Journal: bioRxiv
Article Title: MiR-7a-Klf4 axis as a regulator and therapeutic target of neuroinflammation and ferroptosis in Alzheimer’s disease
doi: 10.1101/2025.03.24.644978
Figure Lengend Snippet: MiR-7a-Klf4 axis regulates microglial activation and neuroinflammation. ( A ) Relative Klf4 mRNA expression by qRT-PCR of N9 cells treated with increasing concentration of AβO (2.5, 5 and 10 μM) (n = 5 replicates). ( B ) Representative confocal images of Klf4 immunofluorescence in N9 cells treated with increasing concentration of AβO (2.5, 5 and 10 μM) and its quantification (n > 500 cells quantified for each group. Scale bar 200 μm). ( C ) Quantification of Klf4 protein expression in N9 cells treated with increasing concentration of LPS (100, 250 and 500 ng/mL) by immunofluorescence (n > 500 cells quantified for each group. Scale bar 200 μm). ( D ) Representative confocal images of Iba1 immunofluorescence for N9 cells transfected with miR-7a mimic and then challenged with AβO (Scale bar 200 μm). ( E ) Relative intracellular ROS levels detected by DCFDA fluorescence in N9 cells treated with miR-7a mimic and AβO (The bar denotes the normalized mean DCFDA fluorescence detected by well scan with > 80 data points. n = 4 biological replicates.). ( F ) qRT-PCR for NF-κB expression of N9 cells treated with miR-7a and AβO (n = 6). ( G ) Quantification of NF-κB protein expression by immunofluorescence (n > 500 cells quantified for each group). RT-PCR of IL6 and TNFα expression ( H ) and iNOS ( I ) of N9 cells treated with miR-7a and AβO (n = 3). ( J ) Quantification of iNOS protein expression by immunofluorescence (n > 500 cells quantified for each group). ( K ) Relative NO levels released to media of N9 cells treated with miR-7a and challenged by AβO, quantified by Griess assay (The bar denotes normalized mean absorbance at 540 nm. n = 4 replicates). ( L ) RT-PCR of NLRP3 expression of LPS primed N9 cells treated with miR-7a and challenged by AβO (n = 3 replicates). ( M ) Quantification of NLRP3 protein expression by immunofluorescence (n > 500 cells quantified for each group). ( N ) RT-qPCR of IL1β and Casp1 expression of LPS primed N9 cells treated with miR-7a and challenged AβO (n =3 and 6 replicates). o , Schematic of sandwich ELISA and quantification of TNFα, IL6 and IL1β released in the media of N9 cells treated with miR-7a and challenged with AβO (n= 6 biological replicates. Created with BioRender.com). Data are represented as mean ± s.e.m. All the statistics were performed using One-way ANOVA with Bonferroni post hoc test *p < 0.05).
Article Snippet: The cell media was subjected to sandwich ELISA for protein quantification of TNFα, IL6 and IL1β using Abclonal ELISA kit as per the manufacturers protocol.
Techniques: Activation Assay, Expressing, Quantitative RT-PCR, Concentration Assay, Immunofluorescence, Transfection, Fluorescence, Reverse Transcription Polymerase Chain Reaction, Griess Assay, Sandwich ELISA
