Journal: eLife

Article Title: PTEN controls glandular morphogenesis through a juxtamembrane β-Arrestin1/ARHGAP21 scaffolding complex

doi: 10.7554/eLife.24578

Figure Lengend Snippet: Values in PTEN -/- cells are expressed as fold change relative to HCT116 control - β-Arrestin1 = 0.78 ± 0.06; p=NS (0.06); ARHGAP21 = 1.53 ± 0.09; *p=0.03. Orange bars indicate PTEN -/- cells. 10.7554/eLife.24578.013 Figure 1—figure supplement 2—source data 1. . Beta-Arestin1 and ARHGAP21 expression in HCT116 clones -Source data.

Article Snippet: Furthermore, short tandem repeat (STR) profiling ( ) conducted by LGC Standards, Middlesex, UK confirmed authenticity by 100% and 94% matches, respectively, between study parental Caco-2 and HCT116 cells and original American Type Culture Collection (ATCC) derivatives.

Techniques: Control, Expressing, Clone Assay

Journal: eLife

Article Title: PTEN controls glandular morphogenesis through a juxtamembrane β-Arrestin1/ARHGAP21 scaffolding complex

doi: 10.7554/eLife.24578

Figure Lengend Snippet: Fold change of membrane β-Arrestin1 after LPA (+) or VO [vehicle only (-)] treatment; HCT116 control cells (+) vs (-)=1.62 ± 0.11; **p=0.01; PTEN -/- (+) vs PTEN -/- (-)=0.94 ± 0.11 vs 0.70 ± 0.10; p=NS; HCT116 (-) vs PTEN -/- (-); p=NS. Values are expressed as fold change relative to HCT116 (-) control. Membrane values were normalized against total lysate ADU values for each protein. 10.7554/eLife.24578.014 Figure 1—figure supplement 4—source data 1. LPA effects on Beta-Arrestin1 in HCT116 clones.

Article Snippet: Furthermore, short tandem repeat (STR) profiling ( ) conducted by LGC Standards, Middlesex, UK confirmed authenticity by 100% and 94% matches, respectively, between study parental Caco-2 and HCT116 cells and original American Type Culture Collection (ATCC) derivatives.

Techniques: Membrane, Control, Clone Assay

Journal: eLife

Article Title: PTEN controls glandular morphogenesis through a juxtamembrane β-Arrestin1/ARHGAP21 scaffolding complex

doi: 10.7554/eLife.24578

Figure Lengend Snippet: ( A,B ) SiRNA β-Arrestin1 knockdown (KD) suppresses Cdc42 - GTP in Caco-2 and HCT116 cells (fold changes = 0.62 ± 0.09;*p=0.03 and 0.51 ± 0.03;**p=0.005 respectively. ( C,D ) SiRNA ARHGAP21 KD enhances Cdc42-GTP in Caco-2 Sh PTEN (Sh PTEN ) and HCT116 PTEN -/- ( PTEN -/- ) cells (fold change = 1.41 ± 0.09 and 1.51 ± 0.11, respectively; *p=0.02 for each. Cdc42-GTP ADU was normalized against total Cdc42. ( E ) SiRNA β-Arrestin1 KD suppresses Cdc42-GTP signal intensity, impairs spindle orientation and inhibits single lumen formation. High-power (HP) spindle views (orange border) enlarge areas within white rectangles and show orientation angles (interrupted white arrows) of spindle planes (double-headed solid white arrows) toward gland centres (GCs). Normal spindle planes are orientated at approximately 90 0 angles relative to gland centres [GCs] . Summary SiRNA effects on spindle angles relative to GCs are shown in . ( F ) Summary SiRNA effects on Cdc42-GTP intensity shown in ( E ) - control vs β-Arrestin1 SiRNA = 24.67 ± 1.45 vs 14.33 ± 0.88 AI units; **p=0.004. β-Arrestin1 KD also suppresses single central lumen formation in 3D Caco-2 cultures (E; ). ( G ) SiRNA ARHGAP21 KD increases Cdc42 - GTP signal intensity ( H ), rescues spindle orientation ( G , ) and central lumen formation in Sh PTEN 3D cultures ( G , ). ( H ) Cdc42-GTP, control vs SiRNA ARHGAP21 KD in Sh PTEN cultures = 10.67 ± 0.67 vs=19.67 ± 0.88 AI units; **p<0.01. Assays at 4 days of culture. Imaging Cdc42-GTP [green], pericentrin ( PCN ) [red], α-Tubulin [green], ARHGAP21 [red], PRKCZ [red], β-Arrestin1 [red] and DAPI [blue]. All experiments conducted in triplicate. All analyses by paired Student’s t test. Scale bars 20 µm. Molecular weights indicated by arrows in blots. 10.7554/eLife.24578.020 Figure 2—source data 1. Source data for . 10.7554/eLife.24578.021 Figure 2—source data 2. Source data for . 10.7554/eLife.24578.022 Figure 2—source data 3. Source data for . 10.7554/eLife.24578.023 Figure 2—source data 4. Source data for .

Article Snippet: Furthermore, short tandem repeat (STR) profiling ( ) conducted by LGC Standards, Middlesex, UK confirmed authenticity by 100% and 94% matches, respectively, between study parental Caco-2 and HCT116 cells and original American Type Culture Collection (ATCC) derivatives.

Techniques: Knockdown, Control, Imaging

Journal: eLife

Article Title: PTEN controls glandular morphogenesis through a juxtamembrane β-Arrestin1/ARHGAP21 scaffolding complex

doi: 10.7554/eLife.24578

Figure Lengend Snippet: ( A ) β-Arrestin1-ARHGAP21 CoIPs in PTEN-expressing and -deficient cells. β-Arrestin1-associated ARHGAP21 shown in top panel against a constant β-Arrestin1 bait signal (second panel). IgG-negative controls. Total β-Arrestin1 and ARHGAP21 in lysates and GAPDH loading controls shown in lower three panels. ( B ) Summary β-Arrestin1-associated ARHGAP21 in PTEN-deficient (colored bars) vs PTEN-expressing cells (clear bars). Values normalized against total ARHGAP21 = 0.35 ± 0.01;**p<0.01 (Sh PTEN ) and 0.39 ± 0.02;**p<0.01 ( PTEN -/ - cells), respectively. ( C ) Schematic of GFP-labeled PTEN constructs used (top to bottom - wild type (wt) PTEN; PTEN - MCBR3 membrane binding mutant; catalytically inactive PTEN C124S; PTEN C124S - A4 (CS-A4) and PTEN C124S -T383A (CS-T383A) mutants that lack or retain β-Arrestin1 binding capacity, respectively , C2 and the C2-MCBR3 membrane binding mutant. ( D ) β-Arrestin1-associated ARHGAP21 in Sh PTEN cells after transfection with GFP-labeled-EV control vs - C2 or -C2-MCBR3 (top panel). β-Arrestin1 bait signal shown in second panel. Total β-Arrestin1 and ARHGAP21 in lysates shown in third and fourth panels. Expression of GFP-labeled C2, C2-MCBR3 and EV and GAPDH loading controls shown in two lowest panels. ( E ) Summary fold change of β-Arrestin1-associated ARHGAP21 vs EV control; C2 = 2.51 ± 0.08;**p<0.01 or C2-MBCR3 = 1.03 ± 0.06; p=NS. β-Arrestin1-associated ARHGAP21 normalized against total ARHGAP21 in lysate. ( F ) Proximity ligation assay (PLA) of β-Arrestin1 interactions with PTEN constructs (red fluorescence) in PTEN -/- cells. Top row - GFP-labeled CS-T383A, CS-A4, C2, C2-MCBR3; Bottom row - positive control - HCT116 cells; negative control - PTEN -/- cells transfected with GFP only. ( G ) β-Arrestin1-ARHGAP21 interactions. Top row PTEN -/- cells transfected with GFP-labeled full-length PTEN , - PTEN -MCBR3, -C2 and -C2-MCBR3; Bottom row - positive control - HCT116 cells; negative control - PTEN -/- cells transfected with GFP only. Scale bars - 20 µm; Molecular weights indicated by arrows in blots. 10.7554/eLife.24578.034 Figure 3—source data 1. Source data for . 10.7554/eLife.24578.035 Figure 3—source data 2. Source data for .

Article Snippet: Furthermore, short tandem repeat (STR) profiling ( ) conducted by LGC Standards, Middlesex, UK confirmed authenticity by 100% and 94% matches, respectively, between study parental Caco-2 and HCT116 cells and original American Type Culture Collection (ATCC) derivatives.

Techniques: Expressing, Labeling, Construct, Membrane, Binding Assay, Mutagenesis, Transfection, Control, Proximity Ligation Assay, Fluorescence, Positive Control, Negative Control

Journal: eLife

Article Title: PTEN controls glandular morphogenesis through a juxtamembrane β-Arrestin1/ARHGAP21 scaffolding complex

doi: 10.7554/eLife.24578

Figure Lengend Snippet: Vs EV control shown in ( F ); CS-T383A = 69.83 ± 3.67; CS-A4 = 9.62 ± 0.99; C2 = 38.3 ± 2.09; C2-MCBR3 = 8.66 ± 0.81 AI units;**p<0.01. EV control vs CSTA4 or C2-MCBR3 = NS. Positive control - PTEN:β-Arrestin1 interactions in HCT116 cells = 101 ± 4.9; negative controls - No antibody (2.12 ± 0.25 and PTEN -/- cells transfected with GFP-EV only = 3.21 ± 0.48). 10.7554/eLife.24578.037 Figure 3—figure supplement 4—source data 1. PLA analysis of PTEN:Beta-Arrestin1 interactions.

Article Snippet: Furthermore, short tandem repeat (STR) profiling ( ) conducted by LGC Standards, Middlesex, UK confirmed authenticity by 100% and 94% matches, respectively, between study parental Caco-2 and HCT116 cells and original American Type Culture Collection (ATCC) derivatives.

Techniques: Control, Positive Control, Transfection

Journal: eLife

Article Title: PTEN controls glandular morphogenesis through a juxtamembrane β-Arrestin1/ARHGAP21 scaffolding complex

doi: 10.7554/eLife.24578

Figure Lengend Snippet: C2 = 44.23 ± 1.92; C2-MCBR3 = 13.47 ± 0.88; wt PTEN = 63.7 ± 2.35; PTEN M-CBR3 = 18.53 ± 0.90 AI units; Positive control - β-Arrestin1:ARHGA21 interactions in HCT116 cells = 122.8 ± 3.8; negative controls - No antibody (0.39 ± 0.09 and PTEN -/- cells transfected with GFP-EV only = 7.58 ± 0.56). ***p<0.001; For C2 and wt PTEN vs EV (***) p<0.001; For PTEN-MCBR3 vs EV; (**) - p<0.01; For EV vs C2-MCBR3 and C2-MCBR3 vs PTEN-MCBR3, p=NS (n = 35 cells for each PLA experimental condition in triplicate). Red, blue and green bars - BRET assays. Statistical analyses were ANOVA with Bonferroni's multiple comparison for BRET assays and ANOVA with Tukey’s post hoc test for densitometry and PLA assays;**p<0.01, ***p<0.001. Molecular weights indicated by arrows in blots. 10.7554/eLife.24578.038 Figure 3—figure supplement 5—source data 1. PLA assay of Beta-Arrestine1:ARHGAP21 interactions.

Article Snippet: Furthermore, short tandem repeat (STR) profiling ( ) conducted by LGC Standards, Middlesex, UK confirmed authenticity by 100% and 94% matches, respectively, between study parental Caco-2 and HCT116 cells and original American Type Culture Collection (ATCC) derivatives.

Techniques: Positive Control, Transfection, Comparison

Journal: eLife

Article Title: PTEN controls glandular morphogenesis through a juxtamembrane β-Arrestin1/ARHGAP21 scaffolding complex

doi: 10.7554/eLife.24578

Figure Lengend Snippet: C2 = 0.76 ± 0.02; **p<0.01; MCBR3 = 1.04 ± 0.06; [MCBR3 vs control = NS]. 10.7554/eLife.24578.053 Figure 4—figure supplement 3—source data 1. Transfection effects on ARHGAP21 in PTEN-/- HCT116 cells.

Article Snippet: Furthermore, short tandem repeat (STR) profiling ( ) conducted by LGC Standards, Middlesex, UK confirmed authenticity by 100% and 94% matches, respectively, between study parental Caco-2 and HCT116 cells and original American Type Culture Collection (ATCC) derivatives.

Techniques: Control, Transfection

Journal: eLife

Article Title: PTEN controls glandular morphogenesis through a juxtamembrane β-Arrestin1/ARHGAP21 scaffolding complex

doi: 10.7554/eLife.24578

Figure Lengend Snippet: 10.7554/eLife.24578.079 Figure 6—figure supplement 2—source data 1. Effects of peptide binding inhibitor on Beta-arrestin1:ARHGAP21 interactions in HCT116 cells

Article Snippet: Furthermore, short tandem repeat (STR) profiling ( ) conducted by LGC Standards, Middlesex, UK confirmed authenticity by 100% and 94% matches, respectively, between study parental Caco-2 and HCT116 cells and original American Type Culture Collection (ATCC) derivatives.

Techniques: Binding Assay

Journal: eLife

Article Title: PTEN controls glandular morphogenesis through a juxtamembrane β-Arrestin1/ARHGAP21 scaffolding complex

doi: 10.7554/eLife.24578

Figure Lengend Snippet: pep24 vs control peptide treatment effects on Cdc42-GTP ADU = 0.44 ± 0.02; **p<0.01 in HCT116 cells.

Article Snippet: Furthermore, short tandem repeat (STR) profiling ( ) conducted by LGC Standards, Middlesex, UK confirmed authenticity by 100% and 94% matches, respectively, between study parental Caco-2 and HCT116 cells and original American Type Culture Collection (ATCC) derivatives.

Techniques: Control

Journal: eLife

Article Title: PTEN controls glandular morphogenesis through a juxtamembrane β-Arrestin1/ARHGAP21 scaffolding complex

doi: 10.7554/eLife.24578

Figure Lengend Snippet: 10.7554/eLife.24578.080 Figure 6—figure supplement 4—source data 1. - Peptide inhibitor treatment effects in Cdc42-GTP in HCT116 cells - source data

Article Snippet: Furthermore, short tandem repeat (STR) profiling ( ) conducted by LGC Standards, Middlesex, UK confirmed authenticity by 100% and 94% matches, respectively, between study parental Caco-2 and HCT116 cells and original American Type Culture Collection (ATCC) derivatives.

Techniques: