Journal: JCI Insight

Article Title: Chemical inhibition of FBXO7 reduces inflammation and confers neuroprotection by stabilizing the mitochondrial kinase PINK1

doi: 10.1172/jci.insight.131834

Figure Lengend Snippet: (A) BEAS-2B cells were pretreated with MG132 (20 μM), leupeptin (100 μM), or DMSO (control, CON) for 30 minutes, and then CHX (40 μg/mL) was added to assay protein decay. (B) BEAS-2B cells were nucleofected with V5-tagged Fbxo7 plasmid at indicated amounts and incubated for 48 hours before immunoblotting. (C) BEAS-2B cells were nucleofected with 4 individual shRNAs separately and incubated for 72 hours before immunoblotting. (D) BEAS-2B cells were nucleofected with control shRNA or Fbxo7 shRNA for 72 hours and then treated with CHX (40 μg/mL) for half-life analysis. (E) In vitro ubiquitylation assays were performed with synthesized PINK1 combined with indicated recombinant proteins.

Article Snippet: Quantitative PCR (qPCR) was performed using TaqMan qPCR reagents: qPCR master mix solution (Applied Biosystems, 4369016) and TaqMan probes for PINK1 (Applied Biosystems, Hs00260868_ml), FBX07 (Applied Biosystems, Hs00201825_ml), and GAPDH (Applied Biosystems, Hs02786624_g1).

Techniques: Control, Plasmid Preparation, Incubation, Western Blot, shRNA, In Vitro, Synthesized, Recombinant

Journal: JCI Insight

Article Title: Chemical inhibition of FBXO7 reduces inflammation and confers neuroprotection by stabilizing the mitochondrial kinase PINK1

doi: 10.1172/jci.insight.131834

Figure Lengend Snippet: (A) Structural analysis of the Fbxo7 FP domain. Docking study of a candidate inhibitor BC1464 within the Fbxo7-FP domain suggests hydrophilic interactions of residues GLN215, LYS235, and LYS266 with BC1464. (B) His pull down Fbxo7 protein was exposed to BC1464 at indicated concentrations. TnT-synthesized PINK1 protein was then incubated with drug-bound Fbxo7 beads overnight. The eluate was subjected to immunoblotting. The relative amounts of PINK1 detected in the pull-downs were normalized to loading and quantified; data are shown as means ± SEM (n = 3). (C) BEAS-2B cells were incubated with BC1464 or the control compound BC1465 at the indicated concentrations for 16 hours before immunoblotting. (D) BEAS-2B cells were pretreated with BC1464 or BC1465 (1 μg/mL) for 16 hours, and the cells were then incubated with CHX (40 μg/mL). The cells were collected at indicated time points for immunoblot analysis. (E) BEAS-2B cells were nucleofected with control or Fbxo7 siRNA (50 pg) and incubated for 72 hours. BC1464 was then administrated at indicated concentration for an additional 18-hour incubation. The cell lysates were subjected to immunoblotting.

Article Snippet: Quantitative PCR (qPCR) was performed using TaqMan qPCR reagents: qPCR master mix solution (Applied Biosystems, 4369016) and TaqMan probes for PINK1 (Applied Biosystems, Hs00260868_ml), FBX07 (Applied Biosystems, Hs00201825_ml), and GAPDH (Applied Biosystems, Hs02786624_g1).

Techniques: Synthesized, Incubation, Western Blot, Control, Concentration Assay

Journal: JCI Insight

Article Title: Chemical inhibition of FBXO7 reduces inflammation and confers neuroprotection by stabilizing the mitochondrial kinase PINK1

doi: 10.1172/jci.insight.131834

Figure Lengend Snippet: (A) Human SH-SY5Y cells stably expressing PINK1-FLAG were treated with the indicated amounts of BC1464 for 3 hours, followed by incubation for 1 hour in the presence of CHX (10 μg/mL) before immunoblotting analysis. (B) Densitometry analysis revealed an IC50 of ~5.2 μg/mL by nonlinear regression (n = 6; interpolated mean with 95% CI bands; Prism v 8.2.1, 2019, sigmoidal 4PL; 32 Degrees of Freedom, R2 = 0.81; Hill coefficient = 2.9). (C and D) Duolink Proximity Ligation Assay of Fbxo7 and PINK1-FLAG in SH-SY5Y cells treated with BC1464 titration. EC50 ~1.4 μM by nonlinear regression (mean ± SEM; **P < 0.01; ***P < 0.001; ****P < 0.0001 vs. vehicle by 1-way ANOVA with Dunnett’s post hoc test). PLA is detected with red fluorescence, and nuclei counterstained using Hoescht 33342. Scale bar: 20 μm. (E–G) SH-SY5Y cells were treated with vehicle or the indicated compound (5 μg/mL) for 16 hours and then lysed for Western blot for ubiquitin phosphorylated at S65 (E) and phosphorylated PKAc (F). (G) Densitometric analysis of the indicated phospho-epitopes. (mean ± SD, n = 3 wells, representative of 2 independent experiments; 1-way ANOVA with Bonferroni’s post hoc test). (H) SH-SY5Y cells were treated with the indicated concentrations of MPP+ or vehicle control in the presence of 5 μg/mL BC1465 or BC1464 for 24 hours. Cell viability was measured using AlamarBlue fluorescence intensity. (I) Mouse E16 primary cortical neurons were treated with the indicated concentrations of MPP+ in the presence of either 5 μg/mL BC1465 or BC1464 for 24 hours, and cell numbers were measured as in H. Data in H and I are shown as mean ± SD; n = 4 independent experiments; 1-way ANOVA with Bonferroni-corrected 2-tailed t test.

Article Snippet: Quantitative PCR (qPCR) was performed using TaqMan qPCR reagents: qPCR master mix solution (Applied Biosystems, 4369016) and TaqMan probes for PINK1 (Applied Biosystems, Hs00260868_ml), FBX07 (Applied Biosystems, Hs00201825_ml), and GAPDH (Applied Biosystems, Hs02786624_g1).

Techniques: Stable Transfection, Expressing, Incubation, Western Blot, Proximity Ligation Assay, Titration, Fluorescence, Ubiquitin Proteomics, Control

Journal: JCI Insight

Article Title: Chemical inhibition of FBXO7 reduces inflammation and confers neuroprotection by stabilizing the mitochondrial kinase PINK1

doi: 10.1172/jci.insight.131834

Figure Lengend Snippet: (A) Primary fibroblasts from a control subject and 2 Parkinson’s disease patients, one with the G2019S mutation and the other with the R1441G mutation of LRRK2, were treated with 6-OHDA for 16 hours in the presence of DMSO, BC1464, or BC1465 and cell death percentage assessed. (B and C) Fibroblasts from a LRRK2-G2019S patient were treated with 6-OHDA or MPP+, with BC1464 or BC1465 added simultaneously or 2–6 hours later. Similar results were obtained for the other fibroblast lines (Supplemental Figure 4). Mean ± SD, n = 3 independent experiments, ANOVA with post hoc 2-tailed t test for A–C. (D) The fibroblasts from each subject were treated with DMSO vehicle, BC1464, and BC1465 and analyzed for PINK1 and FBXO7 mRNA by RT-qPCR (mean ± SD, n = 3 independent experiments, 1-way ANOVA, P > 0.548). (E and F) Late NPCs from SNCA triplication iPSC line were treated with Neurobasal media (Neg), DMSO, BC1464, or BC1465 as indicated and analyzed for cell death using propidium iodide (mean ± SD, n = 3–5 independent experiments, ANOVA with post hoc t test). Scale bar: 100 μm.

Article Snippet: Quantitative PCR (qPCR) was performed using TaqMan qPCR reagents: qPCR master mix solution (Applied Biosystems, 4369016) and TaqMan probes for PINK1 (Applied Biosystems, Hs00260868_ml), FBX07 (Applied Biosystems, Hs00201825_ml), and GAPDH (Applied Biosystems, Hs02786624_g1).

Techniques: Control, Mutagenesis, Quantitative RT-PCR

Journal: Experimental and Therapeutic Medicine

Article Title: Targeting translocator protein protects against myocardial ischemia/reperfusion injury by alleviating mitochondrial dysfunction

doi: 10.3892/etm.2024.12638

Figure Lengend Snippet: TSPO regulates autophagy and mitophagy via related pathways following A/R stimulation. (A) Representative fluorescence images and quantitative analysis of LC3B expression. Scale bar, 20 µm. (B) Western blotting and (C) densitometry analysis of LC3B, Beclin1 and ATG5 expression levels in si-NC and si-TSPO H9c2 cells with or without A/R stimulation. (D) Western blotting and (E) densitometry analysis of p-PI3K, PI3K, p-Akt, Akt, p-mTOR and mTOR expression levels. (F) Western blotting and (G) densitometry analysis of P62, PINK1 and Parkin expression levels. (H) Western blotting and (I) densitometry analysis of Mfn2 and Drp1 protein expression levels. * P<0.05, ** P<0.01, *** P<0.001, **** P<0.0001. TSPO, translocator protein; A/R, anoxia/reoxygenation; si, small interfering; NC, negative control; p-, phosporyalated.

Article Snippet: Membranes were blocked with 5% skimmed milk at room temperature for 1 h, then incubated with the following primary antibodies overnight at 4˚C: GAPDH (cat. no. HRP-60004; ProteinTech Group, Inc.), cleaved-caspase-3 (cat. no. 9661S; Cell Signaling Technology, Inc.), caspase-3 (cat. no. 66470-1-Ig; ProteinTech Group, Inc.), Bax (cat. no. 2772S; Cell Signaling Technology, Inc.), Bcl-2 (cat. no. 26593-1-AP; ProteinTech Group, Inc.), TSPO (cat. no. ab109497; Abcam), LC3B (cat. no. ab51520; Abcam), ATG5 (cat. no. 12994S; Cell Signaling Technology, Inc.), Beclin 1 (cat. no. 66665-1-Ig; ProteinTech Group, Inc.), P62 (23214S; ProteinTech Group, Inc.), PINK1 (cat. no. 23274-1-AP; ProteinTech Group, Inc.), Parkin (cat. no. 66674-1-Ig; ProteinTech Group, Inc.), Mfn2 (cat. no. 12186-1-AP; ProteinTech Group, Inc.), Drp1 (cat. no. 12957-1-AP; ProteinTech Group, Inc.), ATP5B (cat. no. 17247-1-AP; ProteinTech Group, Inc.), PI3K (cat. no. 4257S; Cell Signaling Technology, Inc.), phosphorylated (p)-PI3K (cat. no. 4228S; Cell Signaling Technology, Inc.), Akt (cat. no. 9272S, Cell Signaling Technology, Inc.), p-Akt (cat. no. 4060S; Cell Signaling Technology, Inc.), mTOR (cat. no. 2983S; Cell Signaling Technology, Inc.), p-mTOR (cat. no. 2971S; Cell Signaling Technology, Inc.), PKR-like ER kinase (PERK; cat. no. ab229912; Abcam), p-PERK (cat. no. 3179S; Cell Signaling Technology, Inc.), ATF6 (cat. no. 24169-1-AP; ProteinTech Group, Inc.) and inositol-requiring enzyme 1 (IRE1; cat. no. bs16696R, BIOSS).

Techniques: Fluorescence, Expressing, Western Blot, Negative Control

Journal: Cell Cycle

Article Title: PTEN Physically Interacts with and Regulates E2F1-mediated Transcription in Lung Cancer

doi: 10.1080/15384101.2017.1388970

Figure Lengend Snippet: PTEN-4A is a more Potent Tumor Suppressor than PTEN-WT. (A) In the PTEN-4A mutant protein, the serine-threonine cluster (S380, T382, T383, S385) in the PTEN C-tail is replaced by alanine residues, converting it into an open/active conformer. (B) Lentiviral transduction, followed by puromycin selection, was used to stably express Flag PTEN-WT and Flag PTEN-4A proteins in PTEN deficient NSCLC cell line H1299. The expression levels of both PTEN-WT and PTEN-4A proteins were similar. (C) PTEN-4A inhibited cell proliferation significantly more than PTEN-WT in a standard cell proliferation assays (i). Even in the presence of a proliferative signal such as leptin, PTEN-4A remained a potent inhibitor of cell proliferation (ii). Data are derived from experiments performed in triplicates ± S.E. (n = 3, *p<0.05). (D) Cell proliferation assays on H1299, PTEN-WT and PTEN-4A cells measured by the ECIS method also revealed that PTEN-4A (black line) significantly suppressed cell proliferation as compared to PTEN-WT (pink line). (E) Likewise, PTEN-4A (blue line) significantly inhibited the migratory potential of H1299 lung cancer cells as compared to PTEN-WT (red line).

Article Snippet: 809 pCDNA3 GFP PTEN WT, and pCDNA3 GFP PTEN A4 plasmids were a gift from Dr. William Sellers (Addgene plasmid #10750, #10753, #10744, #10746, #10765, #10759 and #10760) [ 15 , 80 ].

Techniques: Mutagenesis, Transduction, Selection, Stable Transfection, Expressing, Derivative Assay

Journal: Cell Cycle

Article Title: PTEN Physically Interacts with and Regulates E2F1-mediated Transcription in Lung Cancer

doi: 10.1080/15384101.2017.1388970

Figure Lengend Snippet: PTEN-4A Preferentially Localizes to the Nucleus. (A) Total nuclear proteins isolated from H1299 cells stably expressing Flag-tagged PTEN-WT and PTEN-4A revealed that PTEN-4A preferentially localized to the nucleus as compared to PTEN-WT, as examined by immunoblotting with Flag antibodies. (B) Lamin B1, an exclusively nuclear protein, was used as a loading control and to normalize densitometric values obtained for Flag-tagged PTEN-WT and PTEN-4A protein levels in the nucleus. Levels of PTEN-4A was approximately 2.5-fold higher in the nucleus than PTEN-WT. (C) Preferential nuclear localization for PTEN-4A detected by immunofluorescence signals of GFP-tagged PTEN-WT and GFP-tagged PTEN-4A protein expression in 293T cells. Expression of H2B mCherry stained the nucleus red. Data are derived from three independent experiments ± S.E. (n = 3, *p<0.05).

Article Snippet: 809 pCDNA3 GFP PTEN WT, and pCDNA3 GFP PTEN A4 plasmids were a gift from Dr. William Sellers (Addgene plasmid #10750, #10753, #10744, #10746, #10765, #10759 and #10760) [ 15 , 80 ].

Techniques: Isolation, Stable Transfection, Expressing, Western Blot, Immunofluorescence, Staining, Derivative Assay

Journal: Cell Cycle

Article Title: PTEN Physically Interacts with and Regulates E2F1-mediated Transcription in Lung Cancer

doi: 10.1080/15384101.2017.1388970

Figure Lengend Snippet: PTEN-4A Preferentially Inhibits E2F1-mediated Transcription. (A) Transcriptional assays on four E2F1-responsive promoter-luciferase reporter plasmids co-transfected with E2F1 expression plasmid in H1299 cancer cells indicate that the four promoters are all activated by E2F1 expression. (B) PTEN-4A preferentially suppresses (∼3 fold) the transcription from an artificial E2F1 reporter (E2F-Luc) compared to PTEN-WT. (C) PTEN-4A suppresses transcription mediated by the native E2F1 promoter 2.5 fold more than PTEN-WT. (D) Both PTEN-WT and PTEN-4A likewise suppress transcription mediated by the cyclin E1 promoter. However, PTEN-4A suppresses transcription by the cyclin E1-promoter approximately 2 fold more than PTEN-WT. (E) Both PTEN-WT and PTEN-4A suppress cyclin D1-transcription. However, PTEN-4A suppresses transcription by the cyclin D1-promoter 1.5 fold more than PTEN-WT. (F) As compared to PTEN-4A (lane 2), PTEN catalytic mutants, PTEN C124S 4A (lane 3) and PTEN G129E 4A (lane 4) cannot suppress E2F1-mediated transcription as detected by E2F1-Luc reporter activity in H1299 cells. (G) A PTEN-4A mutant lacking the nuclear localization sequence of PTEN (PTEN d32-4A) cannot suppress E2F1-mediated transcription (lanes 2 and 3), compared to PTEN-4A. (H and I) PTEN-WT, PTEN-4A, PTEN C124S-4A, PTEN G129E-4A and PTEN d32-4A containing expression plasmids stably express the proteins upon transient transfection. (J) Expression of GFP-tagged PTEN-4A and PTEN d32-4A followed by immunofluorescence detection indicate that PTEN d32-4A is excluded from the nucleus. The nucleus is stained red by using a H2B mCherry construct. Positive co-localization (yellow color) was counted independently by two researchers using ImageJ after merging Texas Red and GFP field images. Ten fields/transfection were recorded by each of the two researchers and the data obtained are represented from three independent experiments carried out in triplicate, represented as mean ± S.E. (*p value ≤0.05).

Article Snippet: 809 pCDNA3 GFP PTEN WT, and pCDNA3 GFP PTEN A4 plasmids were a gift from Dr. William Sellers (Addgene plasmid #10750, #10753, #10744, #10746, #10765, #10759 and #10760) [ 15 , 80 ].

Techniques: Luciferase, Transfection, Expressing, Plasmid Preparation, Activity Assay, Mutagenesis, Sequencing, Stable Transfection, Immunofluorescence, Staining, Construct

Journal: Cell Cycle

Article Title: PTEN Physically Interacts with and Regulates E2F1-mediated Transcription in Lung Cancer

doi: 10.1080/15384101.2017.1388970

Figure Lengend Snippet: PTEN-4A Physically Interacts with E2F1 Protein and at E2F1 DNA Binding Sites on Chromatin. (A) Both Flag-PTEN-WT and Flag-PTEN-4A proteins physically associated with the HA-E2F1 containing protein complex in co-immunoprecipitation assays performed on cell extracts derived from 293T cells (indicated by arrows). (B) Endogenous PTEN and E2F1 proteins interact in 293T cell nuclear extracts. (C) Chromatin-immunoprecipitation assays indicate that both PTEN-4A and E2F1 associated with E2F1 DNA-binding sites on the chromatin at the native cyclin D1 and cyclin E1 promoters in the PTEN-4A stable cell line.

Article Snippet: 809 pCDNA3 GFP PTEN WT, and pCDNA3 GFP PTEN A4 plasmids were a gift from Dr. William Sellers (Addgene plasmid #10750, #10753, #10744, #10746, #10765, #10759 and #10760) [ 15 , 80 ].

Techniques: Binding Assay, Immunoprecipitation, Derivative Assay, Chromatin Immunoprecipitation, Stable Transfection

Journal: Cell & Bioscience

Article Title: Electric signals counterbalanced posterior vs anterior PTEN signaling in directed migration of Dictyostelium

doi: 10.1186/s13578-021-00580-x

Figure Lengend Snippet: pten null mutation significantly reduced electrotaxis at lower voltages, which was recovered by re-expression of pten . a-c Images selected from time-lapseVideos showing the migrating cells. Red lines and blue arrows are cell trajectories and endpoints over 15 min. d-f Composite trajectories of migrating cells with the starting points placed at the origin. b and e pten − cells showed significantly reduced electrotaxis. c and f The re-expression of wild type pten on pten − (PTEN/ pten − , or pten rescue) completely reversed the electrotaxis defects in the pten − cells. g , h voltage dependence migration directedness and trajectory speed. *, P < 0.05; **, P < 0.01, compared with no EF control; #, P < 0.05; ##, P < 0.01 compared between WT/ pten rescue groups and pten − group, one-way ANOVA. A minimal 150 cells were analyzed from each experimental group. A minimal 3 repeats were conducted for all conditions investigated. EF = 10 V/cm in a–f , with cathode to the right

Article Snippet: For immunocytochemistry staining, cells were fixed using 1% PFA and stained for PTEN-GFP (rabbit anti-GFP, Millipore, AB3080), phospho-PTEN (Ser380/Thr382/383, Cell Signaling, Cat#9549), myosin II (mouse mAb 56–396-5), PHCrac-GFP (Mouse mAb anti-GFP, Thermo Fisher, A-11120 and F-actin (TRITC-phalloidin).

Techniques: Mutagenesis, Expressing, Migration, Control

Journal: Cell & Bioscience

Article Title: Electric signals counterbalanced posterior vs anterior PTEN signaling in directed migration of Dictyostelium

doi: 10.1186/s13578-021-00580-x

Figure Lengend Snippet: PTEN-GFP redistributed to the posterior plasma membrane of electrotaxing cells, which is depended on actin polymerization. a , b Dictyostelium cells did not show the membrane recruitment of PTEN-GFP in the absence of EF. ( c–e ) An applied EF induced asymmetrical redistribution of PTEN-GFP to the posterior of Dictyostelium cells. ( f and g ) Latrunculin A treatment abolished asymmetrical redistribution of PTEN, while plasma membrane recruitment was still maintained (see J, ##: P < 0.01, compared between Latraculin A treated and control. h and i Washout latrunculin A restored the asymmetrical redistribution of PTEN. b, e, g and i representative line scan of fluorescence intensity of PTEN-GFP for marked cells in a, d, f and h , respectively. ( j ) GFP intensities at the plasma membrane were determined relative to that in the cytosol. Values represent the mean ± s.d. **, P < 0.01 compared between the posterior membrane of no EF vs EF treated group; $$, P < 0.01 compared between posterior and anterior membrane within EF treated cells; ##, P < 0.01 compared between anterior within “LatA + EF” and “EF only” group; ns , P > 0.05 compared between the posterior and anterior membrane of “no EF” and “LatA + EF” group. k Membrane fraction and the whole-cell lysates from cells treated with the indicated duration of EFs were analyzed by immunoblotting with antibodies against phospho-PTEN (pS380) or GFP (PTEN-GFP), respectively. l The PTEN proteins were immunopurified from Dictyostelium cells treated with varies duration of EF, and phosphatase activities were measured. *P < 0.05; **P < 0.01 compared with no EF group. n ⩾ 3

Article Snippet: For immunocytochemistry staining, cells were fixed using 1% PFA and stained for PTEN-GFP (rabbit anti-GFP, Millipore, AB3080), phospho-PTEN (Ser380/Thr382/383, Cell Signaling, Cat#9549), myosin II (mouse mAb 56–396-5), PHCrac-GFP (Mouse mAb anti-GFP, Thermo Fisher, A-11120 and F-actin (TRITC-phalloidin).

Techniques: Clinical Proteomics, Membrane, Control, Fluorescence, Western Blot

Journal: Cell & Bioscience

Article Title: Electric signals counterbalanced posterior vs anterior PTEN signaling in directed migration of Dictyostelium

doi: 10.1186/s13578-021-00580-x

Figure Lengend Snippet: PTEN posterior membrane translocation proceeded that of myosin and the electrotactic response of the cells. EFs were applied at time zero in all experiments. a Time-lapse analysis of fluorescence intensity ratio comparing GFP translocation toward posterior vs anterior plasma membrane in EF. The asymmetric redistribution of myosin II-GFP (blue line) showed significant delay compared to that of PTEN-GFP (red line). Such asymmetric relocalization was fully abolished in pten − cells (orange line). EF triggered a sharp shortening of the average pseudopod length at the posterior region (green line), while increased that at the anterior of the electrotaxing WT cells (black line). b Time-lapse analysis of the directional migration of WT (red line) and pten − cells (blue line). EF = 10 V/cm. c - f Comparison of the re-establishment of the PTEN-GFP and myosin II-GFP posterior membrane translocation post latrunculin A washout in EF. g Time-lapse analysis of fluorescence intensity ratio comparing GFP translocation toward posterior vs anterior plasma membrane post latrunculin A washout in EF. The asymmetric redistribution of PTEN-GFP (red line) preceded that of myosin II-GFP (blue line). EF triggered elongation of the average pseudopod length was re-established at the anterior region (black line), but not at the posterior region of the cells (green line). h Membrane fraction cell lysates from the cells treated with the indicated duration of EFs were analyzed by immunoblotting with antibodies against plasma membrane fraction of myosin II. i Band intensity was quantified (n ≥ 3)

Article Snippet: For immunocytochemistry staining, cells were fixed using 1% PFA and stained for PTEN-GFP (rabbit anti-GFP, Millipore, AB3080), phospho-PTEN (Ser380/Thr382/383, Cell Signaling, Cat#9549), myosin II (mouse mAb 56–396-5), PHCrac-GFP (Mouse mAb anti-GFP, Thermo Fisher, A-11120 and F-actin (TRITC-phalloidin).

Techniques: Membrane, Translocation Assay, Fluorescence, Clinical Proteomics, Migration, Comparison, Western Blot

Journal: Cell & Bioscience

Article Title: Electric signals counterbalanced posterior vs anterior PTEN signaling in directed migration of Dictyostelium

doi: 10.1186/s13578-021-00580-x

Figure Lengend Snippet: Myosin II redistributed to the posterior plasma membrane of electrotaxing cells, which is depended on actin polymerization and PTEN signaling. a–e Time-lapse images showing translocation and asymmetric redistribution of Myosin II to the posterior plasma membrane of the WT cells in EF (10 V/cm). f, g Latrunculin A treatment abolished asymmetric membrane translocation of myosin-GFP in EF. EF = 30 V/cm. Similar results were observed in 34 cells from a minimum of three independent experiments. h–k Washout of latrunculin A restored the asymmetrical redistribution of myosin GFP to the posterior plasma membrane of the electrotaxing WT cells. See Movie “Smovie11_myosinGFP_EF_LatA_washout”. EF = 30 V/cm. The same results were observed in 53 cells from 3 independent experiments. l-q Myosin-GFP/ pten − lost plasma membrane redistribution of myosin II in EF (30 V/cm). e, g, k, m, o, q fluorescence intensity line-scan of myosin-GFP along the lines indicated in d, f, j, l, n and p , respectively. r GFP intensities at the plasma membrane were determined relative to that in the cytosol. Values represent the mean ± s.d. ( n ⩾15)

Article Snippet: For immunocytochemistry staining, cells were fixed using 1% PFA and stained for PTEN-GFP (rabbit anti-GFP, Millipore, AB3080), phospho-PTEN (Ser380/Thr382/383, Cell Signaling, Cat#9549), myosin II (mouse mAb 56–396-5), PHCrac-GFP (Mouse mAb anti-GFP, Thermo Fisher, A-11120 and F-actin (TRITC-phalloidin).

Techniques: Clinical Proteomics, Membrane, Translocation Assay, Fluorescence

Journal: Cell & Bioscience

Article Title: Electric signals counterbalanced posterior vs anterior PTEN signaling in directed migration of Dictyostelium

doi: 10.1186/s13578-021-00580-x

Figure Lengend Snippet: PTEN dependent anterior plasma membrane translocation of PH-Crac in EF. a-b PHCrac-GFP was redistributed asymmetrically to the leading edge of the electrotaxing WT cells. EF = 30 V/cm. c Latrunculin A treatment did not affect the EF-triggered asymmetric redistribution of PH-Crac in WT cells. d PH-Crac anterior relocalization was abolished in pten − cells, while evenly distributed global membrane translocation remained. e Time-lapse analysis of fluorescence intensity ratio comparing membrane translocation of PTEN-GFP (posterior vs anterior) and PHCrac-GFP (anterior vs posterior) plasma membrane in EF. The asymmetric relocalization of PHCrac-GFP was fully abolished in pten − cells. f Relative fluorescence intensity analysis further confirmed that the EF-triggered anterior redistribution of PHCrac-GFP was not affected by latrunculin A in WT cells, and significantly increased membrane translocation was recorded at both anterior and posterior of pten − cells. g Membrane fraction cell lysates from the cells treated with the indicated duration of EFs were analyzed by immunoblotting with antibodies against PHCrac-GFP in WT or pten − cells, respectively

Article Snippet: For immunocytochemistry staining, cells were fixed using 1% PFA and stained for PTEN-GFP (rabbit anti-GFP, Millipore, AB3080), phospho-PTEN (Ser380/Thr382/383, Cell Signaling, Cat#9549), myosin II (mouse mAb 56–396-5), PHCrac-GFP (Mouse mAb anti-GFP, Thermo Fisher, A-11120 and F-actin (TRITC-phalloidin).

Techniques: Clinical Proteomics, Membrane, Translocation Assay, Fluorescence, Western Blot

Journal: Cell & Bioscience

Article Title: Electric signals counterbalanced posterior vs anterior PTEN signaling in directed migration of Dictyostelium

doi: 10.1186/s13578-021-00580-x

Figure Lengend Snippet: PTEN mediated electrotaxis via maintenance of biased pseudopod protrusion toward the cathodal forwarding direction. PHCrac-GFP containing pseudopod protrusions were quantified. a-b EF triggered a biased pseudopod redistribution at the anterior of electrotaxing WT cells. Two representative consecutive time points are shown. Time interval = 20 s. c and g Distribution of total pseudopods number of 12 EF-treated WT or pten − cells, respectively. d and h Distribution of total time spent in each category from pseudopods of 12 EF-treated WT or pten − cells, respectively. e and f EF-triggered pseudopod anterior redistribution was fully abolished in pten − cells. Two representative consecutive time points are shown. Time interval = 20 s ( i ) Comparing the average new pseudopods number generated between a single WT and pten − cell, in the absence or presence of EF for 320 s. j Comparing total pseudopods time generated between a single WT and pten − cell, in the absence or presence of EF for 320 s. k Comparison studies of pseudopod number and time in orientation of 12 EF-treated WT or pten − cells, respectively. Recording time = 320 s. All data are confirmed from minimal 3 independent experiments

Article Snippet: For immunocytochemistry staining, cells were fixed using 1% PFA and stained for PTEN-GFP (rabbit anti-GFP, Millipore, AB3080), phospho-PTEN (Ser380/Thr382/383, Cell Signaling, Cat#9549), myosin II (mouse mAb 56–396-5), PHCrac-GFP (Mouse mAb anti-GFP, Thermo Fisher, A-11120 and F-actin (TRITC-phalloidin).

Techniques: Generated, Comparison

Journal: Cell & Bioscience

Article Title: Electric signals counterbalanced posterior vs anterior PTEN signaling in directed migration of Dictyostelium

doi: 10.1186/s13578-021-00580-x

Figure Lengend Snippet: Immunocytochemistry staining for PTEN, myosin II, PH-Crac and F-acin in EF. a–d PTEN-GFP and myosin II colocalized at the posterior plasma membrane of the electrotaxing cells. e–h Phospho-PTEN and PHCrac-GFP asymmetrically redistributed to the posterior and anterior membrane of the electrotaxing cells, respectively. i–l PHCrac-GFP and F-actin colocalized at the anterior plasma membrane of the electrotaxing cells. m F-actin leading-edge recruitment was abolished in pten- cells. n Distribution of F-actin positive pseudopods number of EF-treated WT or pten − cells, respectively

Article Snippet: For immunocytochemistry staining, cells were fixed using 1% PFA and stained for PTEN-GFP (rabbit anti-GFP, Millipore, AB3080), phospho-PTEN (Ser380/Thr382/383, Cell Signaling, Cat#9549), myosin II (mouse mAb 56–396-5), PHCrac-GFP (Mouse mAb anti-GFP, Thermo Fisher, A-11120 and F-actin (TRITC-phalloidin).

Techniques: Immunocytochemistry, Staining, Clinical Proteomics, Membrane