, more CCK1R is available to bind caerulein, leading to greater acinar cell damage. However, this higher CCK1R expression also promotes increased acinar cell proliferation and regeneration, explaining the accelerated pancreatic regeneration observed in Lrg1-/- mice despite the presence of more severe initial damage.

Article Snippet: CCK1R antagonist and LRG1 neutralizing antibody treatment in-vivo L364, 718 (#HY-106301, MedChemExpress, USA), a competitive non-peptide antagonist of CCK1R was administered intraperitoneally in Lrg1−/− mice at a dose of 0.1mg/kg, 30 minutes before each caerulein injection.

Techniques: Binding Assay, Activation Assay, Expressing

Journal: Theranostics

Article Title: LRG1 inhibition promotes acute pancreatitis recovery by inducing cholecystokinin Type 1 receptor expression via Akt.

doi: 10.7150/thno.110116

Figure Lengend Snippet: Figure 1. Loss of LRG1 does not affect the structure and function of the normal mouse pancreas. (A) Immunofluorescence staining of LRG1 (red), CD31 or NG2 (green), and DAPI (blue) in the normal mouse pancreas. (B) Pancreas weight to body weight ratio of wild-type and Lrg1-/- mice. (C) H&E staining showing vasculature of wild-type

Article Snippet: For immunofluorescence staining, paraffin (6μm) or cryosections (8μm) were subjected to antigen retrieval using sodium citrate buffer before being stained with primary antibodies against LRG1 (#13224-1-AP, Proteintech, USA), CD31 (#550274, BD Biosciences, USA or #ab28364, Abcam, UK), AN2 (#130-100-468, Miltenyi Biotec, Germany), Glucagon (#ab92517, Abcam, UK), Insulin (#ab ab7842, Abcam, UK), Amylase (#sc-46657, Santa-Cruz Biotechnology, USA), Cytokeratin 19 (#PAB12676, Abnova, USA), Myeloperoxidase (MPO) (#ab9535; Abcam, UK or #AF3667, R&D Systems, USA) and Ki67 (#ab15580; Abcam, UK) followed by staining with DAPI, Alexa 488, Alexa 594 and Alexa 657 secondary antibodies (Thermo Fisher Scientific, USA).

Techniques: Immunofluorescence, Staining

Journal: Theranostics

Article Title: LRG1 inhibition promotes acute pancreatitis recovery by inducing cholecystokinin Type 1 receptor expression via Akt.

doi: 10.7150/thno.110116

Figure Lengend Snippet: Figure 2. LRG1 is highly induced in humans and mice during AP. (A) ELISA analysis of LRG1 levels in the serum of healthy controls (n = 10) and AP patients (n = 18). (B) Correlation analysis with regression line (95% confidence intervals) of serum LRG1 and CRP in AP patients. (C) Schematic diagram of caerulein-induced AP in mice. (D)

Article Snippet: For immunofluorescence staining, paraffin (6μm) or cryosections (8μm) were subjected to antigen retrieval using sodium citrate buffer before being stained with primary antibodies against LRG1 (#13224-1-AP, Proteintech, USA), CD31 (#550274, BD Biosciences, USA or #ab28364, Abcam, UK), AN2 (#130-100-468, Miltenyi Biotec, Germany), Glucagon (#ab92517, Abcam, UK), Insulin (#ab ab7842, Abcam, UK), Amylase (#sc-46657, Santa-Cruz Biotechnology, USA), Cytokeratin 19 (#PAB12676, Abnova, USA), Myeloperoxidase (MPO) (#ab9535; Abcam, UK or #AF3667, R&D Systems, USA) and Ki67 (#ab15580; Abcam, UK) followed by staining with DAPI, Alexa 488, Alexa 594 and Alexa 657 secondary antibodies (Thermo Fisher Scientific, USA).

Techniques: Enzyme-linked Immunosorbent Assay

Journal: Theranostics

Article Title: LRG1 inhibition promotes acute pancreatitis recovery by inducing cholecystokinin Type 1 receptor expression via Akt.

doi: 10.7150/thno.110116

Figure Lengend Snippet: Figure 3. LRG1 is regulated by IL-6 in pancreatic acinar cells during AP. (A) ELISA analysis of IL-6 levels in the serum of healthy controls (n = 10) and AP patients (n = 18). (B) Correlation analysis with regression line (95% confidence intervals) of serum LRG1 and IL-6 in AP patients. (C) ELISA analysis of serum IL-6 levels in mice subjected to caerulein-induced AP. (D) qRT-PCR analysis of Il6 in mouse pancreas at various time points during AP progression. (E) qRT-PCR analysis of Il6 in isolated acinar cells 24 hours following the first caerulein injection. (F) Western blot (left) and densitometry analysis (right) of phosphorylated and total STAT3, and LRG1 in wild-type acinar cells subjected to DMSO or STATTIC treatment with or without addition of IL-6. (G) Schematic indicating organization of the mouse LRG1 promoter containing two putative STAT3 transcription factor binding sites. (H) DNA agarose gel (left) and quantitative analysis (right) of chromatin immunoprecipitation assay for STAT3 and LRG1 promoter association in the presence or absence of IL-6 in primary acinar cells. All images are representative. Data are presented as mean ± s.e.m. Significance was determined by one-way ANOVA followed by Holm-Sidak’s multiple comparison test or unpaired, two-tailed Student’s t-test of n ≥ 3 mice or independent experiments unless stated otherwise; *: p < 0.05, **: p < 0.01.

Article Snippet: For immunofluorescence staining, paraffin (6μm) or cryosections (8μm) were subjected to antigen retrieval using sodium citrate buffer before being stained with primary antibodies against LRG1 (#13224-1-AP, Proteintech, USA), CD31 (#550274, BD Biosciences, USA or #ab28364, Abcam, UK), AN2 (#130-100-468, Miltenyi Biotec, Germany), Glucagon (#ab92517, Abcam, UK), Insulin (#ab ab7842, Abcam, UK), Amylase (#sc-46657, Santa-Cruz Biotechnology, USA), Cytokeratin 19 (#PAB12676, Abnova, USA), Myeloperoxidase (MPO) (#ab9535; Abcam, UK or #AF3667, R&D Systems, USA) and Ki67 (#ab15580; Abcam, UK) followed by staining with DAPI, Alexa 488, Alexa 594 and Alexa 657 secondary antibodies (Thermo Fisher Scientific, USA).

Techniques: Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Isolation, Injection, Western Blot, Binding Assay, Agarose Gel Electrophoresis, Chromatin Immunoprecipitation, Comparison, Two Tailed Test

Journal: Theranostics

Article Title: LRG1 inhibition promotes acute pancreatitis recovery by inducing cholecystokinin Type 1 receptor expression via Akt.

doi: 10.7150/thno.110116

Figure Lengend Snippet: Figure 4. LRG1 deficiency exacerbates caerulein-induced AP. (A) H&E images demonstrating the overall pancreatic damage in saline or caerulein-treated wild-type and Lrg1-/- mice (Inter- and intralobular damage (asterisk), infiltrated inflammatory cells (arrowhead), and edema (arrow)). Scale bar: 50μm, scale bar for boxed regions: 25μm. Histopathological grading of (B) overall pancreatic damage (C) lobular damage, (D) inflammatory cell infiltration, and (E) edema in H&E-stained wild-type and Lrg1-/- pancreatic tissues. (F) Immunofluorescent staining against MPO (red) and DAPI (blue) (left) and quantification (right) of MPO+ inflammatory cells (arrow) in wild-type and Lrg1-/- pancreas. Scale bar: 20μm. qRT-PCR analysis of the mRNA levels of inflammatory cytokines, (G) Tnfa, (H) Il6, and (I) Cxcl1 in wild-type and Lrg1-deficient pancreas. (J) Analysis of serum amylase activity in wild-type and Lrg1-/- mice. qRT-PCR analysis of mRNA levels of pancreatic (K) Amy2 and (L) Krt19. All analyses were performed 24 hours after the induction of AP. Images are representative. Data are presented as the mean ± s.e.m. Significance was determined by one-way ANOVA followed by Holm-Sidak’s multiple comparison test or unpaired, two-tailed Student’s t-test of n ≥ 6 mice; *: p < 0.05, **: p < 0.01, ***: p < 0.001.

Article Snippet: For immunofluorescence staining, paraffin (6μm) or cryosections (8μm) were subjected to antigen retrieval using sodium citrate buffer before being stained with primary antibodies against LRG1 (#13224-1-AP, Proteintech, USA), CD31 (#550274, BD Biosciences, USA or #ab28364, Abcam, UK), AN2 (#130-100-468, Miltenyi Biotec, Germany), Glucagon (#ab92517, Abcam, UK), Insulin (#ab ab7842, Abcam, UK), Amylase (#sc-46657, Santa-Cruz Biotechnology, USA), Cytokeratin 19 (#PAB12676, Abnova, USA), Myeloperoxidase (MPO) (#ab9535; Abcam, UK or #AF3667, R&D Systems, USA) and Ki67 (#ab15580; Abcam, UK) followed by staining with DAPI, Alexa 488, Alexa 594 and Alexa 657 secondary antibodies (Thermo Fisher Scientific, USA).

Techniques: Saline, Staining, Quantitative RT-PCR, Activity Assay, Comparison, Two Tailed Test

Journal: Theranostics

Article Title: LRG1 inhibition promotes acute pancreatitis recovery by inducing cholecystokinin Type 1 receptor expression via Akt.

doi: 10.7150/thno.110116

Figure Lengend Snippet: Figure 5. Non-myeloid cell-derived LRG1 protects against AP-induced damage. (A) Schematic diagram of bone marrow transplantation with wild-type mice reconstituted with wild-type BMCs (Wild-type Wild-type), Lrg1-/- mice reconstituted with wild-type BMCs (Wild-type Lrg1-/-), wild-type mice reconstituted with Lrg1-/- BMCs (Lrg1-/- Wild-type) and Lrg1-/- mice reconstituted with Lrg1-/- BMCs (Lrg1-/- Lrg1-/-). (B) H&E staining and (C) histopathological scoring of overall pancreatic damage. Scale bar: 100μm, scale bar of boxed region: 50μm. (D) Immunofluorescent staining against MPO (red) and DAPI (blue) (left) and quantification (right) of infiltrated MPO+ inflammatory cells (arrow) in the pancreas. Scale bar: 25μm. qRT-PCR analysis of mRNA levels of (E) Cxcl1, (F) Ccl2, (G) Tnfa, (H) Il6 and (I) Amy2 in the pancreas. (J) Western blot (top) and densitometry analysis (bottom) for cleaved caspase 3 levels in primary wild-type or Lrg1-/- acinar cells subjected to saline or caerulein treatment. qRT-PCR analysis of mRNA levels of (K) Tnfa, (L) Il6, and (M) Cxcl1 in primary acinar cells isolated from wild-type or Lrg1-/- mice subjected to saline or caerulein treatment. All analyses were performed 24 hours after the induction of AP. Images are representative. Data are presented as the mean ± s.e.m. Significance was determined by one-way ANOVA followed by Holm-Sidak’s multiple comparisons test of n ≥ 4 mice; *: p < 0.05, **: p < 0.01, ***: p < 0.001.

Article Snippet: For immunofluorescence staining, paraffin (6μm) or cryosections (8μm) were subjected to antigen retrieval using sodium citrate buffer before being stained with primary antibodies against LRG1 (#13224-1-AP, Proteintech, USA), CD31 (#550274, BD Biosciences, USA or #ab28364, Abcam, UK), AN2 (#130-100-468, Miltenyi Biotec, Germany), Glucagon (#ab92517, Abcam, UK), Insulin (#ab ab7842, Abcam, UK), Amylase (#sc-46657, Santa-Cruz Biotechnology, USA), Cytokeratin 19 (#PAB12676, Abnova, USA), Myeloperoxidase (MPO) (#ab9535; Abcam, UK or #AF3667, R&D Systems, USA) and Ki67 (#ab15580; Abcam, UK) followed by staining with DAPI, Alexa 488, Alexa 594 and Alexa 657 secondary antibodies (Thermo Fisher Scientific, USA).

Techniques: Derivative Assay, Transplantation Assay, Staining, Quantitative RT-PCR, Western Blot, Saline, Isolation

Journal: Theranostics

Article Title: LRG1 inhibition promotes acute pancreatitis recovery by inducing cholecystokinin Type 1 receptor expression via Akt.

doi: 10.7150/thno.110116

Figure Lengend Snippet: Figure 6. Lrg1-deletion benefits AP recovery in mice in AP. (A) H&E staining and (B) histopathological grading demonstrating overall pancreatic damage. Scale bar: 50μm, Scale bar of boxed regions: 25μm. qRT-PCR analysis of mRNA levels of (C) Tnfa, (D) Il6, and (E) Il1. (F) Serum amylase activity in saline or caerulein-treated wild-type and Lrg1-deficient mice. qRT-PCR analysis of (G) Amy2 mRNA levels in the pancreas. qRT-PCR analysis of mRNA levels of anti-inflammatory cytokines (H) Il10 (I) Nfkbia and proliferative markers (J) Cyclin B, Ccnb, (K) Cyclin D, Ccnd, and (L) Cyclin E, Ccne in the pancreas. (M) Immunofluorescent staining against Ki67 (red), AMY (green), and DAPI (blue) (left) and quantification (right) of Ki67+ proliferating acinar cells (arrow) in the pancreas. Scale bar: 20μm. All images are representative. Figures (A)-(G) were performed in the pancreas of saline or caerulein-treated wild-type and Lrg1-deficient mice at 3- and 7-day post the induction of AP. Figures (H)-(I) and (J)-(M) were performed in the pancreas of saline- or caerulein-injected wild-type and Lrg1-/- mice 24 hours and 3 days post the induction of AP respectively. Data are presented as the mean ± s.e.m. Significance was determined by one-way ANOVA followed by Holm-Sidak’s multiple comparisons test or unpaired, two-tailed Student’s t-test of n ≥ 4 mice; *: p < 0.05, **: p < 0.01, ***: p < 0.001, n.s.: not significant, p > 0.05.

Article Snippet: For immunofluorescence staining, paraffin (6μm) or cryosections (8μm) were subjected to antigen retrieval using sodium citrate buffer before being stained with primary antibodies against LRG1 (#13224-1-AP, Proteintech, USA), CD31 (#550274, BD Biosciences, USA or #ab28364, Abcam, UK), AN2 (#130-100-468, Miltenyi Biotec, Germany), Glucagon (#ab92517, Abcam, UK), Insulin (#ab ab7842, Abcam, UK), Amylase (#sc-46657, Santa-Cruz Biotechnology, USA), Cytokeratin 19 (#PAB12676, Abnova, USA), Myeloperoxidase (MPO) (#ab9535; Abcam, UK or #AF3667, R&D Systems, USA) and Ki67 (#ab15580; Abcam, UK) followed by staining with DAPI, Alexa 488, Alexa 594 and Alexa 657 secondary antibodies (Thermo Fisher Scientific, USA).

Techniques: Staining, Quantitative RT-PCR, Activity Assay, Saline, Injection, Two Tailed Test

Journal: Theranostics

Article Title: LRG1 inhibition promotes acute pancreatitis recovery by inducing cholecystokinin Type 1 receptor expression via Akt.

doi: 10.7150/thno.110116

Figure Lengend Snippet: Figure 7. LRG1 regulates acinar cell function through AKT-mediated CCK1R expression. (A) qRT-PCR analysis of cholecystokinin A receptor (Cck1r) mRNA levels and (B) Western blot (top) and densitometry analysis (bottom) of CCK1R protein levels in the pancreas of adult wild-type and Lrg1-/- mice. (C) Western blot (left) and densitometry analysis (right) of phosphorylated and total levels of ALK5, AKT, ERK, and SMAD2 in the pancreatic of wild-type and Lrg1-deficient mice. (D) Western blot (left) and densitometry analysis (right) of phosphorylated and total levels of ALK5 and AKT protein in primary acinar cells isolated from wild-type or Lrg1-/- mice in the presence or absence of rhLRG1. (E) qRT-PCR analysis Cck1r mRNA levels and (F) Western blot (left) and densitometry analysis (right) of CCK1R protein levels in primary acinar cells isolated from wild-type or Lrg1-/- mice in the presence or absence of rhLRG1. (G) Western blot (left) and densitometry analysis (right) of pALK5, ALK5, pAKT, and AKT levels in primary acinar cells isolated from Lrg1-/- mice subjected to the treatment with ALK5 (SB 431542) or AKT(MK2206) specific inhibitor. (H) qRT-PCR analysis of Cck1r mRNA levels and (I) Western blot (left) and densitometry analysis (right) of CCK1R protein levels in primary acinar cells isolated from Lrg1-/- mice subjected to the treatment with ALK5 or AKT inhibitor. All images are representative. Data are presented as the mean ± s.e.m. Significance was determined by one-way ANOVA followed by Holm-Sidak’s multiple comparisons test or unpaired, two-tailed Student’s t-test of n ≥ 4 mice or independent experiments; *: p < 0.05, **: p < 0.01, ***: p < 0.001.

Article Snippet: For immunofluorescence staining, paraffin (6μm) or cryosections (8μm) were subjected to antigen retrieval using sodium citrate buffer before being stained with primary antibodies against LRG1 (#13224-1-AP, Proteintech, USA), CD31 (#550274, BD Biosciences, USA or #ab28364, Abcam, UK), AN2 (#130-100-468, Miltenyi Biotec, Germany), Glucagon (#ab92517, Abcam, UK), Insulin (#ab ab7842, Abcam, UK), Amylase (#sc-46657, Santa-Cruz Biotechnology, USA), Cytokeratin 19 (#PAB12676, Abnova, USA), Myeloperoxidase (MPO) (#ab9535; Abcam, UK or #AF3667, R&D Systems, USA) and Ki67 (#ab15580; Abcam, UK) followed by staining with DAPI, Alexa 488, Alexa 594 and Alexa 657 secondary antibodies (Thermo Fisher Scientific, USA).

Techniques: Cell Function Assay, Expressing, Quantitative RT-PCR, Western Blot, Isolation, Two Tailed Test

Journal: Theranostics

Article Title: LRG1 inhibition promotes acute pancreatitis recovery by inducing cholecystokinin Type 1 receptor expression via Akt.

doi: 10.7150/thno.110116

Figure Lengend Snippet: Figure 8. LRG1 regulates AP pathology in a CCK1R-dependent manner. (A) Schematic diagram of CCK1R antagonist L364,718 administration strategy. (B) H&E staining and (C) histopathological scoring of overall pancreatic damage in vehicle or L364,718-treated Lrg1-/- mice 24 hours after the induction of AP. Scale bar: 100μm, scale bar of boxed regions: 50μm. (D) Immunofluorescent staining against MPO (red) and DAPI (blue) (left) and quantification (right) of infiltrated MPO+ inflammatory cells (arrow) of the pancreas tissues of caerulein-treated Lrg1-/- mice following the treatment with either vehicle control or L364,718. Scale bar: 25μm. qRT-PCR analysis of pancreatic mRNA levels of (E) Il6 and (F) Cxcl1 (G) Ccl2 in vehicle or L364,718 treated Lrg1-deficient mice following the induction of AP. (H) Serum amylase activity of the vehicle or L364,718 treated Lrg1-/- mice 24 hours following the AP induction. qRT-PCR analysis of pancreatic mRNA levels of (I) Amy2 and (J) Krt19. All images are representative. Data are presented as the mean ± s.e.m. Significance was determined by unpaired, two-tailed Student’s t-test of n ≥ 5 mice; *: p < 0.05, **: p < 0.01, ***: p < 0.001.

Article Snippet: For immunofluorescence staining, paraffin (6μm) or cryosections (8μm) were subjected to antigen retrieval using sodium citrate buffer before being stained with primary antibodies against LRG1 (#13224-1-AP, Proteintech, USA), CD31 (#550274, BD Biosciences, USA or #ab28364, Abcam, UK), AN2 (#130-100-468, Miltenyi Biotec, Germany), Glucagon (#ab92517, Abcam, UK), Insulin (#ab ab7842, Abcam, UK), Amylase (#sc-46657, Santa-Cruz Biotechnology, USA), Cytokeratin 19 (#PAB12676, Abnova, USA), Myeloperoxidase (MPO) (#ab9535; Abcam, UK or #AF3667, R&D Systems, USA) and Ki67 (#ab15580; Abcam, UK) followed by staining with DAPI, Alexa 488, Alexa 594 and Alexa 657 secondary antibodies (Thermo Fisher Scientific, USA).

Techniques: Staining, Control, Quantitative RT-PCR, Activity Assay, Two Tailed Test

Journal: Theranostics

Article Title: LRG1 inhibition promotes acute pancreatitis recovery by inducing cholecystokinin Type 1 receptor expression via Akt.

doi: 10.7150/thno.110116

Figure Lengend Snippet: Figure 9. LRG1 inhibition promotes pancreatic recovery in AP. (A) Schematic diagram of LRG1 antibody treatment strategy. (B) Western blot (left) and densitometry analysis (right) of pancreatic phosphorylated and total ALK5 and AKT levels in AP mice treated with IgG or LRG1 antibody. (C) qRT-PCR analysis of pancreatic Cck1r mRNA levels in AP mice treated with IgG or LRG1 antibody. (D) Western blot (top) and densitometry analysis (bottom) of pancreatic CCK1R protein levels in AP mice treated with IgG or LRG1 antibody. (E) H&E staining demonstrating inflammatory cell infiltration (arrowhead) and (F) histopathological grading of the pancreas of AP mice subjected to IgG or LRG1 antibody treatment. Scale bar: 100μm, Scale bar of boxed regions: 50μm. qRT-PCR analysis of pancreatic (G) Amy2, (H) Il10, (I) Nfkbia, and (J) Ccnb (K) Ccne levels in IgG or LRG1 antibody-treated mice at Day 2 following AP induction. (L) Immunofluorescent staining against Ki67 (red), AMY (green), and DAPI (blue) (top) and quantification (bottom) of Ki67+ acinar cells (arrow) in IgG and LRG1 antibody-treated mice at Day 3 post-AP induction. Scale bar: 20μm. Data are presented as the mean ± s.e.m. All images are representative and all experiments are conducted 2 days post-AP induction unless specified. Significance was determined by unpaired, two-tailed Student’s t-test of n ≥ 5 mice; *: p < 0.05, **: p < 0.01.

Article Snippet: For immunofluorescence staining, paraffin (6μm) or cryosections (8μm) were subjected to antigen retrieval using sodium citrate buffer before being stained with primary antibodies against LRG1 (#13224-1-AP, Proteintech, USA), CD31 (#550274, BD Biosciences, USA or #ab28364, Abcam, UK), AN2 (#130-100-468, Miltenyi Biotec, Germany), Glucagon (#ab92517, Abcam, UK), Insulin (#ab ab7842, Abcam, UK), Amylase (#sc-46657, Santa-Cruz Biotechnology, USA), Cytokeratin 19 (#PAB12676, Abnova, USA), Myeloperoxidase (MPO) (#ab9535; Abcam, UK or #AF3667, R&D Systems, USA) and Ki67 (#ab15580; Abcam, UK) followed by staining with DAPI, Alexa 488, Alexa 594 and Alexa 657 secondary antibodies (Thermo Fisher Scientific, USA).

Techniques: Inhibition, Western Blot, Quantitative RT-PCR, Staining, Two Tailed Test

Journal: Theranostics

Article Title: LRG1 inhibition promotes acute pancreatitis recovery by inducing cholecystokinin Type 1 receptor expression via Akt.

doi: 10.7150/thno.110116

Figure Lengend Snippet: Figure 10. Mechanistic summary of the role of LRG1 in pancreatic injury and regeneration. In wild-type acinar cells, the binding of caerulein to CCK1R on the surface of acinar cells triggers a cascade of signaling events, including the activation of PKC, STAT3, and JNK pathways. This leads to acinar cell apoptosis and the production of pro-inflammatory cytokines such as TNF-α and IL-6, which amplify the inflammatory response initiated by the initial acinar cell injury. Concurrently, IL-6 signals through its receptor, IL-6R, to activate the transcription factor STAT3, which subsequently induces LRG1 expression in acinar cells. LRG1, in turn, antagonizes the TGFβRII/ALK5/AKT-mediated expression of CCK1R, a trophic factor for acinar cells, thereby limiting pancreatic regeneration. In Lrg1-/- acinar cells, the inhibitory effect of LRG1 on the TGFβRII/ALK5/AKT pathway is absent, resulting in elevated CCK1R expression compared to wild-type acinar cells. Consequently, more CCK1R is available to bind caerulein, leading to greater acinar cell damage. However, this higher CCK1R expression also promotes increased acinar cell proliferation and regeneration, explaining the accelerated pancreatic regeneration observed in Lrg1-/- mice despite the presence of more severe initial damage.

Article Snippet: For immunofluorescence staining, paraffin (6μm) or cryosections (8μm) were subjected to antigen retrieval using sodium citrate buffer before being stained with primary antibodies against LRG1 (#13224-1-AP, Proteintech, USA), CD31 (#550274, BD Biosciences, USA or #ab28364, Abcam, UK), AN2 (#130-100-468, Miltenyi Biotec, Germany), Glucagon (#ab92517, Abcam, UK), Insulin (#ab ab7842, Abcam, UK), Amylase (#sc-46657, Santa-Cruz Biotechnology, USA), Cytokeratin 19 (#PAB12676, Abnova, USA), Myeloperoxidase (MPO) (#ab9535; Abcam, UK or #AF3667, R&D Systems, USA) and Ki67 (#ab15580; Abcam, UK) followed by staining with DAPI, Alexa 488, Alexa 594 and Alexa 657 secondary antibodies (Thermo Fisher Scientific, USA).

Techniques: Binding Assay, Activation Assay, Expressing

Journal: Theranostics

Article Title: LRG1 inhibition promotes acute pancreatitis recovery by inducing cholecystokinin Type 1 receptor expression via Akt.

doi: 10.7150/thno.110116

Figure Lengend Snippet: Figure 1. Loss of LRG1 does not affect the structure and function of the normal mouse pancreas. (A) Immunofluorescence staining of LRG1 (red), CD31 or NG2 (green), and DAPI (blue) in the normal mouse pancreas. (B) Pancreas weight to body weight ratio of wild-type and Lrg1-/- mice. (C) H&E staining showing vasculature of wild-type

Article Snippet: Blots were probed with LRG1 antibody (rabbit monoclonal, #13224-1-AP, Proteintech,USA), phospho-PKC δ antibody (mouse monoclonal (#sc-365969, Santa-Cruz Biotechnology, USA), phospho-PKC epsilon antibody (rabbit polyclonal, #ab63387, Abcam, UK), PKC antibody (mouse monoclonal, #sc-17769, Santa-Cruz Biotechnology, USA), phospho-STAT3 (rabbit monoclonal, #9145, Cell Signaling Technology, USA), STAT3 (rabbit monoclonal, #12640, Cell Signaling Technology, USA), phospho-SAPK/JNK (rabbit monoclonal, #9255, Cell Signaling Technology, USA), SAPK/JNK (rabbit monoclonal, #9145, Cell Signaling Technology, USA), cleaved caspase 3 antibody (rabbit monoclonal, #9664, Cell Signaling Technology, USA), phospho-PKCnu antibody (rabbit polyclonal, #orb4440, Biorbyt, UK), PKCnu antibody (rabbit polyclonal, #bs-4157R, Bioss Inc, USA), CCK1R antibody (rabbit polyclonal, #bs-11514R, Bioss Inc, USA), phospho-Akt antibody (rabbit monoclonal, #4060; Cell Signaling Technology), Akt antibody (rabbit monoclonal, #9272; Cell Signaling Technology), phospho-p44/42 MAPK (ERK1/2) antibody (rabbit monoclonal, #4370, Cell Signaling Technology, USA), p44/42 MAPK (ERK1/2) antibody (rabbit monoclonal, #4695, Cell Signaling Technology, USA), phospho-Smad2 antibody (rabbit monoclonal, #3108, Cell Signaling Technology, USA), Smad2 antibody (rabbit monoclonal, #5339, Cell Signaling Technology, USA), HSP60 antibody (rabbit monoclonal, #12165, Cell Signaling Technology, USA), RPLP0 antibody (rabbit polyclonal, #11290-2-AP, Proteintech,USA), GAPDH antibody (mouse monoclonal, #sc-32233, Santa-Cruz Biotechnology, USA), followed by horseradish peroxidase–conjugated secondary antibodies (Bethyl Laboratories, USA).

Techniques: Immunofluorescence, Staining

Journal: Theranostics

Article Title: LRG1 inhibition promotes acute pancreatitis recovery by inducing cholecystokinin Type 1 receptor expression via Akt.

doi: 10.7150/thno.110116

Figure Lengend Snippet: Figure 2. LRG1 is highly induced in humans and mice during AP. (A) ELISA analysis of LRG1 levels in the serum of healthy controls (n = 10) and AP patients (n = 18). (B) Correlation analysis with regression line (95% confidence intervals) of serum LRG1 and CRP in AP patients. (C) Schematic diagram of caerulein-induced AP in mice. (D)

Article Snippet: Blots were probed with LRG1 antibody (rabbit monoclonal, #13224-1-AP, Proteintech,USA), phospho-PKC δ antibody (mouse monoclonal (#sc-365969, Santa-Cruz Biotechnology, USA), phospho-PKC epsilon antibody (rabbit polyclonal, #ab63387, Abcam, UK), PKC antibody (mouse monoclonal, #sc-17769, Santa-Cruz Biotechnology, USA), phospho-STAT3 (rabbit monoclonal, #9145, Cell Signaling Technology, USA), STAT3 (rabbit monoclonal, #12640, Cell Signaling Technology, USA), phospho-SAPK/JNK (rabbit monoclonal, #9255, Cell Signaling Technology, USA), SAPK/JNK (rabbit monoclonal, #9145, Cell Signaling Technology, USA), cleaved caspase 3 antibody (rabbit monoclonal, #9664, Cell Signaling Technology, USA), phospho-PKCnu antibody (rabbit polyclonal, #orb4440, Biorbyt, UK), PKCnu antibody (rabbit polyclonal, #bs-4157R, Bioss Inc, USA), CCK1R antibody (rabbit polyclonal, #bs-11514R, Bioss Inc, USA), phospho-Akt antibody (rabbit monoclonal, #4060; Cell Signaling Technology), Akt antibody (rabbit monoclonal, #9272; Cell Signaling Technology), phospho-p44/42 MAPK (ERK1/2) antibody (rabbit monoclonal, #4370, Cell Signaling Technology, USA), p44/42 MAPK (ERK1/2) antibody (rabbit monoclonal, #4695, Cell Signaling Technology, USA), phospho-Smad2 antibody (rabbit monoclonal, #3108, Cell Signaling Technology, USA), Smad2 antibody (rabbit monoclonal, #5339, Cell Signaling Technology, USA), HSP60 antibody (rabbit monoclonal, #12165, Cell Signaling Technology, USA), RPLP0 antibody (rabbit polyclonal, #11290-2-AP, Proteintech,USA), GAPDH antibody (mouse monoclonal, #sc-32233, Santa-Cruz Biotechnology, USA), followed by horseradish peroxidase–conjugated secondary antibodies (Bethyl Laboratories, USA).

Techniques: Enzyme-linked Immunosorbent Assay

Journal: Theranostics

Article Title: LRG1 inhibition promotes acute pancreatitis recovery by inducing cholecystokinin Type 1 receptor expression via Akt.

doi: 10.7150/thno.110116

Figure Lengend Snippet: Figure 3. LRG1 is regulated by IL-6 in pancreatic acinar cells during AP. (A) ELISA analysis of IL-6 levels in the serum of healthy controls (n = 10) and AP patients (n = 18). (B) Correlation analysis with regression line (95% confidence intervals) of serum LRG1 and IL-6 in AP patients. (C) ELISA analysis of serum IL-6 levels in mice subjected to caerulein-induced AP. (D) qRT-PCR analysis of Il6 in mouse pancreas at various time points during AP progression. (E) qRT-PCR analysis of Il6 in isolated acinar cells 24 hours following the first caerulein injection. (F) Western blot (left) and densitometry analysis (right) of phosphorylated and total STAT3, and LRG1 in wild-type acinar cells subjected to DMSO or STATTIC treatment with or without addition of IL-6. (G) Schematic indicating organization of the mouse LRG1 promoter containing two putative STAT3 transcription factor binding sites. (H) DNA agarose gel (left) and quantitative analysis (right) of chromatin immunoprecipitation assay for STAT3 and LRG1 promoter association in the presence or absence of IL-6 in primary acinar cells. All images are representative. Data are presented as mean ± s.e.m. Significance was determined by one-way ANOVA followed by Holm-Sidak’s multiple comparison test or unpaired, two-tailed Student’s t-test of n ≥ 3 mice or independent experiments unless stated otherwise; *: p < 0.05, **: p < 0.01.

Article Snippet: Blots were probed with LRG1 antibody (rabbit monoclonal, #13224-1-AP, Proteintech,USA), phospho-PKC δ antibody (mouse monoclonal (#sc-365969, Santa-Cruz Biotechnology, USA), phospho-PKC epsilon antibody (rabbit polyclonal, #ab63387, Abcam, UK), PKC antibody (mouse monoclonal, #sc-17769, Santa-Cruz Biotechnology, USA), phospho-STAT3 (rabbit monoclonal, #9145, Cell Signaling Technology, USA), STAT3 (rabbit monoclonal, #12640, Cell Signaling Technology, USA), phospho-SAPK/JNK (rabbit monoclonal, #9255, Cell Signaling Technology, USA), SAPK/JNK (rabbit monoclonal, #9145, Cell Signaling Technology, USA), cleaved caspase 3 antibody (rabbit monoclonal, #9664, Cell Signaling Technology, USA), phospho-PKCnu antibody (rabbit polyclonal, #orb4440, Biorbyt, UK), PKCnu antibody (rabbit polyclonal, #bs-4157R, Bioss Inc, USA), CCK1R antibody (rabbit polyclonal, #bs-11514R, Bioss Inc, USA), phospho-Akt antibody (rabbit monoclonal, #4060; Cell Signaling Technology), Akt antibody (rabbit monoclonal, #9272; Cell Signaling Technology), phospho-p44/42 MAPK (ERK1/2) antibody (rabbit monoclonal, #4370, Cell Signaling Technology, USA), p44/42 MAPK (ERK1/2) antibody (rabbit monoclonal, #4695, Cell Signaling Technology, USA), phospho-Smad2 antibody (rabbit monoclonal, #3108, Cell Signaling Technology, USA), Smad2 antibody (rabbit monoclonal, #5339, Cell Signaling Technology, USA), HSP60 antibody (rabbit monoclonal, #12165, Cell Signaling Technology, USA), RPLP0 antibody (rabbit polyclonal, #11290-2-AP, Proteintech,USA), GAPDH antibody (mouse monoclonal, #sc-32233, Santa-Cruz Biotechnology, USA), followed by horseradish peroxidase–conjugated secondary antibodies (Bethyl Laboratories, USA).

Techniques: Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Isolation, Injection, Western Blot, Binding Assay, Agarose Gel Electrophoresis, Chromatin Immunoprecipitation, Comparison, Two Tailed Test

Journal: Theranostics

Article Title: LRG1 inhibition promotes acute pancreatitis recovery by inducing cholecystokinin Type 1 receptor expression via Akt.

doi: 10.7150/thno.110116

Figure Lengend Snippet: Figure 4. LRG1 deficiency exacerbates caerulein-induced AP. (A) H&E images demonstrating the overall pancreatic damage in saline or caerulein-treated wild-type and Lrg1-/- mice (Inter- and intralobular damage (asterisk), infiltrated inflammatory cells (arrowhead), and edema (arrow)). Scale bar: 50μm, scale bar for boxed regions: 25μm. Histopathological grading of (B) overall pancreatic damage (C) lobular damage, (D) inflammatory cell infiltration, and (E) edema in H&E-stained wild-type and Lrg1-/- pancreatic tissues. (F) Immunofluorescent staining against MPO (red) and DAPI (blue) (left) and quantification (right) of MPO+ inflammatory cells (arrow) in wild-type and Lrg1-/- pancreas. Scale bar: 20μm. qRT-PCR analysis of the mRNA levels of inflammatory cytokines, (G) Tnfa, (H) Il6, and (I) Cxcl1 in wild-type and Lrg1-deficient pancreas. (J) Analysis of serum amylase activity in wild-type and Lrg1-/- mice. qRT-PCR analysis of mRNA levels of pancreatic (K) Amy2 and (L) Krt19. All analyses were performed 24 hours after the induction of AP. Images are representative. Data are presented as the mean ± s.e.m. Significance was determined by one-way ANOVA followed by Holm-Sidak’s multiple comparison test or unpaired, two-tailed Student’s t-test of n ≥ 6 mice; *: p < 0.05, **: p < 0.01, ***: p < 0.001.

Article Snippet: Blots were probed with LRG1 antibody (rabbit monoclonal, #13224-1-AP, Proteintech,USA), phospho-PKC δ antibody (mouse monoclonal (#sc-365969, Santa-Cruz Biotechnology, USA), phospho-PKC epsilon antibody (rabbit polyclonal, #ab63387, Abcam, UK), PKC antibody (mouse monoclonal, #sc-17769, Santa-Cruz Biotechnology, USA), phospho-STAT3 (rabbit monoclonal, #9145, Cell Signaling Technology, USA), STAT3 (rabbit monoclonal, #12640, Cell Signaling Technology, USA), phospho-SAPK/JNK (rabbit monoclonal, #9255, Cell Signaling Technology, USA), SAPK/JNK (rabbit monoclonal, #9145, Cell Signaling Technology, USA), cleaved caspase 3 antibody (rabbit monoclonal, #9664, Cell Signaling Technology, USA), phospho-PKCnu antibody (rabbit polyclonal, #orb4440, Biorbyt, UK), PKCnu antibody (rabbit polyclonal, #bs-4157R, Bioss Inc, USA), CCK1R antibody (rabbit polyclonal, #bs-11514R, Bioss Inc, USA), phospho-Akt antibody (rabbit monoclonal, #4060; Cell Signaling Technology), Akt antibody (rabbit monoclonal, #9272; Cell Signaling Technology), phospho-p44/42 MAPK (ERK1/2) antibody (rabbit monoclonal, #4370, Cell Signaling Technology, USA), p44/42 MAPK (ERK1/2) antibody (rabbit monoclonal, #4695, Cell Signaling Technology, USA), phospho-Smad2 antibody (rabbit monoclonal, #3108, Cell Signaling Technology, USA), Smad2 antibody (rabbit monoclonal, #5339, Cell Signaling Technology, USA), HSP60 antibody (rabbit monoclonal, #12165, Cell Signaling Technology, USA), RPLP0 antibody (rabbit polyclonal, #11290-2-AP, Proteintech,USA), GAPDH antibody (mouse monoclonal, #sc-32233, Santa-Cruz Biotechnology, USA), followed by horseradish peroxidase–conjugated secondary antibodies (Bethyl Laboratories, USA).

Techniques: Saline, Staining, Quantitative RT-PCR, Activity Assay, Comparison, Two Tailed Test

Journal: Theranostics

Article Title: LRG1 inhibition promotes acute pancreatitis recovery by inducing cholecystokinin Type 1 receptor expression via Akt.

doi: 10.7150/thno.110116

Figure Lengend Snippet: Figure 5. Non-myeloid cell-derived LRG1 protects against AP-induced damage. (A) Schematic diagram of bone marrow transplantation with wild-type mice reconstituted with wild-type BMCs (Wild-type Wild-type), Lrg1-/- mice reconstituted with wild-type BMCs (Wild-type Lrg1-/-), wild-type mice reconstituted with Lrg1-/- BMCs (Lrg1-/- Wild-type) and Lrg1-/- mice reconstituted with Lrg1-/- BMCs (Lrg1-/- Lrg1-/-). (B) H&E staining and (C) histopathological scoring of overall pancreatic damage. Scale bar: 100μm, scale bar of boxed region: 50μm. (D) Immunofluorescent staining against MPO (red) and DAPI (blue) (left) and quantification (right) of infiltrated MPO+ inflammatory cells (arrow) in the pancreas. Scale bar: 25μm. qRT-PCR analysis of mRNA levels of (E) Cxcl1, (F) Ccl2, (G) Tnfa, (H) Il6 and (I) Amy2 in the pancreas. (J) Western blot (top) and densitometry analysis (bottom) for cleaved caspase 3 levels in primary wild-type or Lrg1-/- acinar cells subjected to saline or caerulein treatment. qRT-PCR analysis of mRNA levels of (K) Tnfa, (L) Il6, and (M) Cxcl1 in primary acinar cells isolated from wild-type or Lrg1-/- mice subjected to saline or caerulein treatment. All analyses were performed 24 hours after the induction of AP. Images are representative. Data are presented as the mean ± s.e.m. Significance was determined by one-way ANOVA followed by Holm-Sidak’s multiple comparisons test of n ≥ 4 mice; *: p < 0.05, **: p < 0.01, ***: p < 0.001.

Article Snippet: Blots were probed with LRG1 antibody (rabbit monoclonal, #13224-1-AP, Proteintech,USA), phospho-PKC δ antibody (mouse monoclonal (#sc-365969, Santa-Cruz Biotechnology, USA), phospho-PKC epsilon antibody (rabbit polyclonal, #ab63387, Abcam, UK), PKC antibody (mouse monoclonal, #sc-17769, Santa-Cruz Biotechnology, USA), phospho-STAT3 (rabbit monoclonal, #9145, Cell Signaling Technology, USA), STAT3 (rabbit monoclonal, #12640, Cell Signaling Technology, USA), phospho-SAPK/JNK (rabbit monoclonal, #9255, Cell Signaling Technology, USA), SAPK/JNK (rabbit monoclonal, #9145, Cell Signaling Technology, USA), cleaved caspase 3 antibody (rabbit monoclonal, #9664, Cell Signaling Technology, USA), phospho-PKCnu antibody (rabbit polyclonal, #orb4440, Biorbyt, UK), PKCnu antibody (rabbit polyclonal, #bs-4157R, Bioss Inc, USA), CCK1R antibody (rabbit polyclonal, #bs-11514R, Bioss Inc, USA), phospho-Akt antibody (rabbit monoclonal, #4060; Cell Signaling Technology), Akt antibody (rabbit monoclonal, #9272; Cell Signaling Technology), phospho-p44/42 MAPK (ERK1/2) antibody (rabbit monoclonal, #4370, Cell Signaling Technology, USA), p44/42 MAPK (ERK1/2) antibody (rabbit monoclonal, #4695, Cell Signaling Technology, USA), phospho-Smad2 antibody (rabbit monoclonal, #3108, Cell Signaling Technology, USA), Smad2 antibody (rabbit monoclonal, #5339, Cell Signaling Technology, USA), HSP60 antibody (rabbit monoclonal, #12165, Cell Signaling Technology, USA), RPLP0 antibody (rabbit polyclonal, #11290-2-AP, Proteintech,USA), GAPDH antibody (mouse monoclonal, #sc-32233, Santa-Cruz Biotechnology, USA), followed by horseradish peroxidase–conjugated secondary antibodies (Bethyl Laboratories, USA).

Techniques: Derivative Assay, Transplantation Assay, Staining, Quantitative RT-PCR, Western Blot, Saline, Isolation

Journal: Theranostics

Article Title: LRG1 inhibition promotes acute pancreatitis recovery by inducing cholecystokinin Type 1 receptor expression via Akt.

doi: 10.7150/thno.110116

Figure Lengend Snippet: Figure 6. Lrg1-deletion benefits AP recovery in mice in AP. (A) H&E staining and (B) histopathological grading demonstrating overall pancreatic damage. Scale bar: 50μm, Scale bar of boxed regions: 25μm. qRT-PCR analysis of mRNA levels of (C) Tnfa, (D) Il6, and (E) Il1. (F) Serum amylase activity in saline or caerulein-treated wild-type and Lrg1-deficient mice. qRT-PCR analysis of (G) Amy2 mRNA levels in the pancreas. qRT-PCR analysis of mRNA levels of anti-inflammatory cytokines (H) Il10 (I) Nfkbia and proliferative markers (J) Cyclin B, Ccnb, (K) Cyclin D, Ccnd, and (L) Cyclin E, Ccne in the pancreas. (M) Immunofluorescent staining against Ki67 (red), AMY (green), and DAPI (blue) (left) and quantification (right) of Ki67+ proliferating acinar cells (arrow) in the pancreas. Scale bar: 20μm. All images are representative. Figures (A)-(G) were performed in the pancreas of saline or caerulein-treated wild-type and Lrg1-deficient mice at 3- and 7-day post the induction of AP. Figures (H)-(I) and (J)-(M) were performed in the pancreas of saline- or caerulein-injected wild-type and Lrg1-/- mice 24 hours and 3 days post the induction of AP respectively. Data are presented as the mean ± s.e.m. Significance was determined by one-way ANOVA followed by Holm-Sidak’s multiple comparisons test or unpaired, two-tailed Student’s t-test of n ≥ 4 mice; *: p < 0.05, **: p < 0.01, ***: p < 0.001, n.s.: not significant, p > 0.05.

Article Snippet: Blots were probed with LRG1 antibody (rabbit monoclonal, #13224-1-AP, Proteintech,USA), phospho-PKC δ antibody (mouse monoclonal (#sc-365969, Santa-Cruz Biotechnology, USA), phospho-PKC epsilon antibody (rabbit polyclonal, #ab63387, Abcam, UK), PKC antibody (mouse monoclonal, #sc-17769, Santa-Cruz Biotechnology, USA), phospho-STAT3 (rabbit monoclonal, #9145, Cell Signaling Technology, USA), STAT3 (rabbit monoclonal, #12640, Cell Signaling Technology, USA), phospho-SAPK/JNK (rabbit monoclonal, #9255, Cell Signaling Technology, USA), SAPK/JNK (rabbit monoclonal, #9145, Cell Signaling Technology, USA), cleaved caspase 3 antibody (rabbit monoclonal, #9664, Cell Signaling Technology, USA), phospho-PKCnu antibody (rabbit polyclonal, #orb4440, Biorbyt, UK), PKCnu antibody (rabbit polyclonal, #bs-4157R, Bioss Inc, USA), CCK1R antibody (rabbit polyclonal, #bs-11514R, Bioss Inc, USA), phospho-Akt antibody (rabbit monoclonal, #4060; Cell Signaling Technology), Akt antibody (rabbit monoclonal, #9272; Cell Signaling Technology), phospho-p44/42 MAPK (ERK1/2) antibody (rabbit monoclonal, #4370, Cell Signaling Technology, USA), p44/42 MAPK (ERK1/2) antibody (rabbit monoclonal, #4695, Cell Signaling Technology, USA), phospho-Smad2 antibody (rabbit monoclonal, #3108, Cell Signaling Technology, USA), Smad2 antibody (rabbit monoclonal, #5339, Cell Signaling Technology, USA), HSP60 antibody (rabbit monoclonal, #12165, Cell Signaling Technology, USA), RPLP0 antibody (rabbit polyclonal, #11290-2-AP, Proteintech,USA), GAPDH antibody (mouse monoclonal, #sc-32233, Santa-Cruz Biotechnology, USA), followed by horseradish peroxidase–conjugated secondary antibodies (Bethyl Laboratories, USA).

Techniques: Staining, Quantitative RT-PCR, Activity Assay, Saline, Injection, Two Tailed Test

Journal: Theranostics

Article Title: LRG1 inhibition promotes acute pancreatitis recovery by inducing cholecystokinin Type 1 receptor expression via Akt.

doi: 10.7150/thno.110116

Figure Lengend Snippet: Figure 7. LRG1 regulates acinar cell function through AKT-mediated CCK1R expression. (A) qRT-PCR analysis of cholecystokinin A receptor (Cck1r) mRNA levels and (B) Western blot (top) and densitometry analysis (bottom) of CCK1R protein levels in the pancreas of adult wild-type and Lrg1-/- mice. (C) Western blot (left) and densitometry analysis (right) of phosphorylated and total levels of ALK5, AKT, ERK, and SMAD2 in the pancreatic of wild-type and Lrg1-deficient mice. (D) Western blot (left) and densitometry analysis (right) of phosphorylated and total levels of ALK5 and AKT protein in primary acinar cells isolated from wild-type or Lrg1-/- mice in the presence or absence of rhLRG1. (E) qRT-PCR analysis Cck1r mRNA levels and (F) Western blot (left) and densitometry analysis (right) of CCK1R protein levels in primary acinar cells isolated from wild-type or Lrg1-/- mice in the presence or absence of rhLRG1. (G) Western blot (left) and densitometry analysis (right) of pALK5, ALK5, pAKT, and AKT levels in primary acinar cells isolated from Lrg1-/- mice subjected to the treatment with ALK5 (SB 431542) or AKT(MK2206) specific inhibitor. (H) qRT-PCR analysis of Cck1r mRNA levels and (I) Western blot (left) and densitometry analysis (right) of CCK1R protein levels in primary acinar cells isolated from Lrg1-/- mice subjected to the treatment with ALK5 or AKT inhibitor. All images are representative. Data are presented as the mean ± s.e.m. Significance was determined by one-way ANOVA followed by Holm-Sidak’s multiple comparisons test or unpaired, two-tailed Student’s t-test of n ≥ 4 mice or independent experiments; *: p < 0.05, **: p < 0.01, ***: p < 0.001.

Article Snippet: Blots were probed with LRG1 antibody (rabbit monoclonal, #13224-1-AP, Proteintech,USA), phospho-PKC δ antibody (mouse monoclonal (#sc-365969, Santa-Cruz Biotechnology, USA), phospho-PKC epsilon antibody (rabbit polyclonal, #ab63387, Abcam, UK), PKC antibody (mouse monoclonal, #sc-17769, Santa-Cruz Biotechnology, USA), phospho-STAT3 (rabbit monoclonal, #9145, Cell Signaling Technology, USA), STAT3 (rabbit monoclonal, #12640, Cell Signaling Technology, USA), phospho-SAPK/JNK (rabbit monoclonal, #9255, Cell Signaling Technology, USA), SAPK/JNK (rabbit monoclonal, #9145, Cell Signaling Technology, USA), cleaved caspase 3 antibody (rabbit monoclonal, #9664, Cell Signaling Technology, USA), phospho-PKCnu antibody (rabbit polyclonal, #orb4440, Biorbyt, UK), PKCnu antibody (rabbit polyclonal, #bs-4157R, Bioss Inc, USA), CCK1R antibody (rabbit polyclonal, #bs-11514R, Bioss Inc, USA), phospho-Akt antibody (rabbit monoclonal, #4060; Cell Signaling Technology), Akt antibody (rabbit monoclonal, #9272; Cell Signaling Technology), phospho-p44/42 MAPK (ERK1/2) antibody (rabbit monoclonal, #4370, Cell Signaling Technology, USA), p44/42 MAPK (ERK1/2) antibody (rabbit monoclonal, #4695, Cell Signaling Technology, USA), phospho-Smad2 antibody (rabbit monoclonal, #3108, Cell Signaling Technology, USA), Smad2 antibody (rabbit monoclonal, #5339, Cell Signaling Technology, USA), HSP60 antibody (rabbit monoclonal, #12165, Cell Signaling Technology, USA), RPLP0 antibody (rabbit polyclonal, #11290-2-AP, Proteintech,USA), GAPDH antibody (mouse monoclonal, #sc-32233, Santa-Cruz Biotechnology, USA), followed by horseradish peroxidase–conjugated secondary antibodies (Bethyl Laboratories, USA).

Techniques: Cell Function Assay, Expressing, Quantitative RT-PCR, Western Blot, Isolation, Two Tailed Test

Journal: Theranostics

Article Title: LRG1 inhibition promotes acute pancreatitis recovery by inducing cholecystokinin Type 1 receptor expression via Akt.

doi: 10.7150/thno.110116

Figure Lengend Snippet: Figure 8. LRG1 regulates AP pathology in a CCK1R-dependent manner. (A) Schematic diagram of CCK1R antagonist L364,718 administration strategy. (B) H&E staining and (C) histopathological scoring of overall pancreatic damage in vehicle or L364,718-treated Lrg1-/- mice 24 hours after the induction of AP. Scale bar: 100μm, scale bar of boxed regions: 50μm. (D) Immunofluorescent staining against MPO (red) and DAPI (blue) (left) and quantification (right) of infiltrated MPO+ inflammatory cells (arrow) of the pancreas tissues of caerulein-treated Lrg1-/- mice following the treatment with either vehicle control or L364,718. Scale bar: 25μm. qRT-PCR analysis of pancreatic mRNA levels of (E) Il6 and (F) Cxcl1 (G) Ccl2 in vehicle or L364,718 treated Lrg1-deficient mice following the induction of AP. (H) Serum amylase activity of the vehicle or L364,718 treated Lrg1-/- mice 24 hours following the AP induction. qRT-PCR analysis of pancreatic mRNA levels of (I) Amy2 and (J) Krt19. All images are representative. Data are presented as the mean ± s.e.m. Significance was determined by unpaired, two-tailed Student’s t-test of n ≥ 5 mice; *: p < 0.05, **: p < 0.01, ***: p < 0.001.

Article Snippet: Blots were probed with LRG1 antibody (rabbit monoclonal, #13224-1-AP, Proteintech,USA), phospho-PKC δ antibody (mouse monoclonal (#sc-365969, Santa-Cruz Biotechnology, USA), phospho-PKC epsilon antibody (rabbit polyclonal, #ab63387, Abcam, UK), PKC antibody (mouse monoclonal, #sc-17769, Santa-Cruz Biotechnology, USA), phospho-STAT3 (rabbit monoclonal, #9145, Cell Signaling Technology, USA), STAT3 (rabbit monoclonal, #12640, Cell Signaling Technology, USA), phospho-SAPK/JNK (rabbit monoclonal, #9255, Cell Signaling Technology, USA), SAPK/JNK (rabbit monoclonal, #9145, Cell Signaling Technology, USA), cleaved caspase 3 antibody (rabbit monoclonal, #9664, Cell Signaling Technology, USA), phospho-PKCnu antibody (rabbit polyclonal, #orb4440, Biorbyt, UK), PKCnu antibody (rabbit polyclonal, #bs-4157R, Bioss Inc, USA), CCK1R antibody (rabbit polyclonal, #bs-11514R, Bioss Inc, USA), phospho-Akt antibody (rabbit monoclonal, #4060; Cell Signaling Technology), Akt antibody (rabbit monoclonal, #9272; Cell Signaling Technology), phospho-p44/42 MAPK (ERK1/2) antibody (rabbit monoclonal, #4370, Cell Signaling Technology, USA), p44/42 MAPK (ERK1/2) antibody (rabbit monoclonal, #4695, Cell Signaling Technology, USA), phospho-Smad2 antibody (rabbit monoclonal, #3108, Cell Signaling Technology, USA), Smad2 antibody (rabbit monoclonal, #5339, Cell Signaling Technology, USA), HSP60 antibody (rabbit monoclonal, #12165, Cell Signaling Technology, USA), RPLP0 antibody (rabbit polyclonal, #11290-2-AP, Proteintech,USA), GAPDH antibody (mouse monoclonal, #sc-32233, Santa-Cruz Biotechnology, USA), followed by horseradish peroxidase–conjugated secondary antibodies (Bethyl Laboratories, USA).

Techniques: Staining, Control, Quantitative RT-PCR, Activity Assay, Two Tailed Test

Journal: Theranostics

Article Title: LRG1 inhibition promotes acute pancreatitis recovery by inducing cholecystokinin Type 1 receptor expression via Akt.

doi: 10.7150/thno.110116

Figure Lengend Snippet: Figure 9. LRG1 inhibition promotes pancreatic recovery in AP. (A) Schematic diagram of LRG1 antibody treatment strategy. (B) Western blot (left) and densitometry analysis (right) of pancreatic phosphorylated and total ALK5 and AKT levels in AP mice treated with IgG or LRG1 antibody. (C) qRT-PCR analysis of pancreatic Cck1r mRNA levels in AP mice treated with IgG or LRG1 antibody. (D) Western blot (top) and densitometry analysis (bottom) of pancreatic CCK1R protein levels in AP mice treated with IgG or LRG1 antibody. (E) H&E staining demonstrating inflammatory cell infiltration (arrowhead) and (F) histopathological grading of the pancreas of AP mice subjected to IgG or LRG1 antibody treatment. Scale bar: 100μm, Scale bar of boxed regions: 50μm. qRT-PCR analysis of pancreatic (G) Amy2, (H) Il10, (I) Nfkbia, and (J) Ccnb (K) Ccne levels in IgG or LRG1 antibody-treated mice at Day 2 following AP induction. (L) Immunofluorescent staining against Ki67 (red), AMY (green), and DAPI (blue) (top) and quantification (bottom) of Ki67+ acinar cells (arrow) in IgG and LRG1 antibody-treated mice at Day 3 post-AP induction. Scale bar: 20μm. Data are presented as the mean ± s.e.m. All images are representative and all experiments are conducted 2 days post-AP induction unless specified. Significance was determined by unpaired, two-tailed Student’s t-test of n ≥ 5 mice; *: p < 0.05, **: p < 0.01.

Article Snippet: Blots were probed with LRG1 antibody (rabbit monoclonal, #13224-1-AP, Proteintech,USA), phospho-PKC δ antibody (mouse monoclonal (#sc-365969, Santa-Cruz Biotechnology, USA), phospho-PKC epsilon antibody (rabbit polyclonal, #ab63387, Abcam, UK), PKC antibody (mouse monoclonal, #sc-17769, Santa-Cruz Biotechnology, USA), phospho-STAT3 (rabbit monoclonal, #9145, Cell Signaling Technology, USA), STAT3 (rabbit monoclonal, #12640, Cell Signaling Technology, USA), phospho-SAPK/JNK (rabbit monoclonal, #9255, Cell Signaling Technology, USA), SAPK/JNK (rabbit monoclonal, #9145, Cell Signaling Technology, USA), cleaved caspase 3 antibody (rabbit monoclonal, #9664, Cell Signaling Technology, USA), phospho-PKCnu antibody (rabbit polyclonal, #orb4440, Biorbyt, UK), PKCnu antibody (rabbit polyclonal, #bs-4157R, Bioss Inc, USA), CCK1R antibody (rabbit polyclonal, #bs-11514R, Bioss Inc, USA), phospho-Akt antibody (rabbit monoclonal, #4060; Cell Signaling Technology), Akt antibody (rabbit monoclonal, #9272; Cell Signaling Technology), phospho-p44/42 MAPK (ERK1/2) antibody (rabbit monoclonal, #4370, Cell Signaling Technology, USA), p44/42 MAPK (ERK1/2) antibody (rabbit monoclonal, #4695, Cell Signaling Technology, USA), phospho-Smad2 antibody (rabbit monoclonal, #3108, Cell Signaling Technology, USA), Smad2 antibody (rabbit monoclonal, #5339, Cell Signaling Technology, USA), HSP60 antibody (rabbit monoclonal, #12165, Cell Signaling Technology, USA), RPLP0 antibody (rabbit polyclonal, #11290-2-AP, Proteintech,USA), GAPDH antibody (mouse monoclonal, #sc-32233, Santa-Cruz Biotechnology, USA), followed by horseradish peroxidase–conjugated secondary antibodies (Bethyl Laboratories, USA).

Techniques: Inhibition, Western Blot, Quantitative RT-PCR, Staining, Two Tailed Test

Journal: Theranostics

Article Title: LRG1 inhibition promotes acute pancreatitis recovery by inducing cholecystokinin Type 1 receptor expression via Akt.

doi: 10.7150/thno.110116

Figure Lengend Snippet: Figure 10. Mechanistic summary of the role of LRG1 in pancreatic injury and regeneration. In wild-type acinar cells, the binding of caerulein to CCK1R on the surface of acinar cells triggers a cascade of signaling events, including the activation of PKC, STAT3, and JNK pathways. This leads to acinar cell apoptosis and the production of pro-inflammatory cytokines such as TNF-α and IL-6, which amplify the inflammatory response initiated by the initial acinar cell injury. Concurrently, IL-6 signals through its receptor, IL-6R, to activate the transcription factor STAT3, which subsequently induces LRG1 expression in acinar cells. LRG1, in turn, antagonizes the TGFβRII/ALK5/AKT-mediated expression of CCK1R, a trophic factor for acinar cells, thereby limiting pancreatic regeneration. In Lrg1-/- acinar cells, the inhibitory effect of LRG1 on the TGFβRII/ALK5/AKT pathway is absent, resulting in elevated CCK1R expression compared to wild-type acinar cells. Consequently, more CCK1R is available to bind caerulein, leading to greater acinar cell damage. However, this higher CCK1R expression also promotes increased acinar cell proliferation and regeneration, explaining the accelerated pancreatic regeneration observed in Lrg1-/- mice despite the presence of more severe initial damage.

Article Snippet: Blots were probed with LRG1 antibody (rabbit monoclonal, #13224-1-AP, Proteintech,USA), phospho-PKC δ antibody (mouse monoclonal (#sc-365969, Santa-Cruz Biotechnology, USA), phospho-PKC epsilon antibody (rabbit polyclonal, #ab63387, Abcam, UK), PKC antibody (mouse monoclonal, #sc-17769, Santa-Cruz Biotechnology, USA), phospho-STAT3 (rabbit monoclonal, #9145, Cell Signaling Technology, USA), STAT3 (rabbit monoclonal, #12640, Cell Signaling Technology, USA), phospho-SAPK/JNK (rabbit monoclonal, #9255, Cell Signaling Technology, USA), SAPK/JNK (rabbit monoclonal, #9145, Cell Signaling Technology, USA), cleaved caspase 3 antibody (rabbit monoclonal, #9664, Cell Signaling Technology, USA), phospho-PKCnu antibody (rabbit polyclonal, #orb4440, Biorbyt, UK), PKCnu antibody (rabbit polyclonal, #bs-4157R, Bioss Inc, USA), CCK1R antibody (rabbit polyclonal, #bs-11514R, Bioss Inc, USA), phospho-Akt antibody (rabbit monoclonal, #4060; Cell Signaling Technology), Akt antibody (rabbit monoclonal, #9272; Cell Signaling Technology), phospho-p44/42 MAPK (ERK1/2) antibody (rabbit monoclonal, #4370, Cell Signaling Technology, USA), p44/42 MAPK (ERK1/2) antibody (rabbit monoclonal, #4695, Cell Signaling Technology, USA), phospho-Smad2 antibody (rabbit monoclonal, #3108, Cell Signaling Technology, USA), Smad2 antibody (rabbit monoclonal, #5339, Cell Signaling Technology, USA), HSP60 antibody (rabbit monoclonal, #12165, Cell Signaling Technology, USA), RPLP0 antibody (rabbit polyclonal, #11290-2-AP, Proteintech,USA), GAPDH antibody (mouse monoclonal, #sc-32233, Santa-Cruz Biotechnology, USA), followed by horseradish peroxidase–conjugated secondary antibodies (Bethyl Laboratories, USA).

Techniques: Binding Assay, Activation Assay, Expressing