Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Macrophage migration inhibitory factor mediates joint capsule fibrosis via facilitating phospholipid metabolite PGE2 production in fibroblasts

doi: 10.1007/s00018-025-05800-y

Figure Lengend Snippet: Effects of CREB on the MIF-induced COX2 expression and PGE2 production. A , Western blot analysis of p-CREB following joint capsule fibroblasts treated with 2 µg/mL MIF for 0, 15, 30, 60, and 120 min. B , Quantitative results of A. C , Western blot analysis of p-CREB in joint capsule fibroblasts following siRNA2 knockdown of CD74 for 48 h and 2 µg/mL MIF stimulation for 15 min. D , Quantitative results of C. E , Evaluation of CREB knockdown efficiency using qRT-PCR after transfection for 48 h and siRNA1 was selected for subsequent experiments. F–H , Following siRNA1 knockdown of CREB for 48 h and 2 μg/ml MIF stimulation for 24 h, COX2 expression in joint capsule fibroblasts was assessed by qRT-PCR ( F ) and Western blot ( G ). Quantitative results of G as shown in ( H ). I and J , PGE2 production in supernatant ( I ) and lysate ( J ) of joint capsule fibroblasts were tested by ELISA accordingly. Each experiment was conducted three times. Error bars denote standard deviation. * P < 0.05 compared with Control group or 0 min group

Article Snippet: CREB gene silencing (RiboBio, Guangzhou, China) was similarly performed using different siRNA sequences (siRNA1, siRNA2, siRNA3) following the same procedures.

Techniques: Expressing, Western Blot, Knockdown, Quantitative RT-PCR, Transfection, Enzyme-linked Immunosorbent Assay, Standard Deviation, Control

Journal: Scientific reports

Article Title: Decreased expression of insulin-degrading enzyme increases gluconeogenesis and glucose production in cultured hepatocytes administered with glucagon.

doi: 10.1038/s41598-025-03790-2

Figure Lengend Snippet: Fig. 1. Effects of liver-specific depletion of IDE on the glucagon signaling pathway. Livers from fasted 3-month-old WT and L-IDE-KO mice were excised and protein lysates were prepared by homogenizing the tissues in lyses buffer. (A) Representative western blot of liver lysates (40 µg protein/sample) from WT (white bars) or L-IDEKO (black bars). (B) Densitometric analyses of the data in panel A of the glucagon receptor (GCGR), (C) p-CREB, (D) CREB and (E) the ratio p-CREB versus total CREB protein. Data are expressed relative to WT. Mean ± SEM for n = 4 independent experiments per genotype. *p value < 0.05 versus WT by Students´ T-test.

Article Snippet: TaqMan® Gene Expression assay references (from Applied Biosystems, USA) were as follows: Mm01247058_m1 for phosphoenolpyruvate carboxykinase (Pck1), Mm00839363_m1 for glucose-6 phosphatase (G6pc), Mm00433546_m1 for glucagon receptor (Gcgr) and Mm00501607_m1 for cAMP Response Element-Binding Protein (Creb1).

Techniques: Western Blot

Journal: Scientific reports

Article Title: Decreased expression of insulin-degrading enzyme increases gluconeogenesis and glucose production in cultured hepatocytes administered with glucagon.

doi: 10.1038/s41598-025-03790-2

Figure Lengend Snippet: Fig. 2. Effects of genetic depletion of IDE on glucagon signaling and expression of gluconeogenic genes in primary mouse hepatocytes. Primary hepatocytes isolated from fasted 3-month-old WT and L-IDE-KO mice were treated with glucagon (50 ng/mL) at the indicated times followed by quantification of protein levels of the glucagon signaling pathway. (A) Representative western blot (40 µg protein/sample) depicting WT (white bars) and L-IDE-KO (black bars) mouse primary hepatocytes treated with glucagon. Densitometric analyses of the data in panel A of the GCGR (B), p-CREB (C), CREB (D) and the ratio p-CREB versus total CREB protein (E). Data are expressed relative to WT. Mean ± SEM for n = 3 independent experiments per genotype. *p value < 0.05 versus WT by two-way ANOVA. #p value < 0.05 versus untreated cells (time 0) by two-way ANOVA. Primary hepatocytes isolated from WT and L-IDE-KO mice were treated with glucagon as above followed extraction and quantification of mRNA levels of Pck1 (F) and G6pc (G). Data are mean ± SEM (relative to control) for n = 3–6 independent experiments in triplicate per genotype and condition. *p value < 0.05 versus WT by Students’ T-test.

Article Snippet: TaqMan® Gene Expression assay references (from Applied Biosystems, USA) were as follows: Mm01247058_m1 for phosphoenolpyruvate carboxykinase (Pck1), Mm00839363_m1 for glucose-6 phosphatase (G6pc), Mm00433546_m1 for glucagon receptor (Gcgr) and Mm00501607_m1 for cAMP Response Element-Binding Protein (Creb1).

Techniques: Expressing, Isolation, Western Blot, Extraction, Control

Journal: Scientific reports

Article Title: Decreased expression of insulin-degrading enzyme increases gluconeogenesis and glucose production in cultured hepatocytes administered with glucagon.

doi: 10.1038/s41598-025-03790-2

Figure Lengend Snippet: Fig. 4. Effects of IDE deficiency on glucagon signaling and expression of gluconeogenic genes in a cell line of hepatocytes. AML12 cells were serum-starved for 18 h followed by incubation with glucagon (50 ng/mL) at the indicated times and the effects of Ide deficiency on glucagon signaling and expression of gluconeogenic genes were examined. (A) Representative western blots depicting control (white bars) and shRNA-IDE (black bars) hepatocytes treated with glucagon. Densitometric analysis of data in panel A for IDE (B), GCGR (C) and CREB (D). Data are mean ± SEM. n = 3 per group. #p value < 0.05 versus untreated cells (time 0) or *p value < 0.05 versus control cells by two-way ANOVA. Gene expression levels of Gcgr (E) or Creb1 (F). Data are mean ± SEM. n = 3 per group. #p value < 0.05 versus untreated cells (time 0) or *p value < 0.05 versus control cells by two-way ANOVA. (G) cAMP levels after 30 min of glucagon stimulation in control and IDE-deficient cells. Data are mean ± SEM. n = 3 per group. *p value < 0.05 versus control cells by Students´ T-test. (H) Representative western blots of p-PKA substrates for control and shRNA-IDE cells treated with glucagon (50 ng/mL) at the indicated times. (I) Densitometric analysis of data in panel H for p-PKA substrates. Data are mean ± SEM. n = 3 per group. #p value < 0.05 versus untreated cells (time 0) or *p value < 0.05 versus control cells by two-way ANOVA. (J) Densitometric analysis of data in panel A for p-CREB and the ratio p-CREB/CREB (K). Data are mean ± SEM. n = 3 per group. #p value < 0.05 versus untreated cells (time 0) or *p value < 0.05 versus control cells by two-way ANOVA. Gene expression levels of G6pc (L) or Pck1 (M). Data are mean ± SEM. n = 3 per group. #p value < 0.05 versus untreated cells (time 0) or *p value < 0.05 versus control cells by two-way ANOVA.

Article Snippet: TaqMan® Gene Expression assay references (from Applied Biosystems, USA) were as follows: Mm01247058_m1 for phosphoenolpyruvate carboxykinase (Pck1), Mm00839363_m1 for glucose-6 phosphatase (G6pc), Mm00433546_m1 for glucagon receptor (Gcgr) and Mm00501607_m1 for cAMP Response Element-Binding Protein (Creb1).

Techniques: Expressing, Incubation, Western Blot, Control, shRNA, Gene Expression

Journal: bioRxiv

Article Title: Dibutyryl cyclic AMP downregulates tenascin-C in neurons and astrocytes and reduces AAV-mediated gene expression in DRG neurons

doi: 10.1101/2025.05.13.653846

Figure Lengend Snippet: ( A ) Representative snapshots from Imaris software of DRGs visualized using the Zeiss Lightsheet Z.1 following ECi clearing. Scale bar: 400 µm. ( B ) Representative confocal images of DRG sections showing varying signal intensities in the presence and absence of db-cAMP. For intensity measurements, all stained sections were imaged using the same laser power, gain, and airy units. Scale bar: 400 µm. ( C ) Quantification of (A). Bar graphs display the total number of GFP-positive or V5-positive cells in the DRG. Data are presented as mean ± SEM, analysed by unpaired t-test, ns > 0.5, *p < 0.05, n = 6 DRGs/group. ( D ) Quantification of (B). Bar graphs show the total GFP-positive or V5-positive signal per DRG. Data are presented as mean ± SEM, analysed by unpaired t-test, **p < 0.01, ****p < 0.0001, n = 8 DRGs/group, with three sections evaluated per DRG to obtain the final value. ( E, F ) Immunoblotting results for GFP, PKA C-α, β-actin, and vinculin in DRG samples from animals injected with GFP only and those co-injected with db-cAMP 13 weeks post-injection. Values were normalised to vinculin. Two housekeeping proteins were utilised to enhance the reliability and accuracy of the results. Results are presented as mean density values ± SEM; n = 3 DRGs, with western blots repeated four times in separate experiments; ns > 0.5, *p < 0.05, **p < 0.01, analysed by unpaired t-test. ( G ) Heat maps illustrate Log 2 fold changes (as Log 2 (2^-ΔΔCt)) for the expression of Creb1 and Itg α 9 , normalised to the uninjured, uninjected control. Log 2 (2^-ΔΔCt) values were determined through qRT-PCR analysis. Results are presented as a heat map.

Article Snippet: For qPCR, TaqMan® gene expression assays (Life Technologies by Thermo Fisher Scientific, Waltham, MA, USA) were used for CREB1 (Rn00578828_g1), Itga9 (Rn01746751_m1) and GAPDH (Rn01775763_g1), all purchased from Applied Biosystems and used according to the manufacturer’s recommendations.

Techniques: Software, Staining, Western Blot, Injection, Expressing, Control, Quantitative RT-PCR