Journal: Scientific Reports
Article Title: Small molecule targeting long noncoding RNA GAS5 administered intranasally improves neuronal insulin signaling and decreases neuroinflammation in an aged mouse model
doi: 10.1038/s41598-022-27126-6
Figure Lengend Snippet: Depletion of GAS5 reduces insulin receptor levels and insulin signaling in neurons. HT22 cells were transfected with GAS5 siRNA or scrambled control siRNA (Con siRNA) for 48 h. ( a ) RNA was isolated, qPCR was performed, normalized to β-actin expression and relative quantification (RQ) was determined for GAS5 and IR levels using control as reference (n = 3). Statistical analysis was performed by one-way ANOVA, ***p < 0.001. ( b ) Cell lysate was harvested, and western blot was performed using antibodies against IR, pAKT, AKT, pTau, Tau, pGSK3β, pGSK3α/β, GSK3α/β, and β-actin. Graph shows relative densitometric analysis of individual bands as indicated with phosphorylated protein normalized to total protein (n = 3). Statistical analysis was performed by two-tail Student’s t-test, ***p < 0.001. ( c ) HT22 cells were transfected with GAS5 siRNA for 24 h followed by treatment with 100 nM insulin for 24 h. Whole cell lysates were analyzed using automated WES with antibodies against IR, p-p85, p85, p110α, GAPDH and the virtual blots generated by WES software COMPASS are shown. The graph shows chemiluminescence peaks normalized to GAPDH generated by COMPASS (n = 3). Statistical analysis was performed by two-tail Student’s t-test, ***p < 0.001. ( d ) QPCR was performed, normalized to β-actin expression and relative quantification (RQ) determined for IL1β, IL6, GILZ, and AChE levels using control as reference (n = 3). Statistical analysis was performed by one-way ANOVA,**p < 0.01, ***p < 0.001, and ns not significant.
Article Snippet: Membranes were probed with pAKT (Ser 473, Cell Signaling #4058), AKT (Cell Signaling #2962), pGSK3β (Tyr 216/279, ThermoFisher #44-604G), GSK3β (Cell Signaling #9315), pGSK3α/β (Abcam #75,745), GSK3α/β (Abcam #131,356), pTau (Ser202, Cell Signaling #39,357), Tau DACO (Agilent A002401-2), pTau (AT8, Invitrogen MN1020B), Total tau (SantaCruz sc-32274) and β-Actin (Sigma #A3854).
Techniques: Transfection, Control, Isolation, Expressing, Western Blot, Generated, Software
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