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:

Journal: BioMed Research International

Article Title: Gene Expression Profiling of Clostridium botulinum under Heat Shock Stress

doi: 10.1155/2013/760904

Figure Lengend Snippet: The COG functional categorization of differentially expressed genes in response to heat shock stress in Clostridium botulinum ATCC 3502. The number in parentheses showed the total number of genes classified in each COG functional category in Clostridium botulinum ATCC 3502. The bar diagram represented the percentage of differentially expressed genes (including upregulated and down-regulated genes) relative to the total number of genes in each COG functional category. The number beside the bar diagram indicated the percentage of differentially expressed genes in each COG functional category relative to the total number of differentially expressed genes under heat shock stress.

Article Snippet: In addition, previous studies showed that C. botulinum ATCC 3502 alters the protein expression of botulinum toxin cluster genes ha-33 under temperature shift from 37°C to 45°C, indicating that ha-33 is a heat shock protein [ , ].

Techniques: Functional Assay

Journal: BioMed Research International

Article Title: Gene Expression Profiling of Clostridium botulinum under Heat Shock Stress

doi: 10.1155/2013/760904

Figure Lengend Snippet: The top 40 differentially expressed genes in response to heat shock stress in Clostridium botulinum ATCC 3502.

Article Snippet: In addition, previous studies showed that C. botulinum ATCC 3502 alters the protein expression of botulinum toxin cluster genes ha-33 under temperature shift from 37°C to 45°C, indicating that ha-33 is a heat shock protein [ , ].

Techniques: Modification, Membrane, Chemotaxis Assay, Transduction, Control