DOI: 10.5061/dryad.ns5qs
title: Data from: PTEN controls glandular morphogenesis through a juxtamembrane β-Arrestin1/ARHGAP21 scaffolding complex

description: PTEN controls three-dimensional (3D) glandular morphogenesis by coupling
 juxtamembrane signalling to mitotic spindle machinery. While molecular
 mechanisms remain unclear, PTEN interacts through its C2 membrane-binding
 domain with the scaffold protein β-Arrestin1. Because β-Arrestin1 binds
 and suppresses the Cdc42 GTPase-activating protein ARHGAP21, we
 hypothesize that PTEN controls Cdc42-dependent morphogenic processes
 through a β-Arrestin1-ARHGAP21 complex. Here we show that PTEN knockdown
 (KD) impairs β-Arrestin1 membrane localization, β-Arrestin1-ARHGAP21
 interactions, Cdc42 activation, mitotic spindle orientation and 3D
 glandular morphogenesis. Effects of PTEN-deficiency were phenocopied by
 β-Arrestin1 KD or inhibition of β-Arrestin1-ARHGAP21 interactions.
 Conversely, silencing of ARHGAP21 enhanced Cdc42 activation and rescued
 aberrant morphogenic processes of PTEN-deficient cultures. Expression of
 the PTEN C2 domain mimicked effects of full-length PTEN but a
 membrane-binding defective mutant of the C2 domain abrogated these
 properties. Our results show that PTEN controls multicellular assembly
 through a membrane-associated regulatory protein complex composed of
 β-Arrestin1, ARHGAP21 and Cdc42.
description: Figure 1B Membrane
 ARHGAP10 and β-Arrestin1 [ADU]). Experiments in
 triplicate.Data for Figure 1B in
 Excel file. Membrane ARHGAP10 and β-Arrestin1 [ADU]). Experiments in
 triplicate.Figure
 1b.xlsxFigure 1D Membrane
 β-Arrestin1 intensityData for Fig 1D in Excel
 file. Membrane β-Arrestin1 intensity (AI) in Caco-2 and Caco-2 ShPTEN cell
 monolayers after control or LPA treatment. Experiments in triplicate. In
 each replicate, signal intensity was assessed in 10 cells (C-L). Mean ±
 sem values were plotted.Figure
 1d.xlsxFigure 1F β-Arrestin1
 membrane (ADU) in Caco-2 and Caco-2 ShPTEN cells after control or LPA
 treatmentData for Fig 1F in Excel
 file. β-Arrestin1 membrane (ADU) in Caco-2 and Caco-2 ShPTEN cells after
 control or LPA treatment. Experiments in triplicate.figure
 1f.xlsxFigure 1 supplement
 2Data
 for Fig 1 Supplement 2 in Excel file. Arbitrary intensity values for
 membrane β-Arrestin1 after LPA treatment in PTEN+/+ and PTEN-/- HCT116
 cell monolayers. Confocal imaging conducted in 10 cells for each replicate
 (n=3) for each experimental condition.Figure 1 supplement
 4Data
 for Figure 1 Supplement 4 in Excel file. Membrane β-Arrestin1 ADU in
 PTEN+/+ and PTEN-/- HCT116 cells after LPA treatment. Experiments in
 triplicatefigure 1 supplement
 4.xlsxFigure
 2BData for Figure 2B in
 Excel file. CDC42-GTP ADU in Caco-2 cells after β-Arrestin1 or NT SiRNA.
 Experiments in triplicate.figure
 2b.xlsxFigure
 2DData for Figure 2D in
 Excel file. CDC42-GTP (ADU) in Caco-2ShPTEN cells after ARHGAP10 or NT
 SiRNA transfections. Experiments in triplicate.Figure
 2d.xlsxFigure 2 supplement
 1Data
 for Figure 2 Supplement 1 in Excel file. CDC42-GTP ADU in PTEN+/+ cells
 after NT or β-Arrestin SiRNA transfection. Experiments conducted in
 triplicate.Figure 2 supplement
 2Data
 for Figure 2 Supplement 2 in Excel file. CDC42-GTP ADU in PTEN-/- HCT116
 cells after NT or ARHGAP SiRNA transfections. Experiments conducted in
 triplicate.Figure 2 supplement
 3Data
 for Figure 2 Supplement 3 in Excel file. CDC42-GTP intensity (AI) in
 Caco-2 glands. Intensity was assessed in 10 randomly selected glands for
 each replicate (n=3) for each experimental condition.Figure 2 supplement
 4Data
 for Figure 2 supplement 4 in Excel file. Spindle angles in Caco-2 cells
 after NT or β-Arrestin1 SiRNA knock down. Spindle angles of 15 mitotic
 figures assessed for each experimental conditionFigure 2 supplement
 5Data
 for Figure 2 supplement 5 in Excel file. Caco-2 glands with single lumens
 after transfection. Assays in 50 glands per replicate (N=3). Results
 expressed as a percentage.figure 2 supplement
 5.xlsxFigure 2 supplement
 6Data
 for Figure 2 supplement 6 in Excel file. CDC42-GTP AI in Caco-2 ShPTEN
 glands. Assays in 10 glands for each replicate (n=3) in each experimental
 conditionFigure 2 supplement
 7Data
 for Figure 2 Supplement 7 in Excel file. Spindle angles in Caco-2 ShPTEN
 cells after NT or ARHGAP10 SiRNA transfection. Assays in 15 glands for
 each experimental condition.Figure 2 supplement
 8Data
 for Figure 2 supplement 8 in Excel file. Caco-2ShPTEN glands with single
 central lumens after transfection. 50 glands counted per replicate (n=3).
 Those with single lumens expressed as a percentage.figure 2 supplement
 8.xlsxFigure
 3BData for Figure 3B in
 Excel file. β-Arrestin1:ARHGAP binding ADU in Caco-2 or Caco-2 ShPTEN
 cells. Experiments in triplicate.figure
 3b.xlsxFigure
 3CData for Figure 3C in
 Excel file. β-Arrestin1:ARHGAP binding ADU in PTEN+/+ and PTEN-/- HCT116
 cellsfigure
 3c.xlsxFigure
 3EData for Figure 3E in
 Excel file. β-Arrestin1:ARHGAP10 binding ADU in Caco-2 ShPTEN cells after
 transfection. Experiments in triplicate.figure
 3e.xlsxFigure
 3GData for Figure 3G in
 Excel file. β-Arrestin1:ARHGAP10 binding ADU in PTEN-/- HCT116 cells.
 Experiments in triplicate.figure
 3g.xlsxFigure
 4BData for Figure 4B in
 Excel file. Membrane β-Arrestin1 (ADU) in Caco-2ShPTEN cells after
 transfection. Experiments in triplicate.figure
 4b.xlsxFigure
 4CData for Figure 4C.
 Membrane ARHGAP10 ADU in Caco-2ShPTEN cells. Experiments in
 triplicatefigure
 4c.xlsxFigure
 4EData for Figure 4E in
 Excel file. Membrane β-Arrestin1 (ADU) in PTEN-/-HCT116 cells after
 transfection. Experiments in triplicate.figure
 4e.xlsxFigure
 4FData for Figure 4F in
 Excel file. Membrane ARHGAP10 (ADU) in PTEN-/-HCT116 cells. Experiments in
 triplicate.figure
 4f.xlsxFigure
 4HData for Figure 4H in
 Excel file. Membrane β-Arrestin1:ARHGAP10 binding (ADU) in Caco-2 ShPTEN
 cells after transfection. Experiments in triplicate.figure
 4h.xlsxFigure
 4JData for Figure 4J in
 Excel file.Membrane β-Arrestin1:ARHGAP10 binding ADU in PTEN-/-HCT116
 cells. Experiments in triplicatefigure
 4J.xlsxFigure
 5BData for Figure 5B in
 Excel file. Membrane β-Arrestin1 intensity (AI) in Caco-2 ShPTEN glands
 after transfection. Intensity assessed in 10 glands for each replicate
 (n=3) for each transfection.figure
 5b.xlsxFigure
 5DData for Figure 5D in
 Excel file. Membrane ARHGAP10 intensity (AI) in Caco-2 ShPTEN glands after
 transfection. Intensity assessed in 10 glands for each replicate (n=3) for
 each transfection.figure
 5d.xlsxFIGURE
 5FData for Figure 5F in
 Excel file. Spindle angle in Caco-2 ShPTEN glands after transfection with
 EV or C2 or MCBR3 constructs. Spinlde angles of 15 mitotic figures per
 transfection.Figure 5 Supplement
 2Data
 for Figure 5 Supplement 2 in Excel file. Caco-2 ShPTEN glands with single
 central lumens. Lumens counted in 50 glands and edpressed as a
 percentagefigure 5 supplement
 2.xlsxFigure
 6BData for Figure 6B in
 Excel file. β-Arrestin1:ARHGAP10 binding ADU in Caco-2 cells after
 treatment. Experiments in triplicate.figure
 6b.xlsxFigure
 6DData for Figure 6D in
 Excel file. CDC42-GTP ADU in Caco-2 cells after treatment. Experiments in
 triplicate.figure
 6d.xlsxFigure
 6FData for Figure 6F in
 Excel file. β-Arrestin1:ARHGAP10 binding ADU in PTEN+/+ HCT116 cells after
 treatment. Experiments in triplicate.figure
 6f.xlsxFigure
 6HData for Figure 6H in
 Excel file. CDC42-GTP (ADU) in PTEN+/+ HCT116 cells after treatment.
 Experiments in triplicate.figure
 6h.xlsxFigure
 6JData for Figure 6J in
 Excel file. Spindle angles in Caco-2 glands after treatment. Spindle
 angles calculated for 15 mitotic figures per treatment.Figure
 6LData for Figure 6L in
 Excel file. Caco-2 glands with single central lumens after treatment.
 Lumens assessed in 50 glands and expressed as a
 percentagefigure
 6L.xlsxFigure
 7BData for Figure 7B in
 Excel file. Spindle angles in Organoids after treatment. Spindle angles
 calculated from 30 mitotic figures per treatment.Figure
 7b.xlsxFigure
 7CData for Figure 7C in
 Excel file. Organoids with single central lumen after treatment. In each
 replicate (n=3), in each experimental condition 10 organoids were studied
 and the number with single lumens were expressed as a percentage
 (%).Figure
 7c.xlsx