Journal: Aging Cell

Article Title: MSC ‐Derived Exosomal lnc‐AGT‐3 : A Novel Anti‐Angiogenic Target in Age‐Related Macular Degeneration Through p53 Signaling Pathway

doi: 10.1111/acel.70377

Figure Lengend Snippet: lnc‐AGT‐3 specifically interacts with hnRNP K. (a) qRT‐PCR analysis of lnc‐AGT‐3 distribution relative to nuclear ( U6 ) and cytoplasmic ( β‐actin ) controls ( n = 3). (b) RNA‐FISH showing lnc‐AGT‐3 localization (Cy3, red) with nuclear ( U6 ) and cytoplasmic ( 18S rRNA ) controls. Nuclei counterstained with DAPI (scale bar = 20 μm). (c) lnc‐AGT‐3 ‐sense and lnc‐AGT‐3 ‐antisense RNAs were biotinylated, transcribed in vitro, and incubated with HUVEC total cell lysates for RNA pull‐down assays. After silver staining, lnc‐AGT‐3 ‐sense‐specific bands were excised and analyzed using mass spectrometry. (d) Western blot validation of hnRNP K binding to biotinylated lnc‐AGT‐3 from pull‐down assays. (e) RIP assays using hnRNP K antibody confirming RNA‐protein interaction ( n = 3). * p < 0.05 versus IgG; Student t test. (f) Co‐localization of lnc‐AGT‐3 (red, FISH) and hnRNP K (green, IF) in HUVECs (scale bar, 20 μm). (g) The relative expression of hnRNP K was determined in HUVECs after lnc‐AGT‐3 siRNA transfection by qRT‐PCR assays ( n = 3, * p < 0.05 vs. Scr siRNA, Student t test). (h) The relative expression of hnRNP K was determined in HUVECs after lnc‐AGT‐3 siRNA transfection by western blotting ( n = 4). (i) IB assays for the flag‐tagged hnRNP Ks (wild type and various constructed truncations) were performed by in vitro–transcribed, biotinylated lnc‐AGT‐3 .

Article Snippet: Cell lysates were incubated with hnRNP K antibody‐conjugated magnetic beads (Proteintech, 11,426–1‐AP, 5 μg) or control IgG (Merck, PP64B) at 4°C overnight.

Techniques: Quantitative RT-PCR, In Vitro, Incubation, Silver Staining, Mass Spectrometry, Western Blot, Biomarker Discovery, Binding Assay, Expressing, Transfection, Construct

Journal: Aging Cell

Article Title: MSC ‐Derived Exosomal lnc‐AGT‐3 : A Novel Anti‐Angiogenic Target in Age‐Related Macular Degeneration Through p53 Signaling Pathway

doi: 10.1111/acel.70377

Figure Lengend Snippet: lnc‐AGT‐3 influences the p53 pathway by means of hnRNP K. (a, b) qRT‐PCR analysis of p53 (a) and TSP1 (b) mRNA levels following hnRNP K knockdown (50 nM, 24 h; n = 3; * p < 0.05 vs. Scr siRNA, Student t test). (c, d) lnc‐AGT‐3 overexpression vector ( AGT ) or NC vector (Vector) was transfected into HUVECs. The mRNA levels of p53 and TSP1 in HUVECs with or without si‐ hnRNP K (50 nM) ( n = 3, * p < 0.05 vs. Vector + Scr siRNA group; # p < 0.05 between the marked group, “ns” represents no statistical significance; ANOVA with Bonferroni). (e) lnc‐AGT‐3 overexpression vector ( AGT ) or NC vector (Vector) was transfected into HUVECs. The protein levels of p53 and TSP1 in HUVECs with or without si‐ hnRNP K (50 nM) ( n = 4). (f) hnRNP K siRNA (si‐ hnRNP K ) (50 nM) or control siRNA (Scr siRNA) (50 nM) was transfected into HUVECs. IB of p53 ubiquitination in HUVECs was conducted with the treatment of 20 μM MG132 ( n = 4). (g) lnc‐AGT‐3 overexpression vector ( AGT ) or NC vector (Vector) was transfected into HUVECs. IB of p53 ubiquitination in HUVECs with or without si‐ hnRNP K (50 nM) was conducted after the treatment of 20 μM MG132 ( n = 4).

Article Snippet: Cell lysates were incubated with hnRNP K antibody‐conjugated magnetic beads (Proteintech, 11,426–1‐AP, 5 μg) or control IgG (Merck, PP64B) at 4°C overnight.

Techniques: Quantitative RT-PCR, Knockdown, Over Expression, Plasmid Preparation, Transfection, Control, Ubiquitin Proteomics