Fig. 4: V domain of PTPN23 is indispensable for sustaining cell survival via interaction with UBAP1.
From: PTPN23-dependent ESCRT machinery functions as a cell death checkpoint
a Left, representative immunofluorescence images of HeLa cells expressing eGFP-VPS4A (WT or E228Q), mCherry-CHMP4B, and PTPN23. VPS4A (WT or E228Q) cDNAs were transiently expressed in HeLa cells stably expressing mCherry-CHMP4B. Immunofluorescence staining was performed after 36 h of VPS4A transfection. Scale bar: 20 μm. Top right, the density plot of the shortest distance between CHMP4B or PTPN23 spots to eGFP-VPS4A-E228Q surfaces. Bottom right, volume percentages of CHMP4B or PTPN23 spots colocalized above the VPS4A-E228Q surface. The percentage of volume colocalized between PTPN23 and CHMP4B is 60.6 ± 2.3 (mean ± SEM). A total of 54 cells were analyzed, in 3 independent experiments. b Schematic depiction of domain-swapping experiments. c GFP competition growth assays measuring PTPN23 dependency on RN2 cell lines overexpressing indicated chimeric proteins (n = 3 independent experiments). Data are presented as mean ± SEM. d Immunoprecipitation assay to measure the binding between UBAP1 and WT PTPN23 or ALIX V domain-swapped PTPN23 (n = 3 independent experiments). e GFP competition growth assay of UBAP1 knockout in NOMO-1 cells (n = 3 independent experiments). Data are presented as mean ± SEM. f Scatter plot of PTPN23 versus UBAP1 gene knockout effect in 1086 cancer cell lines using DepMap 22Q2 Public dataset from Broad DepMap project. g Left, representative immunofluorescence images of eGFP-VPS4A (WT or E228Q), PTPN23 and UBAP1 in HeLa cells. Scale bar: 20 μm. Top right, density plot of the shortest distance from PTPN23 or UBAP1 to VPS4A-E228Q. Bottom right, volume percentage of PTPN23 or UBAP1 spots colocalized above the VPS4A-E228Q surface. The percentage of volume colocalized between PTPN23 and UBAP1 is 46.9 ± 3.8 (mean ± SEM). A total of 80 cells were analyzed, in 3 independent experiments.
