Figure 3
ATO induces PML intracellular reorganization and downregulation associated with p53 upregulation in human NPM1-mutated AML cells. PML (PG-M3) immunocytochemical staining pattern in NPM1-mutated (OCI/AML3 and IMS-M2) vs NPM1 wild-type (OCI/AML2 and U937) AML cell lines (A) and representative NPM1-mutated (n = 5, NPMc+) vs NPM1 wild-type (n = 4, NPMc−) primary AML patients cells (B). Pt. followed by number indicates patient code. (C) PML immunocytochemical staining pattern changes upon ATO/ATRA treatment in primary NPM1-mutated AML patient cells. AML cells from pt. 613 were treated either with vehicle alone (vehicle control) or different ATO concentrations (1 and 3 μM) for the time indicated, and cytospin preparations stained for PG-M3 (left). See panel B, pt. 613, for comparison with the basal PG-M3 staining pattern. AML cells from pts. 643, 644, 645, and 625 were either left untreated or treated with ATO (3 μM), ATRA (1 μM), and ATO plus ATRA for 24 hours, and cytospin preparations stained for PG-M3 (right). (A-C) APAAP technique; hematoxylin counterstaining. Images were collected using an Olympus B61 microscope and a UPlan FI ×100/1.3 NA oil objective; Camedia 4040, Dp_soft Version 3.2; and Adobe Photoshop 7.0. (D) Western blot analysis of PML protein expression upon ATO/ATRA treatment in 3 representative NPM1-mutated AML patients samples. p53 protein levels in untreated vs treated AML cells are shown for pts. 643 and 645. The same membranes were blotted for histone H3 for loading control. (E) PML (PG-M3, secondary goat anti-mouse Alexa-Fluor 488, green) and Sumo1 (Sumo1, secondary goat anti-rabbit Alexa-Fluor 568, red) immunofluorescence staining upon ATO treatment in 1 representative NPM1-mutated AML patient (upper). Nuclei are stained with DAPI (blue). White arrows indicate representative colocalization of Sumo1 and PML (yellow) in NBs. Images were collected by fluorescence microscope (Olympus, Shinjuku, Tokyo, Japan) using a ×100/1.3 NA oil objective and processed with CellSens Digital Imaging Software (Olympus) and Adobe Photoshop 7.0. Western blot analysis for p53 upon ATO treatment on AML cells from the same patient (pt. 613, lower). Histone H3 was used as loading control.

ATO induces PML intracellular reorganization and downregulation associated with p53 upregulation in human NPM1-mutated AML cells. PML (PG-M3) immunocytochemical staining pattern in NPM1-mutated (OCI/AML3 and IMS-M2) vs NPM1 wild-type (OCI/AML2 and U937) AML cell lines (A) and representative NPM1-mutated (n = 5, NPMc+) vs NPM1 wild-type (n = 4, NPMc−) primary AML patients cells (B). Pt. followed by number indicates patient code. (C) PML immunocytochemical staining pattern changes upon ATO/ATRA treatment in primary NPM1-mutated AML patient cells. AML cells from pt. 613 were treated either with vehicle alone (vehicle control) or different ATO concentrations (1 and 3 μM) for the time indicated, and cytospin preparations stained for PG-M3 (left). See panel B, pt. 613, for comparison with the basal PG-M3 staining pattern. AML cells from pts. 643, 644, 645, and 625 were either left untreated or treated with ATO (3 μM), ATRA (1 μM), and ATO plus ATRA for 24 hours, and cytospin preparations stained for PG-M3 (right). (A-C) APAAP technique; hematoxylin counterstaining. Images were collected using an Olympus B61 microscope and a UPlan FI ×100/1.3 NA oil objective; Camedia 4040, Dp_soft Version 3.2; and Adobe Photoshop 7.0. (D) Western blot analysis of PML protein expression upon ATO/ATRA treatment in 3 representative NPM1-mutated AML patients samples. p53 protein levels in untreated vs treated AML cells are shown for pts. 643 and 645. The same membranes were blotted for histone H3 for loading control. (E) PML (PG-M3, secondary goat anti-mouse Alexa-Fluor 488, green) and Sumo1 (Sumo1, secondary goat anti-rabbit Alexa-Fluor 568, red) immunofluorescence staining upon ATO treatment in 1 representative NPM1-mutated AML patient (upper). Nuclei are stained with DAPI (blue). White arrows indicate representative colocalization of Sumo1 and PML (yellow) in NBs. Images were collected by fluorescence microscope (Olympus, Shinjuku, Tokyo, Japan) using a ×100/1.3 NA oil objective and processed with CellSens Digital Imaging Software (Olympus) and Adobe Photoshop 7.0. Western blot analysis for p53 upon ATO treatment on AML cells from the same patient (pt. 613, lower). Histone H3 was used as loading control.

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