Journal: Frontiers in Immunology

Article Title: The mammosphere-derived epithelial cell secretome modulates neutrophil functions in the bovine model

doi: 10.3389/fimmu.2024.1367432

Figure Lengend Snippet: Antibodies used for immunocytochemistry assays, fluorescent bead-based multiplex assays and Western blots.

Article Snippet: Treatments consisting of either RPMI + 2% FBS (control), MDEC CM, 20 ng/ml bovine granulocyte-macrophage colony-stimulating factor (GM CSF) or 50 ng/ml bovine insulin-like growth factor 2 (IGF2) (both from Kingfisher Biotech, and diluted in RPMI + 2% FBS), were added, and cells were incubated for 1 h at 37°C.

Techniques: Immunocytochemistry, Multiplex Assay, Western Blot, Control

Journal: Frontiers in Immunology

Article Title: The mammosphere-derived epithelial cell secretome modulates neutrophil functions in the bovine model

doi: 10.3389/fimmu.2024.1367432

Figure Lengend Snippet: Primers used for reverse transcriptase (RT)-PCR.

Article Snippet: Treatments consisting of either RPMI + 2% FBS (control), MDEC CM, 20 ng/ml bovine granulocyte-macrophage colony-stimulating factor (GM CSF) or 50 ng/ml bovine insulin-like growth factor 2 (IGF2) (both from Kingfisher Biotech, and diluted in RPMI + 2% FBS), were added, and cells were incubated for 1 h at 37°C.

Techniques: Reverse Transcription

Journal: Frontiers in Immunology

Article Title: The mammosphere-derived epithelial cell secretome modulates neutrophil functions in the bovine model

doi: 10.3389/fimmu.2024.1367432

Figure Lengend Snippet: Proteins in bovine mammosphere-derived epithelial cell (MDEC) conditioned medium (CM) affecting neutrophil functions, as detected by liquid chromatography-mass spectrometry.

Article Snippet: Treatments consisting of either RPMI + 2% FBS (control), MDEC CM, 20 ng/ml bovine granulocyte-macrophage colony-stimulating factor (GM CSF) or 50 ng/ml bovine insulin-like growth factor 2 (IGF2) (both from Kingfisher Biotech, and diluted in RPMI + 2% FBS), were added, and cells were incubated for 1 h at 37°C.

Techniques: Chromatography

Journal: Frontiers in Immunology

Article Title: The mammosphere-derived epithelial cell secretome modulates neutrophil functions in the bovine model

doi: 10.3389/fimmu.2024.1367432

Figure Lengend Snippet: Reducing Chemokine (C-X-C motif) ligand 6 (CXCL6) expression in bovine mammosphere-derived epithelial cells (MDECs) changes the effect of conditioned medium (CM) on neutrophil chemotaxis, while reducing insulin-like growth factor 2 (IGF2) expression in MDECs does not change the effect of conditioned medium (CM) on neutrophil phagocytosis. (A) . Recombinant bovine CXCL6 was added to bovine neutrophils to determine if chemotaxis changed relative to RPMI control medium (dotted line). Cells treated with MDEC CM were included as a control. (B) . CXCL6 siRNA was used to knock down CXCL6 in MDEC CM. Chemotaxis was assessed in neutrophils treated with RPMI control medium, MDEC CM, MDEC CM from cells transfected with CXCL6 siRNA, and MDEC CM from cells transfected with a non-specific (negative) siRNA. (C) . Recombinant bovine IGF2 was added to bovine neutrophils to determine if phagocytosis changed relative to the RPMI control (dotted line). Cells treated with MDEC CM were included as a control. (D) . IGF2 siRNA was used to knock down IGF2 in MDEC CM. Phagocytosis was assessed in neutrophils treated with RPMI control medium, MDEC CM, MDEC CM from cells transfected with IGF2 siRNA and MDEC CM from cells that received a non-specific (negative) siRNA. Each data point on graphs represents the results from one experiment, assessing the effects of CM from one MDEC line on 3 neutrophil preparations. Gray dotted lines on graphs indicate the control conditions expressed as 100%. n = 3 experiments.

Article Snippet: Treatments consisting of either RPMI + 2% FBS (control), MDEC CM, 20 ng/ml bovine granulocyte-macrophage colony-stimulating factor (GM CSF) or 50 ng/ml bovine insulin-like growth factor 2 (IGF2) (both from Kingfisher Biotech, and diluted in RPMI + 2% FBS), were added, and cells were incubated for 1 h at 37°C.

Techniques: Expressing, Derivative Assay, Chemotaxis Assay, Recombinant, Control, Knockdown, Transfection

Journal: PLOS One

Article Title: Cellular repressor of E1A-stimulated genes 1 enhances skeletal muscle performance through the stimulation of muscle differentiation and Akt-mTOR signaling pathway activation

doi: 10.1371/journal.pone.0328485

Figure Lengend Snippet: (A) Western blotting for CREG1 was performed using the serum (1 µL) of wild type (WT) and Tg mice. (B-C) The expression of CREG1 in the soleus muscles of WT and Tg mice was examined using RT-PCR and western blot analysis. Relative levels of Creg1 mRNA (B) and CREG1 protein (C) in the soleus muscles. (D) Relative levels of insulin-like growth factor 2 receptor (IGF2R), phosphorylated Akt Ser 473 (pAkt), total Akt (Akt), phosphorylated mechanistic target of rapamycin (pmTOR) Ser 2448 and total mTOR (mTOR) in the soleus muscles. Data were normalized to those of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). (E) Western blotting for IGF2R, pAkt, Akt, pmTOR, mTOR and GAPDH in the soleus muscles. Representative images are shown. Data are presented as mean ± SEM; n = 4–6 per group. A student's t -test was performed; * P < 0.05, versus WT.

Article Snippet: The membranes were blocked for 1 h at room temperature in nonfat dry milk and then incubated overnight at 4 °C with the following antibodies: anti-CREG1 (ab233282; Abcam, Cambridge, UK), anti-Akt Ser 473 [9271; Cell Signaling Technology (CST), Danvers, MA, USA], anti-Akt (9272; CST), anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (2118; CST), anti- insulin-like growth factor 2 receptor (IGF2R) (14364; CST), anti- mTOR Ser 2448 (2971; CST), anti- mTOR (2972; CST), and anti-α/β-tubulin (2148; CST).

Techniques: Western Blot, Expressing, Muscles, Reverse Transcription Polymerase Chain Reaction

Journal: Communications Biology

Article Title: Paradoxical regulation of IGF2 in promoting lipid metabolism in adipose tissues

doi: 10.1038/s42003-025-08458-1

Figure Lengend Snippet: A , B “U” and inverted “U” shaped relationship between IGF2 levels and the lipid species detected by LC-MS/MS assay utilizing a polynomial fourth order equation to fit the non-linear regression curve, where R² (R-squared) represents the coefficient of determination, and Sy.x represents the standard deviation of the residuals, n = 200. C , D “U” and inverted “U” shaped relationship between IGF2 levels and the lipid species detected by ELISA kit assay utilizing a polynomial fourth order equation to fit the non-linear regression curve, n = 200. E , F Pearson correlation analyses of L-IGF2 levels and H-IGF2 levels with triglyceride conducted by LC-MS/MS assay, n = 200, all p < 0.01. G , H Pearson correlation analyses of L-IGF2 levels and H-IGF2 levels with HDL-c conducted by LC-MS/MS assay, n = 200, all p < 0.05. I , J Pearson correlation analyses of L-IGF2 levels and H-IGF2 levels with triglyceride conducted by ELISA kit assay, n = 200, all p < 0.001. K , L Pearson correlation analyses of L-IGF2 levels and H-IGF2 levels with HDL-c conducted by ELISA kit assay, n = 200, all p < 0.05. M , N Multiple stepwise logistic regression analysis of MetS, HOMA-IR, and other metabolic subgroups (central obesity, hypertension, hyperglycemia, hypertriglyceridemia, and low HDL-c) connected with L-IGF2 and H-IGF2 levels conducted by LC-MS/MS assay and ELISA kit assay, respectively.

Article Snippet: The measurement of serum IGF2 level was conducted with an ELISA kit (E-EL-H6037 for human samples and E-EL-M3078 for mice samples, Elabscience, China).

Techniques: Liquid Chromatography with Mass Spectroscopy, Standard Deviation, Enzyme-linked Immunosorbent Assay

Journal: Communications Biology

Article Title: Paradoxical regulation of IGF2 in promoting lipid metabolism in adipose tissues

doi: 10.1038/s42003-025-08458-1

Figure Lengend Snippet: A The diagram of Ad-IGF2 and its control adenovirus were injected into the eWAT of DIO mice at multi-point (2 × 10 10 pfu/mouse, injected two times for a week, a total of 4 weeks of injection). B In vivo imaging was employed to track the expression sites of the overexpressing adenovirus constructs tagged with GFP, including Ad-IGF2 and Ad-GFP. C , D Representative western blot results of IGF2 protein levels in iWAT, eWAT, liver, skeletal muscle and pancreas tissues. E For a duration of 8 weeks, eWAT and iWAT of the mice were carefully dissected and visually documented using photography. F ELISA results of IGF2 protein levels in the serum. n = 6, ** p < 0.01 by two-tailed, unpaired Student’s t test. G – J The weight of eWAT and iWAT, liver and the whole-body weight of mice were compared, n = 6, * p < 0.05, n.s not significant (unpaired Student’s t test). K Hematoxylin/Eosin representative staining of adipocytes in iWAT, eWAT, prWAT, liver, and muscle tissue. Magnification: 20×. L – N Adipocyte size distribution curves of eWAT, iWAT and prWAT between Ad-GFP group mice (gray) and Ad-IGF2 group mice (blue), respectively. n = 6, Data represent the mean ± SD; * p < 0.05, ** p < 0.01, *** p < 0.001 analyzed by a one-way ANOVA with a Tukey’s multiple comparisons test. O , P The measurement analysis of blood glucose levels was conducted at specific times in obese mice after IGF2 overexpression by GTT and ITT assays, n = 6, * p < 0.05, analyzed by a two-way ANOVA with Bonferroni’s multiple comparisons test. Q , R The area under curve (AUC) analysis of GTT and ITT assays were conducted in obese mice after IGF2 overexpression, n = 6, *** p < 0.001 by two-tailed, unpaired Student’s t test. S , T Plasma TC, TG, HDL-c, LDL-c, ALT, AST, and ALP contents have been identified following injection of Ad-GFP and Ad-IGF2, n = 6, Data represent the mean ± SD; * p < 0.05 (unpaired Student’s t test).

Article Snippet: The measurement of serum IGF2 level was conducted with an ELISA kit (E-EL-H6037 for human samples and E-EL-M3078 for mice samples, Elabscience, China).

Techniques: Control, Injection, In Vivo Imaging, Expressing, Construct, Western Blot, Enzyme-linked Immunosorbent Assay, Two Tailed Test, Staining, Over Expression, Clinical Proteomics

Journal: Communications Biology

Article Title: Paradoxical regulation of IGF2 in promoting lipid metabolism in adipose tissues

doi: 10.1038/s42003-025-08458-1

Figure Lengend Snippet: A The diagram of IGF2-RNAi and its control lentivirus NC-RNAi were injected into the tail vein of 3-4-week-old mice (1 × 10 10 pfu/mouse, injected two times for a week, collectively a 4 weeks of injection). B Representative western blot results of IGF2 protein levels in liver tissues when 16 weeks old. C Representative western blot results of IGF2 protein levels in eWAT, iWAT, liver, skeletal muscle, pancreas and kidney tissues. D ELISA results of IGF2 protein level in serum samples, n = 12, *** p < 0.001 (unpaired Student’s t test). E At 16 weeks, liver tissues, iWAT, and eWAT of mice underwent dissection and photography. The representative images were depicted. F – J The whole-body weight, the liver, iWAT, eWAT ( n = 12) and quadriceps ( n = 6) weight of mice were compared, the p -values have been annotated, n.s not significant (unpaired Student’s t test). K Representative H&E staining of iWAT, eWAT, liver and muscle tissues, and the Oil Red staining of liver and muscle tissues. Magnification: 20×. L , M Adipocyte size distribution curves of eWAT and iWAT between NC-RNAi group mice (gray) and IGF2-RNAi group mice (yellow), respectively. n = 6, * p < 0.05, ** p < 0.01, *** p < 0.001 analyzed by a one-way ANOVA with a Tukey’s multiple comparisons test. N , O Plasma TC, TG, HDL-c, LDL-c, ALT, AST, and ALP contents have been identified following injection of IGF2-RNAi and NC-RNAi, n = 6, * p < 0.05, ** p < 0.01 (unpaired Student’s t test). P , R The levels of blood glucose underwent measurement at specific times by GTT and ITT assays, n = 6, * p < 0.05, ** p < 0.01 analyzed by a two-way ANOVA with Bonferroni’s multiple comparisons test. Q , S The area under curve (AUC) analysis of GTT and ITT assays were conducted in obese mice after IGF2 knockdown, n = 6, Data represent the mean ± SD; *** p < 0.001 by two-tailed, unpaired Student’s t test.

Article Snippet: The measurement of serum IGF2 level was conducted with an ELISA kit (E-EL-H6037 for human samples and E-EL-M3078 for mice samples, Elabscience, China).

Techniques: Control, Injection, Western Blot, Enzyme-linked Immunosorbent Assay, Dissection, Staining, Clinical Proteomics, Knockdown, Two Tailed Test

Journal: Communications Biology

Article Title: Paradoxical regulation of IGF2 in promoting lipid metabolism in adipose tissues

doi: 10.1038/s42003-025-08458-1

Figure Lengend Snippet: A The utilization of Nile red staining and Oil red O staining techniques revealed the presence of lipid droplets in 3T3-L1 adipocytes after IGF2 overexpression and knockdown treatment. B , C Quantification of cellular triglyceride (TG) content and the levels of free fatty acid (FFA) in the medium in 3T3-L1 adipocytes after IGF2 overexpression and knockdown treatment. * p < 0.05, ** p < 0.01. D , E The mRNA levels of genes associated with adipogenesis, lipogenesis, and lipolysis in 3T3-L1 cells transfected with Ad-IGF2 or IGF2-RNAi as well as the corresponding controls by RT-qPCR assays, using Ppia as internal controls, n = 6, Data represent the mean ± SD; * p < 0.05, ** p < 0.01, *** p < 0.001 by two-tailed, unpaired Student’s t test. F Protein expression levels of genes related to adipogenesis, lipogenesis and lipolysis in the 3T3-L1 adipocytes by western blot assays. G Mice adipose tissue has been dissected and isolated for primary adipocyte culture, and the results of 0, 4, 8 and 12 days were induced by the classic “Cocktail” induction differentiation regimen. H Primal cell supernatant was extracted during the above induction differentiation process, and IGF2 concentration was detected by ELISA kit. n = 3. I 3T3-L1 preadipocytes underwent treatment via different concentration gradients of recombinant IGF2 protein powder and induced differentiation. Oil red O staining employment demonstrated that the deposition of lipid droplets within adipocytes.

Article Snippet: The measurement of serum IGF2 level was conducted with an ELISA kit (E-EL-H6037 for human samples and E-EL-M3078 for mice samples, Elabscience, China).

Techniques: Staining, Over Expression, Knockdown, Transfection, Quantitative RT-PCR, Two Tailed Test, Expressing, Western Blot, Isolation, Concentration Assay, Enzyme-linked Immunosorbent Assay, Recombinant