phosphorylated adenosine monophosphate activated protein kinase  (Cell Signaling Technology Inc)

Journal: iScience

Article Title: A stress-induced PI3P complex controls autophagy in erythroid precursors

doi: 10.1016/j.isci.2026.115182

Figure Lengend Snippet: SAR405 inhibits autophagy during erythropoiesis (A) RT-qPCR in WT murine splenic erythroblast cells (Lineage − CD71 + ) 3 days after acute hemolytic anemia. N = 3 mice per group. Error bars represent standard error of mean (SEM). ∗∗∗ p < 0.001 (two-tailed unpaired Student’s t test). (B) RT-qPCR in murine splenic erythroblasts (lineage − CD71 + Kit + ) cells isolated from control (WT) or Berkeley Sickle Cell Disease (SCD) mice ( N = 3 per genotype). Error bars represent standard error of mean (SEM). ∗ p < 0.05; ∗∗ p < 0.01 (two-tailed unpaired Student’s t test). Target mRNA expression levels were normalized to 18S rRNA. (C) Western blotting of Lineage − CD71 + cells isolated from control (PBS-treated) and anemia (PHZ-treated) spleens for autophagy-related proteins. N = 3 WT mice per experimental group. (D) Semi-quantitative densitometry of p -Ampk (Thr172) relative to total Ampk and p -eIF2α (Ser51) relative to total eIF2α ( N = 3 mice). Error bars represent standard error of mean (SEM). ∗ p < 0.05; ∗∗∗ p < 0.001 (two-tailed unpaired Student’s t test). (E) Experimental layout of splenic EPCs isolated from Samd14 fl/fl (WT) and Samd14 fl/fl;Vav−Cre (S14-CKO) mice post PHZ-induced anemia. EPCs were retrovirally infected to express the GFP-LC3-RFP probe (Addgene #84573). After 24 h, cells were treated with VPS34 inhibitor SAR405 for an additional 48 h before analysis by flow cytometry. (F) Schematic representation of autophagic flux measurement using GFP-LC3-RFP probe. (G) Gating strategy for GFP and RFP signal measurement by flow cytometry. Progressively maturing erythroid cells (R1-R4) were gated within a viable RFP-expressing cell population using cell surface markers CD71 and TER119. (H) Quantitation of GFP/RFP median fluorescence intensity ratio calculated within R1 (CD71 low/med TER119 - ), R2 (CD71 high TER119 - ), R3 (CD71 high TER119 + ), and R4 (CD71 low/neg TER119 + ) populations in DMSO or SAR405-treated WT cultures ( n = 3 mice). Error bars represent standard error of mean (SEM). ∗∗∗ p < 0.001 (One-way ANOVA). (I) Quantitation of GFP/RFP median fluorescence intensity ratio calculated within R2 (CD71 high TER119 - ) and R4 (CD71 low/neg TER119 + ) populations in DMSO or SAR405-treated WT and S14-CKO cultures ( n = 3 mice per group). Error bars represent standard deviation (SD). ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001 (two-way ANOVA).

Article Snippet: Anti-phospho-AMPKα (Thr172) (clone 40H9, rabbit monoclonal) , Cell Signaling Technology , Cat.: #2535; RRID: AB_331250.

Techniques: Quantitative RT-PCR, Two Tailed Test, Isolation, Control, Expressing, Western Blot, Infection, Flow Cytometry, Quantitation Assay, Fluorescence, Standard Deviation