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: The Journal of Biological Chemistry

Article Title: The mRNA levels of heat shock factor 1 are regulated by thermogenic signals via the cAMP-dependent transcription factor ATF3

doi: 10.1074/jbc.RA119.012072

Figure Lengend Snippet: ATF3 binds to the HSF1 promoter. A, in silico analyses using the Genomatix and Promo software programs identified a putative ATF3/CREB-binding motif located in the proximal promoter region of HSF1 at −258 to −250 bp. B, EMSA assay using nuclear extracts from brown-like differentiated 10T1/2 cells treated with 10 μm forskolin for 4 h. Nuclear extracts incubated with biotin-labeled DNA probes corresponding to the putative ATF3-binding sequences generated a specific shifted band (lane 2). The shift was detected only in the presence of a probe containing the WT ATF3-binding sequence (probe WT), but not with a mutated one (probe Mut). Supershift was achieved by adding an anti-ATF3 antibody (lane 5) to the nuclear extract–probe mixture. For competition assays, a 100-fold excess of unlabeled WT probe was used. Specific complexes are indicated with arrows. C, ChIP assay was performed in brown-like differentiated cells using either anti-IgG or an anti-ATF3 antibody to assess occupancy at the putative ATF3 site in the HSF1 promoter treated with vehicle or forskolin at the concentration of 10 μm for 4 h. The results are expressed as means ± S.E. from three independent experiments. ***, p value < 0.001. n.s., not significant.

Article Snippet: The ATF3 expression plasmid was obtained from Origene (catalog no. MR201634); the pGL3 luciferase reporter vector (catalog no. E1751) and the pRL Renilla luciferase control reporter vector (catalog no. E2231) were purchased from Promega.

Techniques: In Silico, Software, Binding Assay, Incubation, Labeling, Generated, Sequencing, Concentration Assay

Journal: The Journal of Biological Chemistry

Article Title: The mRNA levels of heat shock factor 1 are regulated by thermogenic signals via the cAMP-dependent transcription factor ATF3

doi: 10.1074/jbc.RA119.012072

Figure Lengend Snippet: ATF3 is induced by cold exposure and pharmacological activation of β-adrenergic signaling in thermogenic tissues in vivo and by cAMP activators in vitro. A, mRNA levels of ATF/CREB family members in BAT and scWAT of 9-week-old male and female mice exposed to room temperature or cold for 6 h (n = 3). B, mRNA levels of ATF/CREB family members in BAT and scWAT of 9-week-old male and female mice treated for 3 h with saline or CL316,243 (1 mg/kg of body weight) resuspended in saline (n = 3). The dotted lines correspond to the levels of the control, set as 1. C, mRNA levels of ATF/CREB family in differentiated mouse SVF cells isolated from scWAT treated for 4 h with 10 μm forskolin. D, time course of the induction of mRNA levels of ATF/CREB family in brown-like differentiated 10T1/2 cells treated either with 10 μm forskolin, 10 μm isoproterenol, or 500 μm IBMX for the indicated time. The results are expressed as means ± S.E. from three independent experiments. *, p value < 0.01; **, p value < 0.005; ***, p value <0.001.

Article Snippet: The ATF3 expression plasmid was obtained from Origene (catalog no. MR201634); the pGL3 luciferase reporter vector (catalog no. E1751) and the pRL Renilla luciferase control reporter vector (catalog no. E2231) were purchased from Promega.

Techniques: Activation Assay, In Vivo, In Vitro, Isolation

Journal: The Journal of Biological Chemistry

Article Title: The mRNA levels of heat shock factor 1 are regulated by thermogenic signals via the cAMP-dependent transcription factor ATF3

doi: 10.1074/jbc.RA119.012072

Figure Lengend Snippet: ATF3 is required for the induction of HSF1 in response to elevation of cAMP levels. A, luciferase assays in HEK 293 cells expressing either a WT-HSF1-luc reporter vector containing the putative proximal ATF3 motif (WT promoter) or a HSF1 mutated luciferase reporter vector containing a mutated ATF3-binding site (MUT promoter) treated with vehicle or 10 μm forskolin for 4 h before harvesting. B, luciferase assay in HEK 293 cells co-transfected with either vector or ATF3 expression plasmid with either a WT (WT promoter) or a mutated HSF1-luc reporter promoter (MUT promoter) construct. C and D, RNA and protein levels of HSF1 and ATF3 in differentiated 10T1/2 brown-like cells expressing either vector, an ATF3 expression plasmid (C), a control siRNA (siLuc), or a siATF3 (D), treated with 10 μm forskolin for 2 h before harvesting. Vinculin was used as a loading control for Western blots. The results are expressed as means ± S.E. from three independent experiments. **, p value < 0.005; **, p value < 0.001. n.s., not significant.

Article Snippet: The ATF3 expression plasmid was obtained from Origene (catalog no. MR201634); the pGL3 luciferase reporter vector (catalog no. E1751) and the pRL Renilla luciferase control reporter vector (catalog no. E2231) were purchased from Promega.

Techniques: Luciferase, Expressing, Plasmid Preparation, Binding Assay, Transfection, Construct, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: The mRNA levels of heat shock factor 1 are regulated by thermogenic signals via the cAMP-dependent transcription factor ATF3

doi: 10.1074/jbc.RA119.012072

Figure Lengend Snippet: Requirement of the ATF3/HSF1 axis in the regulation of the brown fat marker UCP1. A and B, mRNA and protein levels of UCP1 in brown differentiated 10T1/2 cells after overexpression of ATF3 (A) or knockdown of ATF3 using siRNA (B). C, mRNA levels of UCP1 and HSF1 and protein levels of UCP1, HSF1, and ATF3 levels in differentiated 10T1/2 cells overexpressing ATF3 in the presence of either siRNA control (siLuc) or siHSF1. The results are expressed as means ± S.E. from three independent experiments. *, p value < 0.01; **, p value < 0.005; ***, p value <0.001.

Article Snippet: The ATF3 expression plasmid was obtained from Origene (catalog no. MR201634); the pGL3 luciferase reporter vector (catalog no. E1751) and the pRL Renilla luciferase control reporter vector (catalog no. E2231) were purchased from Promega.

Techniques: Marker, Over Expression

Journal: Ecotoxicology and environmental safety

Article Title: Effects of pesticide dichlorvos on liver injury in rats and related toxicity mechanisms.

doi: 10.1016/j.ecoenv.2025.117747

Figure Lengend Snippet: Fig. 4. IRGM mediates DDVP-induced damage in BRL-3A cells. (A) A CCK-8 assay was conducted after BRL-3A cells were treated with the specified concentration of DDVP for 12 h. (B) BRL-3A cells were exposed to 8 μM DDVP for the indicated durations for the CCK-8 assay. (C-D) The expression levels of ALT and AST were determined for each group. (E-F) Representative images illustrating ROS staining and the quantification of positive cells were acquired for every group. (G-H) Representative images of calcein-stained cells and an analysis of relative fluorescence intensity were performed for the cells in each group. (I) The expression levels of MDA and SOD in each group were measured using ELISA. Statistical comparisons were made using two-way ANOVA. (J) Western blot analysis was conducted to assess the expression of SLC7A11, P62, IRGM, and HO-1. Bar = 10 μm. Statistical comparisons were made using two-way ANOVA. * P < .05, * * P < .01, * ** P < .001, ns, not significant (P > .05).

Article Snippet: The anti-IRGM (Cat# 4543) antibody was purchased from ProSci, while the rabbit polyclonal monoclonal antibody against β-tubulin (Cat# B1031) was acquired from Biodragon.

Techniques: CCK-8 Assay, Concentration Assay, Expressing, Staining, Fluorescence, Enzyme-linked Immunosorbent Assay, Western Blot

Journal: Ecotoxicology and environmental safety

Article Title: Effects of pesticide dichlorvos on liver injury in rats and related toxicity mechanisms.

doi: 10.1016/j.ecoenv.2025.117747

Figure Lengend Snippet: Fig. 6. DDVP treatment downregulates hepatic IRGM expression and induces hepatocyte apoptosis. (A) A heatmap illustrating differential gene expression in the apoptosis pathway. (B) Images representing TUNEL staining in liver tissue sections, accompanied by the quantification of TUNEL-positive cells, were determined by counting the TUNEL-positive cells in every area. (C) Western blot analysis of BAX and BCL2 expression. Statistical comparisons were made using two-way ANOVA. (D) Representative immunofluorescence images and quantification of cleaved caspase-3 staining (green) and DAPI (blue) in liver tissue sections. (E) A Venn diagram depicting overlapping significantly differentially expressed genes. (F) qPCR analysis of IRGM mRNA expression levels. (G) Western blot analysis of IRGM protein expression levels. Statistical comparisons were made using the two-tailed Student’s t -test. * P < .05, * * P < .01.

Article Snippet: The anti-IRGM (Cat# 4543) antibody was purchased from ProSci, while the rabbit polyclonal monoclonal antibody against β-tubulin (Cat# B1031) was acquired from Biodragon.

Techniques: Expressing, Gene Expression, TUNEL Assay, Staining, Western Blot, Immunofluorescence, Two Tailed Test

Journal: Ecotoxicology and environmental safety

Article Title: Effects of pesticide dichlorvos on liver injury in rats and related toxicity mechanisms.

doi: 10.1016/j.ecoenv.2025.117747

Figure Lengend Snippet: Fig. 7. AAV8-mediated overexpression of IRGM mitigates DDVP-induced liver injury and incomplete autophagy. (A) Observations were conducted via transmission electron microscopy at magnifications of 6000 × (top, scale bar = 5 µm) and 15000 × (bottom, scale bar = 2 µm). Autophagosomes (highlighted by yellow arrows) are visible in the cells. (B) Haemotoxylin and eosin staining analysis is shown (scale bar = 100 µm). (C) A representative immunofluorescence confocal image il lustrates LC3/P62-positive puncta (green) alongside DAPI (blue). (D-E) Quantitative analysis of the number of LC3-positive and P62-positive spots per cell was performed via ImageJ. (F, K, L) Western blot analysis was conducted to assess the expression of IRGM, P62, BAX, BCL2, NRF2, and HO-1. a represents P < .05 compared with the AAV8-NC+Vehicle group; b represents P < .05 compared with the AAV8-NC+DDVP group; c represents P < .05 compared with the AAV8- IRGM+Vehicle group. Statistical comparisons were made using two-way ANOVA. (G, I) Representative images of TUNEL-stained liver tissue sections, along with quantification of positive cells, as calculated by counting TUNEL-positive cells in each area. (H, J) Representative immunofluorescence images and quantification of cleaved caspase-3 staining (green) and DAPI (blue) in liver tissue sections. * P < .05, * * P < .01, * ** P < .001, ns, not significant (P > .05). Group sizes: AAV8- NC+Vehicle (n = 6 rats), AAV8-NC+DDVP (n = 6), AAV8-IRGM+Vehicle (n = 8), AAV8-IRGM+DDVP (n = 8).

Article Snippet: The anti-IRGM (Cat# 4543) antibody was purchased from ProSci, while the rabbit polyclonal monoclonal antibody against β-tubulin (Cat# B1031) was acquired from Biodragon.

Techniques: Over Expression, Transmission Assay, Electron Microscopy, Staining, Immunofluorescence, Western Blot, Expressing, TUNEL Assay

Journal: Journal of Chromatography. B, Analytical Technologies in the Biomedical and Life Sciences

Article Title: Plasma proteomics of pancreatic cancer patients by multi-dimensional liquid chromatography and two-dimensional difference gel electrophoresis (2D-DIGE): Up-regulation of leucine-rich alpha-2-glycoprotein in pancreatic cancer

doi: 10.1016/j.jchromb.2007.01.029

Figure Lengend Snippet: Elevated level of plasma LRG in pancreatic cancer. The localizations of the five LRG spots are indicated by arrows 1–5 on the 2D image of the 150 mM NaCl sample (A). The boxed area was transferred to a nitrocellulose membrane and scanned with a laser scanner to obtain the LRG spots on the membrane (B). The scanned membrane was reacted with an anti-LRG antibody and the antibody–antigen complexes were detected with an ECL system (C). The fluorescent signals of the LRG spots on the 2D-PAGE gels were compared between non-cancer bearing healthy donors (D) and pancreatic cancer patients (E). LRG levels in individuals were examined by SDS-PAGE/Western blotting with an anti-LRG antibody (F). N1–N10: plasma samples from non-cancer bearing healthy donors; P1–P10: plasma samples from pancreatic cancer patients. After the ECL reaction, the membrane was stripped and stained with Coomassie Brilliant Blue (CBB) (F).

Article Snippet: Localization of the leucine-rich alpha-2-glycoprotein (LRG) was examined by specific antibodies against LRG (500× dilution; Abnova Co. Ltd., Taipei City, Taiwan).

Techniques: Clinical Proteomics, Membrane, SDS Page, Western Blot, Staining

Journal: Journal of Chromatography. B, Analytical Technologies in the Biomedical and Life Sciences

Article Title: Plasma proteomics of pancreatic cancer patients by multi-dimensional liquid chromatography and two-dimensional difference gel electrophoresis (2D-DIGE): Up-regulation of leucine-rich alpha-2-glycoprotein in pancreatic cancer

doi: 10.1016/j.jchromb.2007.01.029

Figure Lengend Snippet: LRG levels of plasma samples from validation set 2 examined by SDS-PAGE/Western blotting with an anti-LRG antibody. N11–N15: samples from non-cancer bearing healthy donors; PT1–PT4: samples from chronic pancreatitis patients; P11–P18: samples from pancreatic cancer patients. Following the ECL reaction, the membrane was stripped and stained with Coomassie Brilliant Blue (CBB) (A). The ECL signals for the LRG proteins were normalized to the intensity of the CBB-stained membrane, and the relative LRG signal was calculated (B). N: non-cancer bearing healthy donors, PT: chronic pancreatitis patients, P: pancreatic cancer patients. Gray rhombuses indicate individual data. Black circles and bars indicate data average and standard error for each group, respectively. Comparisons between means were performed using Student's t -test; there were no significant differences.

Article Snippet: Localization of the leucine-rich alpha-2-glycoprotein (LRG) was examined by specific antibodies against LRG (500× dilution; Abnova Co. Ltd., Taipei City, Taiwan).

Techniques: Clinical Proteomics, Biomarker Discovery, SDS Page, Western Blot, Membrane, Staining