Fig 1: Effects of ASO and eGLP1-ASO conjugates on gene expression and protein levels in vitro in cell lines and primary mouse islet cells.(A) MALAT1 RNA measured in GLP1R-HEK293 cells treated overnight with increasing concentrations of either MALAT1-ASO (○) or eGLP1-MALAT1-ASO (•), and data were normalized to MALAT1 expression in untreated cells (Ctrl). Data are plotted as mean ± SD of six replicates. (B) MALAT1 and FOXO1 transcripts measured in primary mouse islets treated overnight with 1 μM MALAT1-ASO and 1 μM FOXO1-ASO (open bars), 1 μM eGLP1-MALAT1-ASO, and 1 μM eGLP1-FOXO1-ASO (closed bars), as indicated in the graph. Data were normalized to corresponding expression in untreated islets (Ctrl). Representative data from one of three independent experiments. (C) MALAT1 RNA measured in human islets treated with 1 μM MALAT1-ASO (○) and 1 μM eGLP1-MALAT1-ASO (•) and compared with expression in untreated islets (x). (D) FOXO1 mRNA measured in mouse islets treated for 96 hours with 1 μM eGLP1-Ctrl-ASO (x), 1 μM FOXO1-ASO (□), or 1 μM eGLP1-FOXO1-ASO (▪). Data were normalized to FOXO1 expression measured in untreated islets (Ctrl) and plotted as dot plots for individual animals and geometrical mean ± 95% CI. (E) FOXO1 protein levels in the islets by Western blot for the untreated and islets treated with 1 μM eGLP1-FOXO1-ASO for 96 hours. (F) Relative protein levels were quantified by normalizing the band intensity to α-tubulin, and only one biological replicate per treatment was evaluated (therefore having no statistical analysis).
Fig 2: GLP1R-dependent uptake of ASO and knockdown of gene expression in mice treated with eGLP1-ASO conjugates in vivo.(A) Representative pancreatic sections stained for ASO by IHC and MALAT1 RNA by ISH from mice treated for 2 weeks with three subcutaneous injections of (a) saline, (b) MALAT1-ASO (1 μmol/kg), or (c) eGLP1-MALAT1-ASO (1 μmol/kg). (B) ASO uptake by IHC and MALAT1 RNA levels by ISH in pancreatic sections from mice treated for 2 weeks with three intravenous injections of (a) saline or (b) eGLP1-MALAT1-ASO (1 μmol/kg). (C) MALAT1 gene expression by ISH in (a) wild-type (WT) and (b) GLP1R knockout (KO) mice 72 hours after a single subcutaneous dose of saline or eGLP1-MALAT1-ASO (1 μmol/kg). (D) Pancreatic section from wild-type mice stained using fluorescence in situ probes for MALAT1 (purple, arrows), insulin (green), and glucagon (red). Pancreatic sections were collected 72 hours after one subcutaneous administration of (a) saline or (b) MALAT1-ASO and (c) eGLP1-control-ASO or (d) eGLP1-MALAT1-ASO, and all compounds were dosed at 1 μmol/kg. Scale bars, 200 μm. Islets are circled in blue in (A) to (C).
Fig 3: Representative confocal images of GLP1R and EEA1 following 30 min incubation with vehicle control (a), 100 nM exenatide (b), or 100 nM 127I2-eGLP1-34S15-ASO (c). Nuclei were counterstained with DAPI. Pearson’s correlation co-efficient for GLP1R colocalization with EEA1 was calculated from cytoplasmic ROIs (d). All analyses are presented as mean ± SEM for 3 independent experiments. For each condition, cells from at least 10 fields of view were analyzed from at least 2 technical replicates per experiment. Scale bar = 10 µm.
Fig 4: NanoSIMS images of GLP1R-HEK293 cells incubated for 30 min with 100 nM or 1 µM 127I2-eGLP1-34S15-ASO maleimide. (a) SIMS images of 100 nM incubation GLP1R-HEK293 cells,12C14N, 34S/13C12C (ASO), 127I/13C12C(GLP1). (b) SIMS images of 1 mM incubation GLP1R-HEK293 cells,12C14N, 34S/13C12C (ASO), 127I/13C12C(GLP1). (c) ROI analysis 100 nM incubation GLP1R-HEK293 cells. (d) ROI analysis 1mM incubation GLP1R-HEK293 cells.
Fig 5: Representative confocal images of GLP1R and LAMP1 following 30 min incubation with vehicle control (a), 100 nM exenatide (b), or 100 nM 127I2-eGLP1-34S15-ASO (c). Nuclei were counterstained with DAPI. Nuclei were counterstained with DAPI. Pearson’s correlation co-efficient for GLP1R colocalization with LAMP1 was calculated from cytoplasmic ROIs (d). All analyses are presented as mean ± SEM for 3 independent experiments. For each condition, cells from at least 10 fields of view were analyzed from at least 2 technical replicates per experiment. Scale bar = 10 µm.
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