Journal: bioRxiv

Article Title: Physiological cerebrospinal fluid like medium reveals autophagy dependency of leukaemia in the central nervous system

doi: 10.64898/2026.03.09.709824

Figure Lengend Snippet: A, Schematic diagram of autophagy pathway and the mechanism of action of bafilomycin A1 to inhibit autophagy. B, C, Relative GFP/RFP ratio of mean fluorescence intensity (MFI) in SEM (C) and REH (D) cells expressing GFP-LC3-RFP-LC3ΔG autophagic flux probe cultured in RPMI, Plasmax (PL), and CSFmax (CSF) for 24 h with or without 50 nM of bafilomycin A1 treatment (n=7 independent experiments). D, Immunoblot analysis showing changes in autophagy markers in human CD19 + (huCD19 + ) SEM cells isolated from the bone marrow (BM) and the brain (CNS) (n=2 biological replicates). E, Percentage of apoptosis in SEM cells cultured in RPMI, Plasmax (PL), and CSFmax (CSF) for 24 h in normoxia (21% O 2 ) with or without 1 μM of MRT403 treatment (n=3 independent experiments). F, Percentage of apoptosis in SEM cells cultured in RPMI, Plasmax (PL), and CSFmax (CSF) for 24 h in normoxia (21% O 2 ) or hypoxia (1% O 2 ) with or without 50 nM of bafilomycin A1 treatment (n=3 independent experiments). G, Immunoblot analysis showing deletion of ULK1 and changes in autophagy markers in SEM cells cultured in RPMI. H, Relative GFP/RFP ratio of mean fluorescence intensity (MFI) in SEM vector control (CTRL) and ULK1 knockout (KO) single clone 1 and 2 cells expressing GFP-LC3-RFP-LC3ΔG autophagic flux probe cultured in RPMI, Plasmax (PL), and CSFmax (CSF) for 24 h (n=3 independent experiments). I, Immunoblot analysis showing deletion of ATG7 and changes in autophagy markers in SEM cells cultured in RPMI. J, Relative GFP/RFP ratio of mean fluorescence intensity (MFI) in SEM CTRL and ATG7 KO single clone 1 and 2 cells expressing GFP-LC3-RFP-LC3ΔG autophagic flux probe cultured in RPMI, Plasmax (PL), and CSFmax (CSF) for 24 h (n=3 independent experiments). Data are shown as the mean ± s.e.m. P-values were calculated with a repeated measure one-way ANOVA with Tukey’s multiple comparison test (B, C, E, F, H, J).

Article Snippet: Similarly, to generate stable ALL cell lines expressing the fluorescent probe GFP-LC3-RFP-LC3ΔG we used PMRX-IP-GFP-LC3-RFP which was a gift from Noboru Mizushima (Addgene Plasmid #84573) [ ].

Techniques: Fluorescence, Expressing, Cell Culture, Western Blot, Isolation, Plasmid Preparation, Control, Knock-Out, Comparison