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