Journal: MedComm

Article Title: Leptin Neutralization Attenuates Inflammaging in Endothelial and Immune Cell Models

doi: 10.1002/mco2.71082

Figure Lengend Snippet: Expression of leptin receptor (LEP‐R) in senescent endothelial cells and differentiated macrophages. (A and B) Representative SA‐β‐Gal staining, along with the relative quantitation, of young and senescent endothelial cells. (C) mRNA expression levels of the senescence markers p16 and p21, of the SASP marker IL‐8, and of LEP‐R presented as fold change in senescent versus young cells. (D and E) Representative Western blots and the relative densitometric analyses of the senescence markers p16 and p21 and of the SASP proteins IL‐6 and IL‐8, as well as of LEP‐R, presented as fold change in senescent versus young cells and normalized using vinculin as internal control. MDA‐MB 231 cells were used as positive control for the expression of leptin receptor for both mRNA and protein assessment. (F) Amplification plots derived from real time PCR of leptin mRNA in endothelial cells (HUVEC), and in MDA‐MB‐231 cell line, used as positive control [ , ]; the vertical axis is in fluorescent units (ΔRn) while the horizontal axis is in PCR cycle numbers. (G–I) mRNA and protein expression levels of both leptin and LEP‐R in THP‐1 monocytes, induced or not to differentiate into macrophages through PMA treatment. MDA‐MB 231 cells were used as positive control for the expression of both leptin and LEP‐R for mRNA and protein assessment. Data are mean ± SD of three independent experiments. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001. For all comparisons, Student's t ‐test was used. EC: endothelial cells. PMA: phorbol‐12‐myristate‐13‐acetate. LEP‐R: leptin receptor.

Article Snippet: Human monocytic THP‐1 cells (ATCC) were cultured in RPMI‐1640 medium supplemented with 10% heat‐inactivated fetal bovine serum (FBS), 1% penicillin/streptomycin, and 1% l ‐glutamine (all from Euroclone).

Techniques: Expressing, Staining, Quantitation Assay, Marker, Western Blot, Control, Positive Control, Amplification, Derivative Assay, Real-time Polymerase Chain Reaction

Journal: MedComm

Article Title: Leptin Neutralization Attenuates Inflammaging in Endothelial and Immune Cell Models

doi: 10.1002/mco2.71082

Figure Lengend Snippet: Leptin neutralization attenuates hyperglycemia‐induced inflammation in macrophages. (A) Experimental design. (B) mRNA expression levels of PAI‐1, IL‐6, and IL‐1α, presented as fold changes over control, in macrophages exposed to hyperglycemia with or without leptin antagonist in the presence of leptin supplementation. (C and D) Representative Western blots and the relative densitometric analyses of IL‐6 and IL‐1β, presented as fold changes over control and normalized using vinculin as internal control, in the same experimental settings. (E) mRNA expression levels of IL‐1α, IL‐6, and MCP‐1 mRNAs presented as fold changes over control, in endothelial cells exposed to hyperglycemia with or without leptin antagonist in the presence of leptin supplementation. (F and G) Representative Western blots and the relative densitometric analyses of p16, p21, IL‐1β, IL‐6, IL‐8, and CXCL1, presented as fold changes over control and normalized using vinculin as internal control, in the same experimental settings. Data are mean ± SD of three independent experiments. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001. For all comparison, ANOVA followed by Holm–Šídák test was used. NG: normal glucose, that is 10 mM glucose; HG: high glucose, that is 30 mM glucose for macrophages and 25 mM glucose for endothelial cells; PMA: phorbol‐12‐myristate‐13‐acetate.

Article Snippet: Human monocytic THP‐1 cells (ATCC) were cultured in RPMI‐1640 medium supplemented with 10% heat‐inactivated fetal bovine serum (FBS), 1% penicillin/streptomycin, and 1% l ‐glutamine (all from Euroclone).

Techniques: Neutralization, Expressing, Control, Western Blot, Comparison