Journal: Cell Insight

Article Title: Cortactin-dependent control of Par1b-regulated epithelial cell polarity in Helicobacter infection

doi: 10.1016/j.cellin.2024.100161

Figure Lengend Snippet: Characterization of cortactin gene knockout in intestinal Caco-2 cells by CRISPR-Cas9. ( A ) Confirmation of cortactin gene knockout in Caco-2Δ cttn cells by fluorescence microscopy using cortactin-specific antibodies (green) and detecting the functional HDR plasmid, expressing RFP (red). Immunostaining of ZO-1 and occludin served as controls. ( B ) Western blot showing a ∼85 kDa protein corresponding to cortactin in Caco-2 wt cells, but not in Caco-2Δ cttn mutant clones 2 and 6. Western blotting with antibodies specific for ZO-1, E-cadherin, Claudin-5, ASPP2 and β-actin served as control. ( C ) Phase-contrast microscopy of representative Caco-2 wt and Caco-2Δ cttn cell lines showing cellular morphologies within respective monolayer. Quantification of cell ( D ) and nuclear ( E ) areas based on F-actin and nuclear staining, respectively, displaying arithmetic means ± SD (standard deviation) as well as individual values (dots). The size differences between Caco-2 wt and Caco-2Δ cttn cells were confirmed to be statistically significant with p ​< ​0.001 (∗∗∗).

Article Snippet: Membranes were blocked with either 5% non-fat milk or 3% BSA and probed with the primary antibodies as follows: mouse α-cortactin (#05–180, Merck-Millipore), rabbit α-Par1b (#HPA074905, Sigma Aldrich), rabbit α-ZO-1 (#61–73000, Invitrogen), rabbit α-E-cadherin (#sc-7870, Santa Cruz), rabbit α-Claudin-5 (#ab15106, Abcam), mouse α-β-actin (#A5441, Sigma Aldrich), mouse α-GFP (#632381, Clontech), rabbit α-omni-probe (α-T7) (#sc-499, Santa Cruz), rabbit α-CagA (#HPP-5003-9, Austral Biologicals), mouse α-PY99 (#sc-7020, Santa Cruz) and α-ASPP2 (#200-401-A19, Rockland).

Techniques: Gene Knockout, CRISPR, Fluorescence, Microscopy, Functional Assay, Plasmid Preparation, Expressing, Immunostaining, Western Blot, Mutagenesis, Clone Assay, Control, Staining, Standard Deviation

Journal: Cell Insight

Article Title: Cortactin-dependent control of Par1b-regulated epithelial cell polarity in Helicobacter infection

doi: 10.1016/j.cellin.2024.100161

Figure Lengend Snippet: H. pylori CagA promotes complex formation of cortactin with ZO-1 . ( A ) Fluorescence microscopy of Caco-2 wt cell monolayer cross-sections (X/Z dimension) without or after infection with H. pylori wt or Δ virB10 mutant. The white dashed lines indicate apical and basal surfaces of the monolayers. Yellow arrows indicate abnormal localization of ZO-1. ( B ) Western blot analysis of protein complex formation in Caco-2 wt cells after H. pylori infection (MOI 100) for 6 h by IP using α-ZO-1 antibodies. Proteins co-immunoprecipitated with ZO-1 were probed using antibodies against cortactin, CagA, β-actin and ASPP2. ( C ) The band intensities of cortactin, β-actin and CagA immunoprecipitated in a complex with ZO-1 were quantified and expressed as relative, protein complex-bound cortactin, β-actin or CagA. Mean intensities ​± ​SD are presented; p ​< ​0.001 (∗∗∗).

Article Snippet: Membranes were blocked with either 5% non-fat milk or 3% BSA and probed with the primary antibodies as follows: mouse α-cortactin (#05–180, Merck-Millipore), rabbit α-Par1b (#HPA074905, Sigma Aldrich), rabbit α-ZO-1 (#61–73000, Invitrogen), rabbit α-E-cadherin (#sc-7870, Santa Cruz), rabbit α-Claudin-5 (#ab15106, Abcam), mouse α-β-actin (#A5441, Sigma Aldrich), mouse α-GFP (#632381, Clontech), rabbit α-omni-probe (α-T7) (#sc-499, Santa Cruz), rabbit α-CagA (#HPP-5003-9, Austral Biologicals), mouse α-PY99 (#sc-7020, Santa Cruz) and α-ASPP2 (#200-401-A19, Rockland).

Techniques: Fluorescence, Microscopy, Infection, Mutagenesis, Western Blot, Immunoprecipitation

Journal: Cell Insight

Article Title: Cortactin-dependent control of Par1b-regulated epithelial cell polarity in Helicobacter infection

doi: 10.1016/j.cellin.2024.100161

Figure Lengend Snippet: H. pylori deregulates the host proteins cortactin, Par1b and ZO-1 in human mucosoids via CagA. ( A ) Fluorescence microscopy of human mucosoids after 6 h infection with either H. pylori wt or Δ cagA mutant. Polarized mucosoids were cultured in a transwell system followed by fixation in PFA and staining with phalloidin to visualize F-actin structures. ( B ) Immunofluorescence microscopy of mucosoid cross-sections (X/Z dimension) after 6 h infection with either H. pylori wt or Δ cagA mutant. ( C ) Z-profiles of Par1b, cortactin, F-actin and DAPI fluorescence from the mucosoid cross-section of representative cells (indicated by yellow arrows in panel B ) show the basal and apical distributions of proteins; a.u. – arbitrary units ( D - E ) Human mucosoids were infected with H. pylori wt or either Δ cagA or Δ virB7 mutants. The samples were immunoprecipitated with α-ZO-1 ( D ) or α-Par1b ( E ) antibodies, and analyzed by Western blotting using antibodies against ZO-1, Par1b, cortactin, CagA and ASPP2.

Article Snippet: Membranes were blocked with either 5% non-fat milk or 3% BSA and probed with the primary antibodies as follows: mouse α-cortactin (#05–180, Merck-Millipore), rabbit α-Par1b (#HPA074905, Sigma Aldrich), rabbit α-ZO-1 (#61–73000, Invitrogen), rabbit α-E-cadherin (#sc-7870, Santa Cruz), rabbit α-Claudin-5 (#ab15106, Abcam), mouse α-β-actin (#A5441, Sigma Aldrich), mouse α-GFP (#632381, Clontech), rabbit α-omni-probe (α-T7) (#sc-499, Santa Cruz), rabbit α-CagA (#HPP-5003-9, Austral Biologicals), mouse α-PY99 (#sc-7020, Santa Cruz) and α-ASPP2 (#200-401-A19, Rockland).

Techniques: Fluorescence, Microscopy, Infection, Mutagenesis, Cell Culture, Staining, Immunofluorescence, Immunoprecipitation, Western Blot

Journal: Cell Insight

Article Title: Cortactin-dependent control of Par1b-regulated epithelial cell polarity in Helicobacter infection

doi: 10.1016/j.cellin.2024.100161

Figure Lengend Snippet: Models highlighting the importance of cortactin in regulating cell polarity through tight junctions (TJ). ( A ) A simplified overview shows that wild-type epithelial monolayers exhibit normal cell polarity provided directly by the Par polarity proteins and indirectly by cortactin. Par1b mainly locates to the basal membrane, as expected. In addition, cortactin seems to regulate monolayer permeability, presumably via the apical junctional complex, in particular by binding to ZO-1. This binding requires serine-phosphorylation of cortactin. ( B ) In cortactin-deficient cells, Par1b mainly locates to the apical membranes leading to disturbed cell polarity. We therefore propose that cortactin exhibits some suppressive activity on Par1b in the TJs. Thus, elevated TEER values were measured that may arise from the absence of cortactin. In addition, the expression of cortactin seems important for proper microvilli formation. ( C ) In H. pylori -infected wild-type monolayers, injected CagA induces Par1b inactivation and serine-phosphorylation of cortactin, associated with strongly enhanced complex formation with ZO-1 in the TJs, which leads to loss of cell polarity. ( D ) During infection with H. pylori, CagA is injected into epithelial cells, which targets Par1b and cell polarity by two different pathways, here named complex-1 and complex-2. In complex-1, CagA binds directly to Par1b via the CRPIA-motif, which triggers loss of cell polarity and may promote cell extrusion. This complex also contains ZO-1 and cortactin. In complex-2, CagA targets ASPP2 at the ABD (ASPP2 Binding Domain) to inhibit the aPKC-containing apical regulatory complex. In this way, aPKC cannot phosphorylate Par3 ( Buti et al., 2020 ) and CagA-bound Par1b ( Saadat et al., 2007 ) any longer, which abrogates their mutual antagonistic activities. We propose that both signaling complexes together result in full disruption of cell polarity and induction of cytoskeletal rearrangements.

Article Snippet: Membranes were blocked with either 5% non-fat milk or 3% BSA and probed with the primary antibodies as follows: mouse α-cortactin (#05–180, Merck-Millipore), rabbit α-Par1b (#HPA074905, Sigma Aldrich), rabbit α-ZO-1 (#61–73000, Invitrogen), rabbit α-E-cadherin (#sc-7870, Santa Cruz), rabbit α-Claudin-5 (#ab15106, Abcam), mouse α-β-actin (#A5441, Sigma Aldrich), mouse α-GFP (#632381, Clontech), rabbit α-omni-probe (α-T7) (#sc-499, Santa Cruz), rabbit α-CagA (#HPP-5003-9, Austral Biologicals), mouse α-PY99 (#sc-7020, Santa Cruz) and α-ASPP2 (#200-401-A19, Rockland).

Techniques: Membrane, Permeability, Binding Assay, Phospho-proteomics, Activity Assay, Expressing, Infection, Injection, Disruption

Journal: Journal of Neuroscience

Article Title: ASPP1/2 Regulate p53-Dependent Death of Retinal Ganglion Cells through PUMA and Fas/CD95 Activation In Vivo

doi: 10.1523/jneurosci.2635-12.2013

Figure Lengend Snippet: Figure2. ASPP1andASPP2areexpressedbyadultRGCs.RetinalimmunofluorescencedemonstratedabundantexpressionofendogenousASPP1(A–G)andASPP2(K–Q)inRGCsvisualizedwith the retrograde tracer Fluorogold. DAPI staining showed that ASPP1 is present in RGC nuclei and cytoplasm (perinuclear) (H–J), while ASPP2 is primarily in the nuclei (R–T). ASPP1 and ASPP2 blockingpeptidesresultedinabsenceofstaining(G,Q),confirmingthespecificityoftheASPP1andASPP2antibodies.Scalebars:A–CandK–M,70m;D–GandN–Q,50m;H–JandR–T,10 m.RPE,Retinalpigmentepithelium;PS,photoreceptorsegments;ONL,outernuclearlayer;OPL,outerplexiformlayer;INL,innernuclearlayer;IPL,innerplexiformlayer;GCL,ganglioncelllayer; FG, Fluorogold.

Article Snippet: Blocking peptides (2.5 g/ml; Bethyl Laboratories) were incubated overnight with ASPP1 or ASPP2 primary antibodies (5:1 ratio) before application onto retinal sections.

Techniques: Staining

Journal: Journal of Neuroscience

Article Title: ASPP1/2 Regulate p53-Dependent Death of Retinal Ganglion Cells through PUMA and Fas/CD95 Activation In Vivo

doi: 10.1523/jneurosci.2635-12.2013

Figure Lengend Snippet: Figure 3. Expression of ASPP family members after optic nerve axotomy. A, The levels or subcellular localization of ASPP1, ASPP2, or the anti-apoptotic member iASPP, visualized by retinal immunohistochemistry and Fluorogold (FG) staining, did not changeat48hafteropticnerveinjury.Scalebars,10m.B,AnalysisofproteinhomogenatesconfirmedthatASPP1,ASPP2,and iASPPlevelsinaxotomizedretinascollectedat48hweresimilartothoseinintact,noninjuredretinas.Thebottomblotisthesame as the top, but probed with an antibody that recognizes -actin used to confirm equal protein loading. C, Densitometric analysis of Western blots, showing the ratio of ASPP proteins relative to -actin, confirmed that there is no significant change in protein expression after injury (Student’s t test, p 0.05).

Article Snippet: Blocking peptides (2.5 g/ml; Bethyl Laboratories) were incubated overnight with ASPP1 or ASPP2 primary antibodies (5:1 ratio) before application onto retinal sections.

Techniques: Expressing, Immunohistochemistry, Staining, Western Blot

Journal: Journal of Neuroscience

Article Title: ASPP1/2 Regulate p53-Dependent Death of Retinal Ganglion Cells through PUMA and Fas/CD95 Activation In Vivo

doi: 10.1523/jneurosci.2635-12.2013

Figure Lengend Snippet: Figure 4. Selective knockdown of retinal ASPP1 or ASPP2 by intravitreal siRNA delivery. Intravitreal delivery of Cy3-tagged siRNA resulted in rapid and effective uptake by RGCs. Lack of Cy3 fluorescence in noninjected control retinas (A–C) contrasted with robust Cy3 labeling in RGCs, visualized with Fluorogold (FG) (D–I), as early as 5 h after siRNA administration. J–M, Intravitreal delivery of siRNA against ASPP1 (siASPP1) led to a significant reduction of retinal ASPP1 protein at 24 h after delivery while control siRNA against GFP (siGFP) had no effect (ANOVA, *p 0.05). siASPP1 did not decrease or increase the protein levels of the other family members, ASPP2 or iASPP, confirming the specificity of the siRNA. Similarly, siRNA against ASPP2 (siASPP2) selectively depletedretinalASPP2proteinlevels(ANOVA,*p0.05)withoutalteringASPP1oriASPPlevels.EndogenouslevelsofbothASPP1andASPP2proteinsreturnedtobasalat48haftersiRNAdelivery. N, O, Immunohistochemistry of axotomized retinas at 24 h after siASPP1 or siASPP2 administration confirmed that siRNA-mediated knockdown of ASPP1/2 occurred in RGCs, visualized with Fluorogold.Scalebars:A–F,50m;G–I,10m;N–O,12m.PS,Photoreceptorsegments;ONL,outernuclearlayer;OPL,outerplexiformlayer;INL,innernuclearlayer;IPL,innerplexiformlayer; GCL, ganglion cell layer.

Article Snippet: Blocking peptides (2.5 g/ml; Bethyl Laboratories) were incubated overnight with ASPP1 or ASPP2 primary antibodies (5:1 ratio) before application onto retinal sections.

Techniques: Knockdown, Fluorescence, Control, Labeling, Immunohistochemistry

Journal: Journal of Neuroscience

Article Title: ASPP1/2 Regulate p53-Dependent Death of Retinal Ganglion Cells through PUMA and Fas/CD95 Activation In Vivo

doi: 10.1523/jneurosci.2635-12.2013

Figure Lengend Snippet: Figure 6. siASPP2-mediated knockdown of PUMA, Fas, and Noxa depends on p53 transcriptional activity. A, Real-time qPCR analysis of rat retinal samples at 6 h after axotomy and siASPP2 administration revealed that ASPP2 knockdown leads to down- regulation of PUMA, Fas/CD95, and Noxa (ANOVA, ***p 0.001; **p 0.01), but not Bax (ANOVA, p 0.5) gene expression. B–E, qPCR of retinal samples from p53-null mice and wild-type littermate controls collected at 6 h after axotomy and siASPP2 injection. Transcript levels of PUMA, Fas/CD95, and Noxa were significantly reduced in noninjured or axotomized p53-null mice with respect to wild-type littermates. Moreover, ASPP2 knockdown effectively reduced PUMA, Fas/CD95, and Noxa gene expres- sion in axotomized retinas from p53 wild-type mice, but not from p53 knock-out mice (B, C, E) (ANOVA, ***p 0.001; **p 0.01). Bax gene expression remained unchanged (D).

Article Snippet: Blocking peptides (2.5 g/ml; Bethyl Laboratories) were incubated overnight with ASPP1 or ASPP2 primary antibodies (5:1 ratio) before application onto retinal sections.

Techniques: Knockdown, Activity Assay, Gene Expression, Injection, Knock-Out

Journal: Cell biology and toxicology

Article Title: ASPP2 deficiency attenuates lipid accumulation through the PPARγ pathway in alcoholic liver injury.

doi: 10.1007/s10565-024-09925-x

Figure Lengend Snippet: Fig. 2 ASPP2 deficiency inhibits the PPARγ and mTOR signaling path- ways. (A and B) Repre- sentative IHC analysis of PPARγ in liver sections, scar bar = 50 µm (A) and quantification analyses (B) of PPARγ positive area (%) in mouse liver tissue from wild-type mice and ASPP2- KD mice. The extended part of the black lines shows the enlarged image from the black box area. (C and D) western blot (C) and quantification analyses (D) of ASPP2, PPARγ, phospho-mTOR, phospho- S6, phospho-p70S6K, and β-actin in mouse liver tissue from wild-type mice and ASPP2-KD mice. (E and F) western blot (E) and quantification analyses (F) of ASPP2, PPARγ, phospho-mTOR, phospho- S6, phospho-p70S6K, and β-actin in primary hepatocytes (Control and ASPP2 siRNA) treated with ethanol for different times. (G and H) western blot (G) and quantification analyses (H) of ASPP2, PPARγ, phospho-mTOR, phospho-S6, phospho- p70S6K, and β-actin in primary hepatocytes (Ad- GFP and Ad-ASPP2) with the indicated treatment. The values represent the means ± SEMs (n = 6 in each group). nsP > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001. Independent- samples T tests between two groups were used for statistical analysis, and one-way ANOVA followed by Bonferroni post hoc tests for multiple comparisons were used for statistical analyses

Article Snippet: Finally, the expression of ASPP2 or PPARγ was observed by microscopy (Leica Microsystems, Mannheim, Germany) after development using a diaminobenzidine kit (Boster, Wuhan, China).

Techniques: Western Blot, Control

Journal: Experimental and Therapeutic Medicine

Article Title: ASPP2 expression predicts the prognosis of patients with hepatocellular carcinoma after transcatheter arterial chemoembolization

doi: 10.3892/etm.2021.9828

Figure Lengend Snippet: Baseline characteristics of the unresectable HCC patients with high or low expression of ASPP2 (n=232).

Article Snippet: The paraffin block was cut into 5-µm sections and then incubated with primary antibody for rat anti-human ASPP2 (BD Pharmingen) in 5% rabbit serum overnight at 4̊C followed by washing and incubation with ABC (avidin-linked biotin complex) rabbit anti-rat Ig.

Techniques: Expressing

Journal: Experimental and Therapeutic Medicine

Article Title: ASPP2 expression predicts the prognosis of patients with hepatocellular carcinoma after transcatheter arterial chemoembolization

doi: 10.3892/etm.2021.9828

Figure Lengend Snippet: Baseline characteristics of the resectable HCC patients with high or low expression of ASPP2 (n=205).

Article Snippet: The paraffin block was cut into 5-µm sections and then incubated with primary antibody for rat anti-human ASPP2 (BD Pharmingen) in 5% rabbit serum overnight at 4̊C followed by washing and incubation with ABC (avidin-linked biotin complex) rabbit anti-rat Ig.

Techniques: Expressing

Journal: Experimental and Therapeutic Medicine

Article Title: ASPP2 expression predicts the prognosis of patients with hepatocellular carcinoma after transcatheter arterial chemoembolization

doi: 10.3892/etm.2021.9828

Figure Lengend Snippet: Expression of Beclin-1 and ASPP2 in tissues from HCC patients with or without administration of TACE. Expression of ASPP2 in HCC tissue from patients with (A) sequential TACE followed by resection or (B) direct resection. (C) The ratio of expression of ASPP2 in the para-carcinoma to carcinoma tissues in the patients with sequential TACE followed by resection or direct resection. n=13, * P<0.05. Expression of Beclin-1 in the HCC tissue from patients with (D) sequential TACE followed by resection or (E) direct resection. (F) Negative correlation between the expression of Beclin-1 and ASPP2 in carcinoma tissue of patients with sequential TACE followed by resection (n=36). Student's t-test and linear correlation analysis were used for statistical analysis. P<0.05 indicates statistical difference. ASPP2, apoptosis-stimulating p53 protein 2; TACE, transcatheter arterial chemoembolization; HCC, hepatocellular carcinoma.

Article Snippet: The paraffin block was cut into 5-µm sections and then incubated with primary antibody for rat anti-human ASPP2 (BD Pharmingen) in 5% rabbit serum overnight at 4̊C followed by washing and incubation with ABC (avidin-linked biotin complex) rabbit anti-rat Ig.

Techniques: Expressing

Journal: Experimental and Therapeutic Medicine

Article Title: ASPP2 expression predicts the prognosis of patients with hepatocellular carcinoma after transcatheter arterial chemoembolization

doi: 10.3892/etm.2021.9828

Figure Lengend Snippet: Multiple logistics regression analysis for the independent risk factor of 1-year mortality rate.

Article Snippet: The paraffin block was cut into 5-µm sections and then incubated with primary antibody for rat anti-human ASPP2 (BD Pharmingen) in 5% rabbit serum overnight at 4̊C followed by washing and incubation with ABC (avidin-linked biotin complex) rabbit anti-rat Ig.

Techniques: Expressing

Journal: Experimental and Therapeutic Medicine

Article Title: ASPP2 expression predicts the prognosis of patients with hepatocellular carcinoma after transcatheter arterial chemoembolization

doi: 10.3892/etm.2021.9828

Figure Lengend Snippet: Survival analysis and expression of ASPP2 in the HCC patients after TACE. (A) The 1-year progression-free survival in unresectable HCC patients at initiation after neoadjuvant TACE combination and resection in all subjects with high expression (dotted line) and low expression of ASPP2 (solid line). (B) The 1-year overall survival in unresectable HCC patients at initiation following neoadjuvant TACE combination and resection in all subjects with high expression (dotted line) and low expression of ASPP2 (solid line). (C) The 1-year progression-free survival in resectable HCC patients following neoadjuvant TACE combination and resection in three groups subjects, including patients who were administered TACE with high expression (red dotted line) and low expression (black dotted line) of ASPP2 or who did not received neoadjunct TACE (black solid line). (D) The 1-year over survival in resectable HCC patients following neoadjuvant TACE combination and resection in three groups subjects, including patients who were administered TACE with high expression and low expression of ASPP2 or not. Survival analysis was used for the statistical analysis. The survival curve is depicted according to the Kaplan-Meier method. Red dotted line, patients administered TACE and with a high expression of ASPP2; Black dotted line, patients administered TACE and with a low expression of ASPP2; Black solid line, patients without TACE administration. ASPP2, apoptosis-stimulating p53 protein 2; TACE, transcatheter arterial chemoembolization; HCC, hepatocellular carcinoma.

Article Snippet: The paraffin block was cut into 5-µm sections and then incubated with primary antibody for rat anti-human ASPP2 (BD Pharmingen) in 5% rabbit serum overnight at 4̊C followed by washing and incubation with ABC (avidin-linked biotin complex) rabbit anti-rat Ig.

Techniques: Expressing

Journal: Nature Communications

Article Title: ASPP2 maintains the integrity of mechanically stressed pseudostratified epithelia during morphogenesis

doi: 10.1038/s41467-022-28590-4

Figure Lengend Snippet: a ASPP2 was detected by indirect immunofluorescence in E3.5 embryos to analyse its localisation pattern. A cross-section through the equatorial plane of a representative embryo is shown (top row), as well as a 3D opacity rendering of the same embryo (bottom row). The F-actin cytoskeleton and nuclei were visualised using Phalloidin and DAPI, respectively. A magnified image of the dashed area is shown on the right. Note how ASPP2 colocalises with F-actin at the apical junctions in cells of the trophectoderm (white arrowheads). The juxtaposed graph shows ASPP2 and F-actin signal intensity along the apical-basal axis of five cell-cell junctions in the TE. Error bars represent ±SD. AJ apical junction, B base of the trophectoderm. Scale bars: 20 and 5 μm (for the magnification). b The localisation pattern of YAP and Par3 was analysed in wild type and ASPP2 RAKA/RAKA embryos by indirect immunofluorescence. A cross-section of representative embryos through the equatorial plane shows the localisation of YAP in the nuclei of the trophectoderm in both wild type and ASPP2 RAKA/RAKA embryos. Maximum intensity projections of these embryos show the localisation of Par3 at the level of apical junctions in the trophectoderm (representative images from six wild type and eight ASPP2 RAKA/RAKA embryos). Scale bar: 20 μm. c ASPP2 knockdown in E3.5 embryos using siRNA against ASPP2 mRNA. ASPP2 knockdown was confirmed by indirect immunofluorescence. Note how signal at the apical junctions is specific to ASPP2 and how YAP is normally localised to the nuclei of TE cells in ASPP2-depleted embryos. Representative images from n = 19 control siRNA-injected embryos and n = 24 ASPP2 siRNA-injected embryos across two independent experiments. Scale bar: 20 μm. Source data are provided as a Source Data file.

Article Snippet: We originally obtained ASPP2 mutant mice in which exons 10–17 were replaced with a neo-r gene from Jackson Laboratory.

Techniques: Immunofluorescence, Knockdown, Control, Injection

Journal: Nature Communications

Article Title: ASPP2 maintains the integrity of mechanically stressed pseudostratified epithelia during morphogenesis

doi: 10.1038/s41467-022-28590-4

Figure Lengend Snippet: a Immunofluorescence of wild type and ASPP2 ΔE4/ΔE4 E6.5 embryos using an anti-Par6 antibody. The phenotypic variability of ASPP2 ΔE4/ΔE4 embryos is illustrated, with embryos either lacking cavities (middle row, five out of nine embryos) or exhibiting smaller cavities (bottom row, four out of nine embryos). The green dashed line highlights the ectopic accumulation of cells in the epiblast of ASPP2 ΔE4/ΔE4 embryos. b Magnification of the corresponding regions shown in panel a . Blue arrowheads highlight the enrichment of F-actin at the apical junctions in the epiblast. Note how F-actin is not enriched at the apical junctions but is instead more homogenously distributed across the apical surface of epiblast cells in ASPP2 ΔE4/ΔE4 embryos (orange arrowhead). The insets within images are 2x magnifications of the corresponding dashed areas. c Quantification of F-actin signal intensity along the apical surface of epiblast cells of wild type ( n = 3 embryos, five measurements per embryo) and ASPP2 ΔE4/ΔE4 embryos ( n = 3 embryos, five measurements per embryo). Measurements were made on cross-sections along the apical domain of individual epiblast cells from apical junction to an apical junction (represented with a blue background in the graph). The 95% confidence interval is represented by the grey area. See material and methods for details. d Immunofluorescence of wild type (representative images from eight embryos) and ASPP2 ΔE4/ΔE4 E5.5 embryos (representative images from five embryos) using an anti-Laminin antibody. e Magnification of the corresponding dashed areas in panel d . f Immunofluorescence of wild type (representative images from seven embryos) and ASPP2 ΔE4/ΔE4 (representative images from two embryos) E6.5 embryos using an anti-SCRIB antibody. g Magnification of the corresponding dashed areas in panel f . Green arrowheads highlight basolateral SCRIB. Note the enrichment of SCRIB at the apical junctions in the epiblast of wild type embryos (blue arrowhead) and its absence in the corresponding localisation in ASPP2 ΔE4/ΔE4 embryos (orange arrowhead). Nuclei and the F-actin cytoskeleton were visualised with DAPI and Phalloidin, respectively. Scale bars: 20 μm. Source data are provided as a Source Data file.

Article Snippet: We originally obtained ASPP2 mutant mice in which exons 10–17 were replaced with a neo-r gene from Jackson Laboratory.

Techniques: Immunofluorescence

Journal: Nature Communications

Article Title: ASPP2 maintains the integrity of mechanically stressed pseudostratified epithelia during morphogenesis

doi: 10.1038/s41467-022-28590-4

Figure Lengend Snippet: a The expression of ASPP2 was conditionally ablated in the epiblast to test for its epiblast-specific requirement ( ASPP2 EpiΔE4/ΔE4 embryos). The ASPP2 expression pattern was analysed by indirect immunofluorescence in ASPP2 EpiWT/ΔE4 (representative images from four embryos) and ASPP2 EpiΔE4/ΔE4 (representative images from four embryos) embryos. ASPP2 proteins were completely absent at the apical junction of epiblast cells in ASPP2 EpiΔE4/ΔE4 embryos. Note that the ASPP2 antibody results in a nonspecific nuclear signal (also seen in Fig. when depleting ASPP2 by siRNA). The dashed area highlights the ectopic accumulation of cells in the epiblast. b Immunofluorescence of wild type (representative images from seven embryos) and ASPP2 RAKA/RAKA (representative images from three embryos) E6.5 embryos using an anti-Par6 antibody. The green dashed line highlights the ectopic accumulation of cells in the epiblast of ASPP2 RAKA/RAKA embryos. c Magnification of the corresponding dashed regions in b . Note the reduced amount of Par6 along the apical domain of epiblast cells in ASPP2 RAKA/RAKA embryos (orange arrowhead). Nuclei and the F-actin cytoskeleton were visualised with DAPI and Phalloidin, respectively. Scale bars: 20 μm.

Article Snippet: We originally obtained ASPP2 mutant mice in which exons 10–17 were replaced with a neo-r gene from Jackson Laboratory.

Techniques: Expressing, Immunofluorescence

Journal: Nature Communications

Article Title: ASPP2 maintains the integrity of mechanically stressed pseudostratified epithelia during morphogenesis

doi: 10.1038/s41467-022-28590-4

Figure Lengend Snippet: a Time-lapse imaging of wild type and ASPP2 ΔE4/ΔE4 embryos. mT/mG-labelled cell membranes were used to manually track cell movement. Yellow dots highlight mother cells at the apical surface of the epiblast immediately prior to a cell division event. Green and magenta dots identify the resulting daughter cells. Note how both daughters reintegrate the epiblast in the wild type whereas one of the two daughters fails to do so in the absence of ASPP2 even after a prolonged period of time ( t = 82.5’). b Diagram illustrating the method used to quantify daughter cell movement following cell divisions. Daughter cell movement was characterised by both the distance travelled ( d ) and the direction of travel (θ) expressed as the angle between the reference vector (the green vector starting from the initial position of the mother cell prior to the division event to the centre of the embryonic region) and the vector characterising absolute daughter cell movement (the red vector starting from the initial position of the mother cell prior to the division event to the final position of the daughter cell). The left panel illustrates the case of a daughter moving basally to reincorporate the epiblast and the right panel describes abnormal daughter cell movement towards the centre of the embryonic region such as seen in ASPP2 ΔE4/ΔE4 embryos. c Graph quantifying cell movement in wild type ( n = 3 embryos, 56 cells) and ASPP2 ΔE4/ΔE4 embryos ( n = 3 embryos, 66 cells). For a given pair of daughter cells, each daughter was defined as ‘apical’ or ‘basal’ depending on their respective position relative to the centre of the embryonic region immediately after a cell division event. d Proportion of daughter cells with an overall apical or basal movement in wild type ( n = 3 embryos, 56 cells) and ASPP2 ΔE4/ΔE4 embryos ( n = 3 embryos, 66 cells). Left panel: Quantification of the proportion of daughter cells with an overall apical (θ from 0° to 90°) or basal movement (θ from 90° to 180°) in wild type and ASPP2 ΔE4/ΔE4 embryos. Right panel: quantification of the proportion of apical and basal daughters with an overall apical (θ from 0° to 90°) or basal movement (θ from 90° to 180°) in wild type and ASPP2 ΔE4/ΔE4 embryos. **** p < 0.0001, NS non-significant (two-sided Fisher’s exact test of independence. The Bonferroni method was used to adjust p values for multiple comparisons. P values from left to right: p = 6.16e-07, p = 1.62e-08, p = 2.83e-09, p = 7.08e-01). Source data are provided as a Source Data file.

Article Snippet: We originally obtained ASPP2 mutant mice in which exons 10–17 were replaced with a neo-r gene from Jackson Laboratory.

Techniques: Imaging, Plasmid Preparation

Journal: Nature Communications

Article Title: ASPP2 maintains the integrity of mechanically stressed pseudostratified epithelia during morphogenesis

doi: 10.1038/s41467-022-28590-4

Figure Lengend Snippet: a Posterior thickening in E7.5 ASPP2 RAKA/RAKA embryos in a BALB/C background. Left panel: the anteroposterior axis was defined using AMOT localisation pattern. Right panel: comparison of tissue thickness in the anterior (three measurements per embryo) and the posterior (three measurements per embryo) of wild type ( n = 5 embryos) and ASPP2 RAKA/RAKA embryos ( n = 5 embryos). For the box plots, the top and bottom lines of each box represent the 75th and 25th percentiles, respectively. The whiskers show the minima to the maxima values and the central line indicates the median. * p < 0.05, **** p < 0.0001 (nested ANOVA, p values from left to right: p = 0.047, p = 3.95e-5). b Cells accumulate in the primitive streak region of ASPP2 RAKA/RAKA embryos. Immunofluorescence of E7.5 wild type (representative images from 55 embryos) and ASPP2 RAKA/RAKA (representative images from 23 embryos) embryos using a T (Brachyury) antibody. c Cells ectopically accumulating in the primitive streak region are unable to apically constrict and do not have enriched F-actin at the apical junctions (area delineated by the dotted orange line) in comparison to wild type (blue arrowheads and magenta dotted lines). Green dotted ROI: the apical surface of the epiblast in the lateral region of the embryo. Lower panel: Magnified regions highlighted in green and orange, respectively. Right panel: quantification of F-actin signal intensity along the apical surface of epiblast cells in the primitive streak region of wild type ( n = 3 embryos, five cells per embryo) and ASPP2 RAKA/RAKA embryos ( n = 3 embryos, five cells per embryo). The 95% confidence interval is represented by the grey area. d – f Airyscan imaging reveals the extent of F-actin disorganisation at the surface of cells accumulating ectopically in the primitive streak region of ASPP2 RAKA/RAKA embryos. d 3D opacity rendering of embryo optical halves, enabling visualisation of the apical surface of epiblast cells in the proamniotic cavity. Note the absence of the typical F-actin mesh pattern at the apical surface of cells in the posterior of ASPP2 RAKA/RAKA embryos (green dotted line). e Cross-section through the primitive streak region, showing enriched F-actin at the apical junctions of wild type (representative image from three embryos) embryos (blue arrowheads) and the formation of F-actin spike-like structures at the contact-free surface of ASPP2 RAKA/RAKA (representative image from three embryos) embryos. f En face view of the epiblast’s apical surface in the posterior of an ASPP2 RAKA/RAKA embryo. Green dotted lines demarcate the disorganised apical region of the posterior and the more organised lateral regions of the epiblast. Right panel: magnification of the epiblast’s apical surface in the posterior of an ASPP2 RAKA/RAKA embryo showing F-actin forming spike-like structures. Nuclei and the F-actin cytoskeleton were visualised with DAPI and Phalloidin, respectively. Scale bars: 50 μm ( a – c ), 20 μm ( e ). Source data are provided as a Source Data file.

Article Snippet: We originally obtained ASPP2 mutant mice in which exons 10–17 were replaced with a neo-r gene from Jackson Laboratory.

Techniques: Comparison, Immunofluorescence, Imaging

Journal: Nature Communications

Article Title: ASPP2 maintains the integrity of mechanically stressed pseudostratified epithelia during morphogenesis

doi: 10.1038/s41467-022-28590-4

Figure Lengend Snippet: a , b FLIM measurements of the FLIPPER-TR tension probe in E6.5 embryos. a Representative FLIM image of an E6.5 embryo. Note that Lifetime smaller than 3.75 and higher than 4.75 are blue and red, respectively. b Mean lifetime values at the apical surface of the epiblast, exVE and emVE ( n = 9 embryos). For the box plots, the top and bottom lines of each box represent the 75th and 25th percentiles, respectively. The whiskers show the minima to the maxima values and the central line indicates the median. **** p < 0.0001, *** p < 0.001, ** p < 0.01 (ANOVA, followed by Tukey’s test. P values from left to right: p = 0, p = 1.52e-4, p = 1.41e-3). c Localisation pattern of SHROOM2 in E6.5 embryos. Blue arrowhead highlights the accumulation of SHROOM2 at the apical junctions in the epiblast. d wild type ( n = 4) and ASPP2 RAKA/RAKA ( n = 2) embryos were grown for 30′ in cylindrical cavities made of biocompatible hydrogels. The localisation pattern of GATA6 and Myosin was then analysed by immunofluorescence. e Magnification of the embryos shown in b . The green dotted line highlights the ectopic accumulation of cells seen in ASPP2 RAKA/RAKA embryos. Note how Myosin is enriched at the apical junctions of wild type epiblast cells (blue arrowheads). The orange arrowhead points to the abnormal distribution of Myosin at the apical surface of these cells. Nuclei and the F-actin cytoskeleton were visualised with DAPI and Phalloidin, respectively. Scale bars: 20 μm. Source data are provided as a Source Data file.

Article Snippet: We originally obtained ASPP2 mutant mice in which exons 10–17 were replaced with a neo-r gene from Jackson Laboratory.

Techniques: Immunofluorescence

Journal: Nature Communications

Article Title: ASPP2 maintains the integrity of mechanically stressed pseudostratified epithelia during morphogenesis

doi: 10.1038/s41467-022-28590-4

Figure Lengend Snippet: a 3D opacity rendering showing the localisation of ASPP2 in E5.5 wild type embryos at the apical junctions of the visceral endoderm where it colocalises with F-actin. b Cross-section (top row) and 3D opacity rendering (bottom row) of the proamniotic cavity showing the localisation pattern of ASPP2 and F-actin at the apical junctions (white arrowhead). c The outer surface of the VE and apical surface of the epiblast were computationally ‘unwrapped’, revealing the enrichment of ASPP2 at specific locations along the apical junctions, often at F-actin-rich tricellular junctions (green arrowheads). a – c Representative images from six embryos. d The interaction between endogenous ASPP2 and the F-actin-binding protein Afadin was examined in Caco-2 cells by co-immunoprecipitation (representative images from three independent experiments). Molecular weights are indicated in kilodaltons. e The localisation pattern of endogenous ASPP2 and Afadin in Caco-2 cells was examined by immunofluorescence (representative images from five independent experiments). The bottom row represents the magnified region highlighted by a dotted box and shows the enrichment of ASPP2 and Afadin at tricellular junctions. ASPP2, Afadin and F-actin signal intensity was quantified across tricellular junctions (graph on the right). f The localisation pattern of Afadin in the proamniotic cavity was analysed by immunofluorescence in E6.5 wild type embryos. The blue arrowhead highlights the colocalisation of Afadin with F-actin at a tricellular junction. g The localisation pattern of F-actin was analysed by time-lapse microscopy in wild type (representative images from ten embryos) and ASPP2 RAKA/RAKA (representative images from six embryos) LifeAct-GFP positive embryos. Note how apical F-actin is disrupted in ASPP2 RAKA/RAKA LifeAct-GFP positive embryos (orange arrowhead). The colour scale represents pixel intensity (grey levels). h At later time points, the ectopic accumulation of cells in the epiblast of ASPP2 RAKA/RAKA LifeAct-GFP positive embryos was evident (dotted line). i Representative FLIM images of ASPP2 EpiWT/ΔE4 ( n = 9 embryos) and ASPP2 EpiΔE4/ΔE4 ( n = 7 embryos) embryos (left) and comparison of mean lifetime values in the epiblast tissue, including delaminating cells (right). The dotted line highlights epiblast cells. For the box plots, the top and bottom lines of each box represent the 75th and 25th percentiles, respectively. The whiskers show the minima to the maxima values and the central line indicates the median. Outliers are represented with black dots. ** p < 0.01 (unpaired two-sided Student’s t -test, p = 4.84e-3). Nuclei and the F-actin cytoskeleton were visualised with DAPI and Phalloidin respectively. Scale bars: 20 μm. Source data are provided as a Source Data file.

Article Snippet: We originally obtained ASPP2 mutant mice in which exons 10–17 were replaced with a neo-r gene from Jackson Laboratory.

Techniques: Binding Assay, Immunoprecipitation, Immunofluorescence, Time-lapse Microscopy, Comparison

Journal: Nature Communications

Article Title: ASPP2 maintains the integrity of mechanically stressed pseudostratified epithelia during morphogenesis

doi: 10.1038/s41467-022-28590-4

Figure Lengend Snippet: a The primitive streak expands comparatively in E7.5 wild type and ASPP2 ΔE4/ΔE4 embryos. Mesoderm cells were labelled by immunofluorescence using an antibody against Brachyury (T). b Patterning proceeds normally in the absence of ASPP2. The ectoderm and cardiac progenitors were labelled in E8.5 wild type and ASPP2 ΔE4/ΔE4 embryos with antibodies against SOX2 and NKX2.5, respectively. ys yolk sack, al allantois, s somites, hf head fold, am amnion, pc proamniotic cavity. c Cardiac progenitors can differentiate into cardiomyocytes in E9.5 ASPP2 ΔE4/ΔE4 embryos. The presence of the contractile machinery (magenta arrowheads) was assessed in wild type and ASPP2 ΔE4/ΔE4 embryos using an antibody against sarcomeric α-actinin. Nuclei and the F-actin cytoskeleton were visualised with DAPI and Phalloidin, respectively. Scale bars: 50 μm ( a , c ), 100 μm ( b ).

Article Snippet: We originally obtained ASPP2 mutant mice in which exons 10–17 were replaced with a neo-r gene from Jackson Laboratory.

Techniques: Immunofluorescence

Journal: Nature Communications

Article Title: ASPP2 maintains the integrity of mechanically stressed pseudostratified epithelia during morphogenesis

doi: 10.1038/s41467-022-28590-4

Figure Lengend Snippet: a Somite architecture is disrupted in ASPP2 ΔE4/ΔE4 embryos. The dotted line highlights the contour of a somite in an ASPP2 ΔE4/ΔE4 embryo. The star indicates the ectopic accumulation of cells in the centre of this somite. Arrowheads point to mitotic figures. b – e Quantification of somite characteristics in wild type ( n = 10 embryos, 58 somites) and ASPP2 ΔE4/ΔE4 ( n = 6 embryos, 35 somites) embryos at E8.5. For the box plots, the top and bottom lines of each box represent the 75th and 25th percentiles, respectively. The whiskers show the minima to the maxima values and the central line indicates the median. Outliers are represented with black dots. * p < 0.05, **** p < 0.0001 (unpaired two-sided Student’s t -test; p = 0 in b , p = 3.093e-05 in c , p = 0.025 in d , p = 1.116e-08 in e ). f Apical-basal polarity is defective in the somites of ASPP2 ΔE4/ΔE4 embryos. Par6 localised apically in wild type somites (arrowhead) whereas it was absent in ASPP2 ΔE4/ΔE4 embryos (star). de definitive endoderm. g Head fold formation is defective in ASPP2 RAKA/RAKA embryos. The organisation of apical F-actin was disorganised locally in the anterior ectoderm of ASPP2 RAKA/RAKA embryos (orange dotted line). F-actin signal intensity along the apical surface of ectoderm cells in disrupted areas in ASPP2 RAKA/RAKA embryos ( n = 3 embryos, five cells per embryo) was compared to wild type cells ( n = 3 embryos, five cells per embryo). Measurements were made on cross-sections along the apical domain of individual ectoderm cells from apical junction to the apical junction (represented with a blue background in the graph). The 95% confidence interval is represented by the grey area. Nuclei and the F-actin cytoskeleton were visualised with DAPI and Phalloidin, respectively. Scale bars: 20 μm ( a , f ), 100 μm ( g ). Source data are provided as a Source Data file.

Article Snippet: We originally obtained ASPP2 mutant mice in which exons 10–17 were replaced with a neo-r gene from Jackson Laboratory.

Techniques: