lentiviruses encoding abi1 shrna  (Santa Cruz Biotechnology)

Journal: iScience

Article Title: Abi1 mediates airway smooth muscle cell proliferation and airway remodeling via Jak2/STAT3 signaling.

doi: 10.1016/j.isci.2022.103833

Figure Lengend Snippet: Figure 1. Expression of Abi1 is upregulated in asthmatic human airway smooth muscle (HASM) cells (A and B) Abi1 mRNA in nonasthmatic HASM cultures (n = 24) and asthmatic HASM cultures (n = 21) was evaluated by RT- qPCR. Abi1 mRNA is upregulated in 10 of 21 asthmatic cultures. (C and D) Abi2 mRNA is similar between nonasthmatic and asthmatic cultures. mRNA normalization is calculated using the following formula: Abi1 RQ (relative quantification) of individual cell culture line divided by the means of Abi1 RQ of all control cell cultures. (E and F) Abi1 protein in HASM cultures was determined using ELISA. Abi1 protein is enhanced in asthmatic ASM cultures with increased Abi1 mRNA expression.

Article Snippet: Briefly, lentiviruses encoding Abi1 shRNA (sc-40306-V) and control shRNA (sc-108080) were purchased from Santa Cruz Biotechnology.

Techniques: Expressing, Quantitative RT-PCR, Cell Culture, Control, Enzyme-linked Immunosorbent Assay

Journal: iScience

Article Title: Abi1 mediates airway smooth muscle cell proliferation and airway remodeling via Jak2/STAT3 signaling.

doi: 10.1016/j.isci.2022.103833

Figure Lengend Snippet: Figure 2. Abi1 regulates HASM proliferation (A) Protein expression of control, stable Abi1 knockdown (KD), and rescue cell cultures from non-asthmatic HASM was evaluated by immunoblotting. Abi1 shRNA inhibits Abi1 protein expression, which is restored by rescue. Data are means G SEM. n = 5. One-way ANOVA was used for statistical analysis. (B and C) Abi1 knockdown (KD) and rescue affect HASM cell numbers (B) and BrdU incorporation (C). Data are normalized to numbers of cells treated with control (Ctrl) shRNA or microplate readings of cells treated with Ctrl shRNA. Data are means G SEM. n = 5. One-way ANOVA was used for statistical analysis. (D) Abi1 overexpression in HASM cells. Data are means G SEM. n = 5. The t test was used for statistical analysis. (E and F) The overexpression (OE) of Abi1 enhances HASM cell numbers (E) and (F). Data are means G SEM. n = 5. The t test was used for statistical analysis. *p < 0.05; **p < 0.01. Data normalization is calculated as follows: Data of treated samples/Data of control samples.

Article Snippet: Briefly, lentiviruses encoding Abi1 shRNA (sc-40306-V) and control shRNA (sc-108080) were purchased from Santa Cruz Biotechnology.

Techniques: Expressing, Control, Knockdown, Western Blot, shRNA, BrdU Incorporation Assay, Over Expression

Journal: iScience

Article Title: Abi1 mediates airway smooth muscle cell proliferation and airway remodeling via Jak2/STAT3 signaling.

doi: 10.1016/j.isci.2022.103833

Figure Lengend Snippet: Figure 3. Abi1 modulates phosphorylation and activation of Jak2 and STAT3 (A and B) Abi1 KD attenuates the PDGF-induced Jak2 (A) and STAT3 (B) phosphorylation in HASM cells. Data are means G SEM. n = 5. Two-way ANOVA was used for statistical analysis. (C) Abi1 deficiency inhibits the PDGF-induced Jak2 kinase activity. Data are means G SEM. n = 5. Two-way ANOVA was used for statistical analysis. (D) The reporter activity of STAT3 is reduced by Abi1 KD. Data are means G SEM. n = 5. Two-way ANOVA was used for statistical analysis. (E and F) Overexpression of Abi1 in HASM cells enhances Jak2 kinase activity and STAT3 activity. Data are means G SEM. n = 5. The t test was used for statistical analysis. *p < 0.05; **p < 0.01. Data normalization is calculated as follows: Data of treated samples/Data of control samples.

Article Snippet: Briefly, lentiviruses encoding Abi1 shRNA (sc-40306-V) and control shRNA (sc-108080) were purchased from Santa Cruz Biotechnology.

Techniques: Phospho-proteomics, Activation Assay, Activity Assay, Over Expression, Control

Journal: iScience

Article Title: Abi1 mediates airway smooth muscle cell proliferation and airway remodeling via Jak2/STAT3 signaling.

doi: 10.1016/j.isci.2022.103833

Figure Lengend Snippet: Figure 4. Stimulation with PDGF promotes the engagement of Abi1 with Jak2 (A) Jak2 constitutively resides on the membrane of HASM cells. PDGF receptor is localized on the membrane. (B) Coimmunoprecipitation (upper panel) and reverse coimmunoprecipitation (lower panel) demonstrate that treatment with PDGF enhances the engagement of Abi1 with Jak2. Data are means G SEM. n = 4. The t test was used for statistical analysis. (C) PDGF stimulation increases FRET signal in cells expressing Abi1-EGFP and Jak2-dsRed. Scale bars, 10 mm **p < 0.01. The images are representative of four independent experiments. Data normalization is calculated as follows: Data of treated samples/Data of control samples.

Article Snippet: Briefly, lentiviruses encoding Abi1 shRNA (sc-40306-V) and control shRNA (sc-108080) were purchased from Santa Cruz Biotechnology.

Techniques: Membrane, Expressing, Control

Journal: iScience

Article Title: Abi1 mediates airway smooth muscle cell proliferation and airway remodeling via Jak2/STAT3 signaling.

doi: 10.1016/j.isci.2022.103833

Figure Lengend Snippet: Figure 5. PDGF induces the spatial redistribution of Abi1 in smooth muscle cells (A) Jak2 is located on the edge of unstimulated HASM cells. PDGF treatment does not affect Jak2 localization. However, Abi1 translocates to the cell periphery upon PDGF stimulation. The arrows indicate a single line scan to analyze the fluorescence intensity for each cell. The inset plots represent fluorescence intensity of the line scan indicated by the arrows. Data are mean G SEM (n = 32 cells from three independent experiments). The t test was used for statistical analysis. (B) The cellular localization of Abi2 is not altered in response to PDGF treatment. Data are mean G SEM (n = 33 cells from three independent experiments). The t test was used for statistical analysis. *p < 0.05. NS, not significant (C) Abi1 KD does not affect the spatial distribution of Jak2 and PDGF receptor b. Data are mean G SEM (n = 33–34 cells from three independent experiments). The t test was used for statistical analysis. NS, not significant. Protein distribution was analyzed using the methods described in method details. The relative intensity ratios (periphery/interior) are calculated using the following formula: ratio of each cell/average ratios of control cells. Scale bar, 10 mm

Article Snippet: Briefly, lentiviruses encoding Abi1 shRNA (sc-40306-V) and control shRNA (sc-108080) were purchased from Santa Cruz Biotechnology.

Techniques: Control

Journal: iScience

Article Title: Abi1 mediates airway smooth muscle cell proliferation and airway remodeling via Jak2/STAT3 signaling.

doi: 10.1016/j.isci.2022.103833

Figure Lengend Snippet: Figure 6. Abi1 regulates STAT3 nuclear import in smooth muscle cells (A) Cells with perinuclear STAT3 localization are considered ‘‘untranslocated cells.’’ Cells with concentrated STAT3 distribution in the nucleus are considered ‘‘translocated cells.’’ The white arrow points to the nucleus without STAT3 translocation. The green arrows point to the nuclei with STAT3 translocation. (B) PDGF stimulation induces STAT3 translocation to the nucleus. Data are means G SEM. n = 5. (C) Abi1 KD inhibits the PDGF-induced STAT3 nuclear import. Ctrl and Abi1 KD cells were treated with PDGF for 2 h followed by immunostaining. Data are means G SEM. n = 4. Two-way ANOVA was used for statistical analysis. (D) Overexpression (OE) of Abi1 is sufficient to induce STAT3 nuclear translocation. Cells were infected with lentivirus encoding the constructs for 2 days followed by staining. Data are means G SEM. n = 4. The t test was used for statistical analysis. **p < 0.01. The percentage of cells with translocated STAT3 was calculated as follows: numbers of cells with translocated STAT3/numbers of total cells observed 3 100.Scale bar, 10 mm

Article Snippet: Briefly, lentiviruses encoding Abi1 shRNA (sc-40306-V) and control shRNA (sc-108080) were purchased from Santa Cruz Biotechnology.

Techniques: Translocation Assay, Immunostaining, Over Expression, Infection, Construct, Staining

Journal: iScience

Article Title: Abi1 mediates airway smooth muscle cell proliferation and airway remodeling via Jak2/STAT3 signaling.

doi: 10.1016/j.isci.2022.103833

Figure Lengend Snippet: Figure 7. Abi1 knockdown inhibits the activity of Jak2/STAT3 and the proliferation in Abi1-up HASM cells (A and B) The basal and the PDGF-induced activity of Jak2 (A) and STAT3 (B) is higher in Abi1-up cells. Data are means G SEM. n = 4 for each group. One-way ANOVA was used for statistical analysis. (C and D) Abi1 KD inhibits the basal and the PDGF-induced activity of Jak2 (C) and STAT3 (D) in Abi1-up cells. Data are means G SEM. n = 4 for each group. Two-way ANOVA was used for statistical analysis (E and F) Abi1 KD reduced the PDGF-induced cell numbers (E) and BrdU incorporation (F). Data are means G SEM. n = 4 for each group. The t test was used for statistical analysis. *p < 0.05. Data normalization is calculated as follows: Data of treated samples/Data of control samples.

Article Snippet: Briefly, lentiviruses encoding Abi1 shRNA (sc-40306-V) and control shRNA (sc-108080) were purchased from Santa Cruz Biotechnology.

Techniques: Knockdown, Activity Assay, BrdU Incorporation Assay, Control

Journal: iScience

Article Title: Abi1 mediates airway smooth muscle cell proliferation and airway remodeling via Jak2/STAT3 signaling.

doi: 10.1016/j.isci.2022.103833

Figure Lengend Snippet: Figure 8. Abi1 mediates the allergen-induced airway smooth muscle thickening and airway hyperresponsiveness in vivo (A) Representative immunofluorescence images of lung sections showing effects of Abi1 conditional knockout (Abi1smko) on increases in airway smooth muscle thickness and proliferation induced by house dust mite (HDM) extracts. Treatment of Abi1lox mice with HDM extracts increases actin positive area and PCNA staining, which are reduced in Abi1smko. Scale bar, 100 mm (B and C) Quantification of actin-positive areas and the intensity of PCNA are evaluated by using NIH ImageJ software. Data are means G SEM (n = 11–12, 6 males and 5–6 females for each group). (D) Airway resistance (Rrs) was measured for Abi1lox mice and conditional KO mice treated with PBS or HDM extracts. (E) The sizes of lungs from Abi1lox mice and Abi1smko mice are similar. Shown are lung images of two Abi1lox mice and two Abi1smko mice. (F) ACh dose-response curves were determined for tracheal rings from Abi1lox mice and conditional KO mice treated with PBS or HDM extracts (n = 11–12, 6 males and 5–6 females for each group). Two-way ANOVA was used for statistical analysis. Data are means G SEM. **p < 0.01; *p < 0.05. Data normalization is calculated as follows: Data of treated samples/Data of control samples.

Article Snippet: Briefly, lentiviruses encoding Abi1 shRNA (sc-40306-V) and control shRNA (sc-108080) were purchased from Santa Cruz Biotechnology.

Techniques: In Vivo, Knock-Out, Staining, Software, Control

Journal: iScience

Article Title: Abi1 mediates airway smooth muscle cell proliferation and airway remodeling via Jak2/STAT3 signaling.

doi: 10.1016/j.isci.2022.103833

Figure Lengend Snippet: Figure 9. Proposed mechanism Stimulation with growth factors such as PDGF induces spatial distribution of Abi1, which facilitates the activation of the Jak2/STAT3 pathway. The association of Abi1 with Jak2 renders the receptor in active state. Abi1 is upregulated in 50% of asthmatic airway smooth muscle cultures, promoting the activation of Jak2 and STAT3, hyperplasia, and airway smooth muscle (ASM) thickening.

Article Snippet: Briefly, lentiviruses encoding Abi1 shRNA (sc-40306-V) and control shRNA (sc-108080) were purchased from Santa Cruz Biotechnology.

Techniques: Activation Assay

Journal: iScience

Article Title: Abi1 mediates airway smooth muscle cell proliferation and airway remodeling via Jak2/STAT3 signaling

doi: 10.1016/j.isci.2022.103833

Figure Lengend Snippet: Expression of Abi1 is upregulated in asthmatic human airway smooth muscle (HASM) cells (A and B) Abi1 mRNA in nonasthmatic HASM cultures (n = 24) and asthmatic HASM cultures (n = 21) was evaluated by RT-qPCR. Abi1 mRNA is upregulated in 10 of 21 asthmatic cultures. (C and D) Abi2 mRNA is similar between nonasthmatic and asthmatic cultures. mRNA normalization is calculated using the following formula: Abi1 RQ (relative quantification) of individual cell culture line divided by the means of Abi1 RQ of all control cell cultures. (E and F) Abi1 protein in HASM cultures was determined using ELISA. Abi1 protein is enhanced in asthmatic ASM cultures with increased Abi1 mRNA expression.

Article Snippet: Lentivirus encoding Abi1 shRNA , Santa Cruz , sc-40306-V.

Techniques: Expressing, Quantitative RT-PCR, Quantitative Proteomics, Cell Culture, Control, Enzyme-linked Immunosorbent Assay

Journal: iScience

Article Title: Abi1 mediates airway smooth muscle cell proliferation and airway remodeling via Jak2/STAT3 signaling

doi: 10.1016/j.isci.2022.103833

Figure Lengend Snippet: Abi1 regulates HASM proliferation (A) Protein expression of control, stable Abi1 knockdown (KD), and rescue cell cultures from non-asthmatic HASM was evaluated by immunoblotting. Abi1 shRNA inhibits Abi1 protein expression, which is restored by rescue. Data are means ± SEM. n = 5. One-way ANOVA was used for statistical analysis. (B and C) Abi1 knockdown (KD) and rescue affect HASM cell numbers (B) and BrdU incorporation (C). Data are normalized to numbers of cells treated with control (Ctrl) shRNA or microplate readings of cells treated with Ctrl shRNA. Data are means ± SEM. n = 5. One-way ANOVA was used for statistical analysis. (D) Abi1 overexpression in HASM cells. Data are means ± SEM. n = 5. The t test was used for statistical analysis. (E and F) The overexpression (OE) of Abi1 enhances HASM cell numbers (E) and (F). Data are means ± SEM. n = 5. The t test was used for statistical analysis. ∗p < 0.05; ∗∗p < 0.01. Data normalization is calculated as follows: Data of treated samples/Data of control samples.

Article Snippet: Lentivirus encoding Abi1 shRNA , Santa Cruz , sc-40306-V.

Techniques: Expressing, Control, Knockdown, Western Blot, shRNA, BrdU Incorporation Assay, Over Expression

Journal: iScience

Article Title: Abi1 mediates airway smooth muscle cell proliferation and airway remodeling via Jak2/STAT3 signaling

doi: 10.1016/j.isci.2022.103833

Figure Lengend Snippet: Abi1 modulates phosphorylation and activation of Jak2 and STAT3 (A and B) Abi1 KD attenuates the PDGF-induced Jak2 (A) and STAT3 (B) phosphorylation in HASM cells. Data are means ± SEM. n = 5. Two-way ANOVA was used for statistical analysis. (C) Abi1 deficiency inhibits the PDGF-induced Jak2 kinase activity. Data are means ± SEM. n = 5. Two-way ANOVA was used for statistical analysis. (D) The reporter activity of STAT3 is reduced by Abi1 KD. Data are means ± SEM. n = 5. Two-way ANOVA was used for statistical analysis. (E and F) Overexpression of Abi1 in HASM cells enhances Jak2 kinase activity and STAT3 activity. Data are means ± SEM. n = 5. The t test was used for statistical analysis. ∗p < 0.05; ∗∗p < 0.01. Data normalization is calculated as follows: Data of treated samples/Data of control samples.

Article Snippet: Lentivirus encoding Abi1 shRNA , Santa Cruz , sc-40306-V.

Techniques: Phospho-proteomics, Activation Assay, Activity Assay, Over Expression, Control

Journal: iScience

Article Title: Abi1 mediates airway smooth muscle cell proliferation and airway remodeling via Jak2/STAT3 signaling

doi: 10.1016/j.isci.2022.103833

Figure Lengend Snippet: Stimulation with PDGF promotes the engagement of Abi1 with Jak2 (A) Jak2 constitutively resides on the membrane of HASM cells. PDGF receptor is localized on the membrane. (B) Coimmunoprecipitation (upper panel) and reverse coimmunoprecipitation (lower panel) demonstrate that treatment with PDGF enhances the engagement of Abi1 with Jak2. Data are means ± SEM. n = 4. The t test was used for statistical analysis. (C) PDGF stimulation increases FRET signal in cells expressing Abi1-EGFP and Jak2-dsRed. Scale bars, 10 μm ∗∗p < 0.01. The images are representative of four independent experiments. Data normalization is calculated as follows: Data of treated samples/Data of control samples.

Article Snippet: Lentivirus encoding Abi1 shRNA , Santa Cruz , sc-40306-V.

Techniques: Membrane, Expressing, Control

Journal: iScience

Article Title: Abi1 mediates airway smooth muscle cell proliferation and airway remodeling via Jak2/STAT3 signaling

doi: 10.1016/j.isci.2022.103833

Figure Lengend Snippet: PDGF induces the spatial redistribution of Abi1 in smooth muscle cells (A) Jak2 is located on the edge of unstimulated HASM cells. PDGF treatment does not affect Jak2 localization. However, Abi1 translocates to the cell periphery upon PDGF stimulation. The arrows indicate a single line scan to analyze the fluorescence intensity for each cell. The inset plots represent fluorescence intensity of the line scan indicated by the arrows. Data are mean ± SEM (n = 32 cells from three independent experiments). The t test was used for statistical analysis. (B) The cellular localization of Abi2 is not altered in response to PDGF treatment. Data are mean ± SEM (n = 33 cells from three independent experiments). The t test was used for statistical analysis. ∗p < 0.05. NS, not significant (C) Abi1 KD does not affect the spatial distribution of Jak2 and PDGF receptor β. Data are mean ± SEM (n = 33–34 cells from three independent experiments). The t test was used for statistical analysis. NS, not significant. Protein distribution was analyzed using the methods described in . The relative intensity ratios (periphery/interior) are calculated using the following formula: ratio of each cell/average ratios of control cells. Scale bar, 10 μm

Article Snippet: Lentivirus encoding Abi1 shRNA , Santa Cruz , sc-40306-V.

Techniques: Fluorescence, Control

Journal: iScience

Article Title: Abi1 mediates airway smooth muscle cell proliferation and airway remodeling via Jak2/STAT3 signaling

doi: 10.1016/j.isci.2022.103833

Figure Lengend Snippet: Abi1 regulates STAT3 nuclear import in smooth muscle cells (A) Cells with perinuclear STAT3 localization are considered “untranslocated cells.” Cells with concentrated STAT3 distribution in the nucleus are considered “translocated cells.” The white arrow points to the nucleus without STAT3 translocation. The green arrows point to the nuclei with STAT3 translocation. (B) PDGF stimulation induces STAT3 translocation to the nucleus. Data are means ± SEM. n = 5. (C) Abi1 KD inhibits the PDGF-induced STAT3 nuclear import. Ctrl and Abi1 KD cells were treated with PDGF for 2 h followed by immunostaining. Data are means ± SEM. n = 4. Two-way ANOVA was used for statistical analysis. (D) Overexpression (OE) of Abi1 is sufficient to induce STAT3 nuclear translocation. Cells were infected with lentivirus encoding the constructs for 2 days followed by staining. Data are means ± SEM. n = 4. The t test was used for statistical analysis. ∗∗p < 0.01. The percentage of cells with translocated STAT3 was calculated as follows: numbers of cells with translocated STAT3/numbers of total cells observed × 100.Scale bar, 10 μm

Article Snippet: Lentivirus encoding Abi1 shRNA , Santa Cruz , sc-40306-V.

Techniques: Translocation Assay, Immunostaining, Over Expression, Infection, Construct, Staining

Journal: iScience

Article Title: Abi1 mediates airway smooth muscle cell proliferation and airway remodeling via Jak2/STAT3 signaling

doi: 10.1016/j.isci.2022.103833

Figure Lengend Snippet: Abi1 knockdown inhibits the activity of Jak2/STAT3 and the proliferation in Abi1-up HASM cells (A and B) The basal and the PDGF-induced activity of Jak2 (A) and STAT3 (B) is higher in Abi1-up cells. Data are means ± SEM. n = 4 for each group. One-way ANOVA was used for statistical analysis. (C and D) Abi1 KD inhibits the basal and the PDGF-induced activity of Jak2 (C) and STAT3 (D) in Abi1-up cells. Data are means ± SEM. n = 4 for each group. Two-way ANOVA was used for statistical analysis (E and F) Abi1 KD reduced the PDGF-induced cell numbers (E) and BrdU incorporation (F). Data are means ± SEM. n = 4 for each group. The t test was used for statistical analysis. ∗p < 0.05. Data normalization is calculated as follows: Data of treated samples/Data of control samples.

Article Snippet: Lentivirus encoding Abi1 shRNA , Santa Cruz , sc-40306-V.

Techniques: Knockdown, Activity Assay, BrdU Incorporation Assay, Control

Journal: iScience

Article Title: Abi1 mediates airway smooth muscle cell proliferation and airway remodeling via Jak2/STAT3 signaling

doi: 10.1016/j.isci.2022.103833

Figure Lengend Snippet: Abi1 mediates the allergen-induced airway smooth muscle thickening and airway hyperresponsiveness in vivo (A) Representative immunofluorescence images of lung sections showing effects of Abi1 conditional knockout (Abi1 smko ) on increases in airway smooth muscle thickness and proliferation induced by house dust mite (HDM) extracts. Treatment of Abi1 −lox mice with HDM extracts increases actin positive area and PCNA staining, which are reduced in Abi1 smko . Scale bar, 100 μm (B and C) Quantification of actin-positive areas and the intensity of PCNA are evaluated by using NIH ImageJ software. Data are means ± SEM (n = 11–12, 6 males and 5–6 females for each group). (D) Airway resistance (Rrs) was measured for Abi1 −lox mice and conditional KO mice treated with PBS or HDM extracts. (E) The sizes of lungs from Abi1 −lox mice and Abi1 smko mice are similar. Shown are lung images of two Abi1 −lox mice and two Abi1 smko mice. (F) ACh dose-response curves were determined for tracheal rings from Abi1 −lox mice and conditional KO mice treated with PBS or HDM extracts (n = 11–12, 6 males and 5–6 females for each group). Two-way ANOVA was used for statistical analysis. Data are means ± SEM. ∗∗p < 0.01; ∗p < 0.05. Data normalization is calculated as follows: Data of treated samples/Data of control samples.

Article Snippet: Lentivirus encoding Abi1 shRNA , Santa Cruz , sc-40306-V.

Techniques: In Vivo, Immunofluorescence, Knock-Out, Staining, Software, Control

Journal: iScience

Article Title: Abi1 mediates airway smooth muscle cell proliferation and airway remodeling via Jak2/STAT3 signaling

doi: 10.1016/j.isci.2022.103833

Figure Lengend Snippet: Proposed mechanism Stimulation with growth factors such as PDGF induces spatial distribution of Abi1, which facilitates the activation of the Jak2/STAT3 pathway. The association of Abi1 with Jak2 renders the receptor in active state. Abi1 is upregulated in ∼50% of asthmatic airway smooth muscle cultures, promoting the activation of Jak2 and STAT3, hyperplasia, and airway smooth muscle (ASM) thickening.

Article Snippet: Lentivirus encoding Abi1 shRNA , Santa Cruz , sc-40306-V.

Techniques: Activation Assay

Journal: iScience

Article Title: Abi1 mediates airway smooth muscle cell proliferation and airway remodeling via Jak2/STAT3 signaling

doi: 10.1016/j.isci.2022.103833

Figure Lengend Snippet:

Article Snippet: Lentivirus encoding Abi1 shRNA , Santa Cruz , sc-40306-V.

Techniques: Virus, Luciferase, shRNA, Control, Recombinant, Plasmid Preparation, Enzyme-linked Immunosorbent Assay, Kinase Assay, Software, Confocal Microscopy, Microscopy

Journal: The Journal of Cell Biology

Article Title: PTEN dephosphorylates Abi1 to promote epithelial morphogenesis

doi: 10.1083/jcb.201910041

Figure Lengend Snippet: Abi1 is a new PTEN substrate. (a) 5-d Pten −/− EBs stably transfected with PTEN G129E or C124S were immunoprecipitated with anti-phosphotyrosine antibody-agarose beads. Analysis of the immunoprecipitates by SDS-PAGE and silver staining followed by mass spectrometry identified that the WAVE regulatory complex (WRC) subunits Cyfip1, Nckap1, and Abi1 were selectively present in the C124S immunoprecipitates. (b) 5-d WT GFP EBs and Pten −/− EBs stably transfected with GFP, wild-type PTEN, PTEN G129E, or C124S were immunoprecipitated (IP) with anti-Abi1 antibody. The WRC subunits Cyfip1, Nckap1, and WAVE2 as well as PTEN were detected by immunoblotting. (c) Direct immunoblot analysis of the above EBs (IP input controls). (d) Substrate-trapping assay. 5-d Pten −/− EB lysates were incubated with N-terminally His-tagged wild-type or mutant PTEN bound to NTA agarose beads with NTA agarose beads only as a negative control. Affinity pull-down products were analyzed by immunoblotting for Abi1. (e) Lysates of 5-d EBs stably transfected with N-terminally Flag-tagged Abi1 were immunoprecipitated with Flag antibody-agarose beads. The immunoprecipitates were incubated either with or without 0.1 µg/µl active PTEN. Immunoblotting was performed using Abi1 phospho-specific antibodies to pY213 and pS216.

Article Snippet: The shRNA vectors targeting specifically mouse Abi1, Abi2, Nckap1, and the control scrambled sequences were obtained from OriGene.

Techniques: Stable Transfection, Transfection, Immunoprecipitation, SDS Page, Silver Staining, Mass Spectrometry, Western Blot, Incubation, Mutagenesis, Negative Control

Journal: The Journal of Cell Biology

Article Title: PTEN dephosphorylates Abi1 to promote epithelial morphogenesis

doi: 10.1083/jcb.201910041

Figure Lengend Snippet: PTEN promotes epiblast differentiation and polarization via down-regulation of the WRC. (a and b) 5-d wild-type GFP and Pten − −/− EBs stably transfected with Nckap1 shRNA or the scrambled control (SC) were analyzed by immunoblotting. Blots were quantified by densitometry, and the ratio of Nanog, Oct4, and keratin 8 to actin was plotted. Mean ± SD. *, P < 0.05 versus WT GFP and Pten −/− Nckap1 knockdown (KD). (c) 5-day EBs were coimmunostained for MPDZ and perlecan. (d) EBs with polarized epiblast epithelium were counted and plotted as a percentage of total EBs examined. n = 1,090–1,166 for each group. Mean ± SD. (e and f) 5-d normal EBs stably transfected either with pCXN2-WAVE2 or only the vector were subjected to immunoblot analysis. Blots were quantified and the ratio of Nanog, Oct4, and keratin 8 to actin was plotted. Mean ± SD. *, P < 0.05 versus the vector control. (g) 5-d EBs were immunostained for MPDZ and perlecan. (h) Formation of the polarized epiblast epithelium in 5-d EBs were quantified by phase-contrast microscopy and plotted as a percentage of total EBs examined. n = 724–750 for each group. Mean ± SD. *, P < 0.01 versus control.

Article Snippet: The shRNA vectors targeting specifically mouse Abi1, Abi2, Nckap1, and the control scrambled sequences were obtained from OriGene.

Techniques: Stable Transfection, Transfection, shRNA, Control, Western Blot, Knockdown, Plasmid Preparation, Microscopy

Journal: The Journal of Cell Biology

Article Title: PTEN dephosphorylates Abi1 to promote epithelial morphogenesis

doi: 10.1083/jcb.201910041

Figure Lengend Snippet: Abi1 is a new PTEN substrate. (a) 5-d Pten −/− EBs stably transfected with PTEN G129E or C124S were immunoprecipitated with anti-phosphotyrosine antibody-agarose beads. Analysis of the immunoprecipitates by SDS-PAGE and silver staining followed by mass spectrometry identified that the WAVE regulatory complex (WRC) subunits Cyfip1, Nckap1, and Abi1 were selectively present in the C124S immunoprecipitates. (b) 5-d WT GFP EBs and Pten −/− EBs stably transfected with GFP, wild-type PTEN, PTEN G129E, or C124S were immunoprecipitated (IP) with anti-Abi1 antibody. The WRC subunits Cyfip1, Nckap1, and WAVE2 as well as PTEN were detected by immunoblotting. (c) Direct immunoblot analysis of the above EBs (IP input controls). (d) Substrate-trapping assay. 5-d Pten −/− EB lysates were incubated with N-terminally His-tagged wild-type or mutant PTEN bound to NTA agarose beads with NTA agarose beads only as a negative control. Affinity pull-down products were analyzed by immunoblotting for Abi1. (e) Lysates of 5-d EBs stably transfected with N-terminally Flag-tagged Abi1 were immunoprecipitated with Flag antibody-agarose beads. The immunoprecipitates were incubated either with or without 0.1 µg/µl active PTEN. Immunoblotting was performed using Abi1 phospho-specific antibodies to pY213 and pS216.

Article Snippet: The shRNA vectors targeting specifically mouse Abi1, Abi2, Nckap1, and the control scrambled sequences were obtained from OriGene.

Techniques: Stable Transfection, Transfection, Immunoprecipitation, SDS Page, Silver Staining, Mass Spectrometry, Western Blot, Incubation, Mutagenesis, Negative Control

Journal: The Journal of Cell Biology

Article Title: PTEN dephosphorylates Abi1 to promote epithelial morphogenesis

doi: 10.1083/jcb.201910041

Figure Lengend Snippet: PTEN-mediated dephosphorylation of Abi1 leads to Abi1 degradation by calpains. (a) Wild-type EBs were cultured for 1–5 d and analyzed by immunoblotting. (b) 2- and 4-d wild-type and Pten −/− EBs were analyzed by immunoblotting. (c) E3.5 and E6.5 embryos were isolated from timed wild-type pregnant mice and analyzed by immunoblotting. (d) The protein level of Abi1 was higher in PTEN-negative (U87) human glioblastoma cells when compared with those that were PTEN positive (LN229). Subsequent restoration of PTEN expression in U87 cells down-regulated Abi1. (e) RT-PCR analysis for Abi1 and WAVE2 in 4-d wild-type and Pten −/− EBs. (f) 4-d EBs were treated with the lysosomal inhibitor chloroquine (CQ), the calpain inhibitor ALLN, or the proteasome inhibitor MG-132 for 16 h. Immunoblotting was performed to detect the WAVE complex subunits. (g) Wild-type, calpain small subunit 1 ( Capns1 )–, and Pten -null ( −/− ) EBs were cultured for 5 d and analyzed by immunoblotting. (h) Immunoblot analysis of 5-d wild-type and calpain 2 ( Capn2 )-null EBs showed that Abi1 and WAVE2 were significantly increased in Capn2 −/− EBs. Intact talin was detected in Capn2 −/− EBs, but not in wild-type EBs. Cleaved talin (the rod domain) was mainly detected in wild-type EBs. (i) Abi1 was immunoprecipitated from 5-d Pten −/− EBs with anti-Abi1 monoclonal antibody. Preimmune mouse IgG (mIgG) served as a control. The immunoprecipitates were incubated with active PTEN for 30 min, washed with PBS, and then treated with active porcine calpain 1 for an additional 30 min. Enzymatic digestion in the reverse order of above was also performed with mouse IgG immunoprecipitates as control. Digestion products were analyzed by immunoblotting using rabbit anti-Abi1 antibody.

Article Snippet: The shRNA vectors targeting specifically mouse Abi1, Abi2, Nckap1, and the control scrambled sequences were obtained from OriGene.

Techniques: De-Phosphorylation Assay, Cell Culture, Western Blot, Isolation, Expressing, Reverse Transcription Polymerase Chain Reaction, Immunoprecipitation, Incubation

Journal: The Journal of Cell Biology

Article Title: PTEN dephosphorylates Abi1 to promote epithelial morphogenesis

doi: 10.1083/jcb.201910041

Figure Lengend Snippet: PTEN regulates epiblast differentiation and polarization via down-regulation of Abi1. (a) 5-d EBs stably transfected with either Abi1 (pCXN2-Abi1) or vector alone (pCXN2) were analyzed by immunoblotting. (b) The blots were quantified by densitometry, and the ratio of pluripotency and differentiation markers relative to actin was plotted. Mean ± SD. *, P < 0.01 versus the pCXN2 group. Krt8, keratin 8; Stx3, syntaxin-3. (c and d) 5-d EBs were analyzed by live phase-contrast microscopy and immunostaining. endo, endoderm; epi, epiblast. EBs with the polarized epiblast epithelium were counted and plotted as a percentage of the total EBs examined. n = 967–1,021 for each group. *, P < 0.01 versus pCXN2. (e) Pten −/− ES cells were stably transfected with Abi1 shRNA (Abi1 KD), the scrambled control (SC), or GFP. 5-d EBs were analyzed by immunoblotting. Wild-type EBs stably expressing GFP served as controls. (f) Immunoblots of 5-d EBs show that knockdown of Abi1 down-regulated Nanog, Oct4, and c-Myc and up-regulated CRB3 and keratin 8. (g) The blots were quantified by densitometry, and the data were plotted. Mean ± SD. *, P < 0.001 versus WT GFP; # , P < 0.01 versus Pten −/− scrambled control. (h and i) Live phase-contrast and immunofluorescence micrographs show epiblast polarization in Abi1-knockdown EBs. EBs with the polarized epiblast epithelium were counted and plotted as a percentage of the total EBs examined. n = 1,025–1,186 for each group. Mean ± SD.

Article Snippet: The shRNA vectors targeting specifically mouse Abi1, Abi2, Nckap1, and the control scrambled sequences were obtained from OriGene.

Techniques: Stable Transfection, Transfection, Plasmid Preparation, Western Blot, Microscopy, Immunostaining, shRNA, Expressing, Immunofluorescence

Journal: The Journal of Cell Biology

Article Title: PTEN dephosphorylates Abi1 to promote epithelial morphogenesis

doi: 10.1083/jcb.201910041

Figure Lengend Snippet: Abi2 is mainly expressed in endoderm and not involved in epiblast polarization. (a) E6.5 mouse embryos were immunostained for Abi2 and basement membrane perlecan. Abi2 was mainly localized to the visceral endoderm (en; arrowheads). epi, epiblast. (b) Immunostaining of 5-d wild-type (WT) and Pten −/− EBs showed localization of Abi2 at the outer endoderm layer. (c) Immunoblots show that shRNA-mediated knockdown of Abi2 in Pten −/− EBs (clone 1 and 2, C1 and C2) did not alter the expression of WAVE2, although both Abi2 and WAVE2 were up-regulated in Pten −/− EBs. (d and e) 5-d EBs were immunostained for the apical polarity marker MPDZ and basement membrane perlecan. Nuclei were stained with DAPI. Quantification of epiblast epithelial formation by live phase microscopy revealed that knockdown of Abi2 failed to rescue epiblast polarity in Pten −/− EBs. Knockdown of both Abi1 and Abi2 did not show any synergistic effect. n = 374–414 for each group. Mean ± SD. *, P < 0.01 versus WT GFP; # , P < 0.01 versus Abi2 KD. SC, the scrambled shRNA.

Article Snippet: The shRNA vectors targeting specifically mouse Abi1, Abi2, Nckap1, and the control scrambled sequences were obtained from OriGene.

Techniques: Immunostaining, Western Blot, shRNA, Expressing, Marker, Staining, Microscopy

Journal: The Journal of Cell Biology

Article Title: PTEN dephosphorylates Abi1 to promote epithelial morphogenesis

doi: 10.1083/jcb.201910041

Figure Lengend Snippet: Pten/Abi1 double-knockout EBs. (a) PCR genotyping was performed with two sets of primers spanning the sgRNA targeting site using DNA extracted from 5-d Pten −/− and Pten −/− ;Abi1 −/− EBs (clones 3-7, 3-17, and 3-13). FKO1, forward knockout primer 1; R2, reverse primer 2; F1, forward primer 1; RKO1, reverve knockout primer 1. (b) Immunoblots show loss of Abi1 expression in 5-d Pten −/− ;Abi1 −/− EBs (clone 3-13). (c) RT-PCR analysis of 5-d Pten −/− ;Abi1 −/− EBs stably transfected with wild-type (WT) human Abi1 (hAbi1) and the mutants Y213F, S216A, and Y213F/S216A (YF/SA) all showed similar expression levels of human Abi1 mRNAs. (d) Wild-type and Pten −/− ;Abi1 −/− EBs stably transfected with Abi1 S216A, S216D, or GFP were cultured for 5 d and analyzed by immunoblotting. Actin served as a loading control. (e) The mRNA level of transfected human Abi1 (hAbi1) in 5-d EBs were analyzed by RT-PCR. 18S RNA served as an internal control. (f and g) 5-d wild-type EBs stably transfected with Abi1 S216A, S216D, or GFP were stained for MPDZ. Basement membrane was stained for perlecan. The formation of a polarized epiblast layer was quantified by live phase-contrast microscopy and plotted as a percentage of total EBs examined. n = 651–707 for each group. Mean ± SD. *, P < 0.05 S216D versus S216A. (h and i) 5-d Pten −/− ;Abi1 −/− EBs stably transfected with Abi1 S216A, S216D, or GFP were stained for MPDZ. Basement membrane was stained for perlecan. Epiblast polarization was quantified and plotted as a percentage of total EBs examined. Mean ± SD. *, P < 0.01 versus the S216A group. n = 544–590 for each group.

Article Snippet: The shRNA vectors targeting specifically mouse Abi1, Abi2, Nckap1, and the control scrambled sequences were obtained from OriGene.

Techniques: Double Knockout, Clone Assay, Knock-Out, Western Blot, Expressing, Reverse Transcription Polymerase Chain Reaction, Stable Transfection, Transfection, Cell Culture, Staining, Microscopy

Journal: The Journal of Cell Biology

Article Title: PTEN dephosphorylates Abi1 to promote epithelial morphogenesis

doi: 10.1083/jcb.201910041

Figure Lengend Snippet: Rescue Pten and Abi1 double-knockout EBs with PTEN dephosphorylation mutant Abi1. (a) 5-d wild-type, Pten −/− , and Pten −/− ; Abi1 −/− EBs were analyzed by immunoblotting. A nonspecific (NS) band appeared in the Abi1 blot using 1% SDS lysis buffer. (b) The blots were analyzed by densitometry, and the ratio of Oct4, c-Myc, and keratin 8 to actin was plotted. Mean ± SD. *, P < 0.01 versus WT. (c) 5-d EBs were coimmunostained for MPDZ and perlecan. (d) Live phase-contrast micrographs show less compaction of 2-d Pten −/− ; Abi1 −/− EBs. (e) Pten −/− ; Abi1 −/− EBs were stably reconstituted with wild-type (wt) Abi1, Abi1 Y213F, S216A, or Y213F/S216A (YF/SA) with wild-type GFP and Pten −/− GFP EBs serving as controls. 5-d EBs were subjected to immunoblot analysis. (f) The blots were quantified by densitometry, and the ratio of Nanog, Oct4, c-Myc, and keratin 8 to actin was plotted. Mean ± SD. *, P < 0.01 versus WT GFP; # ,P < 0.01 versus Pten −/− ; Abi1 −/− (double knockout [dKO]) Abi1wt. (g and h) 5-d EBs were coimmunostained for MPDZ and perlecan. EBs with the polarized epiblast epithelium were counted by live phase microscopy and plotted as a percentage of total EBs examined. n = 662–701 for each group. Mean ± SD. *, P < 0.01 versus WT GFP; # , P < 0.01 versus double-knockout Abi1 wild type.

Article Snippet: The shRNA vectors targeting specifically mouse Abi1, Abi2, Nckap1, and the control scrambled sequences were obtained from OriGene.

Techniques: Double Knockout, De-Phosphorylation Assay, Mutagenesis, Western Blot, Lysis, Stable Transfection, Microscopy

Journal: The Journal of Cell Biology

Article Title: PTEN dephosphorylates Abi1 to promote epithelial morphogenesis

doi: 10.1083/jcb.201910041

Figure Lengend Snippet: Immunofluorescence and chimeric EB analysis. (a) E3.5 embryos were stained for Abi1, WAVE2, and F-actin. Arrowheads define the inner cell mass. E6.5 embryos were stained for Abi1 and basement membrane nidogen. (b) 5-d EBs were immunostained for Abi1 and WAVE2. F-actin was stained with rhodamine-phalloidin. The basement membrane was immunostained for the laminin α1 chain or perlecan. Arrowheads point to the apical surface of polarized epiblast in wild-type EBs and the cell cortex in Pten −/− EBs. The upper right panels show colocalization of Abi1 and WAVE2 with F-actin at the apex of the polarized epiblast in wild-type EBs. The lower right panels show colocalization of Abi1 and WAVE2 with F-actin at the cell cortex in Pten −/− EBs. (c) Isolated epiblast cells were plated on gelatin-coated glass coverslips for 2 h and immunostained for Abi1. F-actin was stained with FITC-phalloidin. (d) Unlabeled wild-type ES cells were mixed with RFP-labeled wild-type, Pten −/− , or Pten −/− with Abi1 knockdown (KD) ES cells in a ratio of 2:1 and cultured in hanging drops for 24 h. The EBs were cultured in suspension for additional 2 d, and cryosections were stained with DAPI to reveal the nucleus. Arrowheads point to an aggregate of Pten −/− RFP cells. (e) The EBs formed in hanging drops were further cultured for 4 d and examined by live phase-contrast and fluorescence microscopy. Both wild-type RFP and Pten −/− Abi1 KD RFP ES cells contributed to the polarized epiblast epithelium (epi), whereas Pten −/− RFP cells formed an aggregate inside the cavity.

Article Snippet: The shRNA vectors targeting specifically mouse Abi1, Abi2, Nckap1, and the control scrambled sequences were obtained from OriGene.

Techniques: Immunofluorescence, Staining, Isolation, Labeling, Cell Culture, Fluorescence, Microscopy