Journal: Kidney international

Article Title: Amiloride blocks lithium entry through the sodium channel thereby attenuating the resultant nephrogenic diabetes insipidus.

doi: 10.1038/ki.2009.91

Figure Lengend Snippet: Figure 1 | mCCDc11 cells: a proper cell model to study lithium-NDI. (a) mCCDc11 cells were grown to confluence, treated for the indicated times (in hours) with 1 nM dDAVP, and subjected to AQP2 immunoblotting or, after blotting, stained with coomassie blue. Non-glycosylated (29 kDa) and complex-glycosylated (40–45 kDa) forms of AQP2, and an a-specific band of 35 kDa, are detected. (b) mCCDc11 cells grown as previously described were treated for 96 h with 1 nM dDAVP, and for the last 24 or 48 h, in the absence () or presence of 1 mM lithium at the basolateral side and 1 or with 10 mM lithium at the apical side. Cells were lysed and immunoblotted for AQP2. Blots were also stained with coomassie blue. Molecular masses (in kDa) are indicated on the left. The signals for non-glycosylated and complex-glycosylated AQP2 were densitometrically quantified and normalized for coomassie blue staining. Mean values of normalized AQP2 expression per condition are given as the percentage of control (±s.e.m.) and were determined from three independent filters per condition. Significant differences (Po0.05) from control () are indicated by an asterisk.

Article Snippet: Polyacrylamide gel electrophoresis, blotting, and blocking of the PVDF membranes were carried out as described.42 The membranes were incubated for 16 h with affinitypurified rabbit AQP2 antibodies (1:3000 dilution),43 affinitypurified rabbit anti-v1 H-ATPase antibodies (1:2000-dilution; gift from Dr S Nielsen, Denmark), rabbit anti-Ser9-GSK3b (1:1000 dilution; Cell Signaling Technology, Beverly, MA, USA), mouse antiGSK3b (1:5000 dilution; BD Transduction Laboratories, San Jose, CA, USA), or with mouse anti-tubulin antibodies (1:100,000 dilution; gift from Dr Kreis, Switzerland) in Tris-buffered saline Tween-20 supplemented with 1% non-fat dried milk.

Techniques: Western Blot, Staining, Expressing, Control

Journal: Kidney international

Article Title: Amiloride blocks lithium entry through the sodium channel thereby attenuating the resultant nephrogenic diabetes insipidus.

doi: 10.1038/ki.2009.91

Figure Lengend Snippet: Figure 2 | ENaC blockers reduce lithium-induced AQP2 downregulation in mCCDc11 cells. (a) Confluent mCCDc11 monolayers were treated for 96 h with 1 nM dDAVP and incubated for the last 48 h in the absence () or presence ( þ ) of lithium and/or with 10 mM amiloride as indicated. At the basolateral and apical side, 1 and 10 mM lithium were used, respectively. (b) Confluent mCCDc11 monolayers were treated described earlier with 10 mM lithium with/without 10 mM benzamil (Li þ Ben) at the apical side for the last 24 h. (c) mCCDc11cells were grown as previously described and treated for the last 12 h in medium containing a lower sodium chloride concentration at the apical side only, with or without lithium (indicated). (a–c) Cells were lysed and immunoblotted for AQP2. Molecular masses (in kDa) are indicated on the left. Semiquantification of the AQP2 signals, normalization, and statistical analysis were carried out as described in the Figure 1 caption. Mean values were determined from three independent filters per condition. Significant differences (Po0.05) are indicated by an asterisk.

Article Snippet: Polyacrylamide gel electrophoresis, blotting, and blocking of the PVDF membranes were carried out as described.42 The membranes were incubated for 16 h with affinitypurified rabbit AQP2 antibodies (1:3000 dilution),43 affinitypurified rabbit anti-v1 H-ATPase antibodies (1:2000-dilution; gift from Dr S Nielsen, Denmark), rabbit anti-Ser9-GSK3b (1:1000 dilution; Cell Signaling Technology, Beverly, MA, USA), mouse antiGSK3b (1:5000 dilution; BD Transduction Laboratories, San Jose, CA, USA), or with mouse anti-tubulin antibodies (1:100,000 dilution; gift from Dr Kreis, Switzerland) in Tris-buffered saline Tween-20 supplemented with 1% non-fat dried milk.

Techniques: Incubation, Concentration Assay

Journal: Kidney international

Article Title: Amiloride blocks lithium entry through the sodium channel thereby attenuating the resultant nephrogenic diabetes insipidus.

doi: 10.1038/ki.2009.91

Figure Lengend Snippet: Figure 4 | Effects of lithium on GSK3b. Confluent mpkCCDcl4 monolayers were treated for 96 h with 1 nM dDAVP. (a) Cells were treated with 1 mM lithium at the basolateral side and 10 mM lithium at the apical side, or with 20 and 1 mM zinc at both sides for the last 48 h and subjected to AQP2, GSK3b, and phospho-GSK3b(Ser9) immunoblotting or, after blotting, stained with coomassie blue. (b) mpkCCDcl4 cells were treated with a specific GSK3-inhibitor (BIO-Acetoxime) for the last 48 h and subjected to AQP2 immunoblotting. Concentrations are expressed in nanomolar. (c) Cells were incubated for the last 48 h in the absence or presence of lithium with or without 10 mM amiloride as indicated. At the basolateral and apical side, 1 and 10 mM lithium were used, respectively. Molecular masses (in kDa) are indicated on the left. Semiquantification, normalization, and statistical analysis were carried out as described in the legend of Figure 1. Significant differences (Po0.05) from control are indicated by an asterisk.

Article Snippet: Polyacrylamide gel electrophoresis, blotting, and blocking of the PVDF membranes were carried out as described.42 The membranes were incubated for 16 h with affinitypurified rabbit AQP2 antibodies (1:3000 dilution),43 affinitypurified rabbit anti-v1 H-ATPase antibodies (1:2000-dilution; gift from Dr S Nielsen, Denmark), rabbit anti-Ser9-GSK3b (1:1000 dilution; Cell Signaling Technology, Beverly, MA, USA), mouse antiGSK3b (1:5000 dilution; BD Transduction Laboratories, San Jose, CA, USA), or with mouse anti-tubulin antibodies (1:100,000 dilution; gift from Dr Kreis, Switzerland) in Tris-buffered saline Tween-20 supplemented with 1% non-fat dried milk.

Techniques: Western Blot, Staining, Incubation, Control

Journal: Kidney international

Article Title: Amiloride blocks lithium entry through the sodium channel thereby attenuating the resultant nephrogenic diabetes insipidus.

doi: 10.1038/ki.2009.91

Figure Lengend Snippet: Figure 5 | Amiloride prevents effects of lithium on AQP2 and H-ATPase expressions in lithium-NDI rats. Wistar rats were fed a normal diet (; n ¼ 6), a diet containing lithium (Li; n ¼ 6), or a diet containing lithium and amiloride (Li þ Am; n ¼ 7). After 4 weeks, one kidney was divided in the cortex, outer medulla, and inner medulla segments and solubilized. An equal amount of protein of the cortex of each rat was immunoblotted for AQP2, H-ATPase, or tubulin (indicated). Molecular masses (in kDa) are indicated on the left.

Article Snippet: Polyacrylamide gel electrophoresis, blotting, and blocking of the PVDF membranes were carried out as described.42 The membranes were incubated for 16 h with affinitypurified rabbit AQP2 antibodies (1:3000 dilution),43 affinitypurified rabbit anti-v1 H-ATPase antibodies (1:2000-dilution; gift from Dr S Nielsen, Denmark), rabbit anti-Ser9-GSK3b (1:1000 dilution; Cell Signaling Technology, Beverly, MA, USA), mouse antiGSK3b (1:5000 dilution; BD Transduction Laboratories, San Jose, CA, USA), or with mouse anti-tubulin antibodies (1:100,000 dilution; gift from Dr Kreis, Switzerland) in Tris-buffered saline Tween-20 supplemented with 1% non-fat dried milk.

Techniques:

Journal: Kidney international

Article Title: Amiloride blocks lithium entry through the sodium channel thereby attenuating the resultant nephrogenic diabetes insipidus.

doi: 10.1038/ki.2009.91

Figure Lengend Snippet: Figure 6 | Amiloride prevents cell conversion in lithium-NDI rats. (a) Of the rats described in the Figure 5 caption, one kidney was removed and fixed. Cryosections were prepared and incubated with rabbit H-ATPase (green) and guinea pig AQP2 (red) antibodies, followed by Alexa-488-conjugated goat-anti-rabbit and Alexa-594-conjugated goat anti-guinea pig antibodies. TOTO-3 (blue) was used to counterstain the sections. Images were produced with confocal laser scanning microscopy. Bars ¼ 10 mm. (b) Of 45 defined areas of the kidney cortex of each control (n ¼ 6), lithium (n ¼ 6), and lithium þ amiloride (n ¼ 7) rats, cells positive for AQP2 or H-ATPase were counted and expressed as the ratio of principal and intercalated cells (±s.e.m.) (total cells control (): 1244; Li: 1393; Li þ Am: 2064). Significant differences *Po0.05.

Article Snippet: Polyacrylamide gel electrophoresis, blotting, and blocking of the PVDF membranes were carried out as described.42 The membranes were incubated for 16 h with affinitypurified rabbit AQP2 antibodies (1:3000 dilution),43 affinitypurified rabbit anti-v1 H-ATPase antibodies (1:2000-dilution; gift from Dr S Nielsen, Denmark), rabbit anti-Ser9-GSK3b (1:1000 dilution; Cell Signaling Technology, Beverly, MA, USA), mouse antiGSK3b (1:5000 dilution; BD Transduction Laboratories, San Jose, CA, USA), or with mouse anti-tubulin antibodies (1:100,000 dilution; gift from Dr Kreis, Switzerland) in Tris-buffered saline Tween-20 supplemented with 1% non-fat dried milk.

Techniques: Incubation, Produced, Confocal Laser Scanning Microscopy, Control

Journal: Science Advances

Article Title: Bi-handed assembly chaperones regulate protein complex assembly through an intramolecular handover mechanism

doi: 10.1126/sciadv.adw9158

Figure Lengend Snippet: ( A ) Diagrams of WT and mutant AAGAB proteins. ( B ) Representative immunoblots showing the expression of the indicated proteins in HeLa cells. ( C ) Flow cytometry measurements showing normalized surface levels of TfR in the indicated HeLa cell lines. Data normalization was performed by setting the mean value of AAGAB KO cells to 100%, and all data points, including the AAGAB KO samples, were normalized to that mean value. Approximately 5000 cells were measured for each sample. Data are presented as mean ± SD of three biological replicates. *** P < 0.001 (compared to AAGAB KO cells); not significant (n.s.) P > 0.05, calculated using one-way analysis of variance (ANOVA) with Holm-Sidak corrections. ( D ) Representative confocal microscopy images showing AP2 puncta (α staining) on the plasma membrane in the indicated HeLa cell lines. The plasma membrane was stained with CF405-conjugated concanavalin A. Images were captured using a 100× oil immersion objective on a Yokogawa/Olympus CV1000 spinning disk confocal microscope. Scale bars, 10 μm. ( E ) Quantification of AP2 puncta on the plasma membrane. Images were captured as in (D) and analyzed using ImageJ. Each dot represents imaging data from an individual cell. Data of all samples were normalized to those of AAGAB KO cells expressing the WT rescue gene. Error bars indicate SD. *** P < 0.001 (compared to AAGAB KO cells); n.s. P > 0.05, calculated using one-way ANOVA with Holm-Sidak corrections. MW, molecular weight.

Article Snippet: Confocal images were captured using a 100× oil immersion objective on a Yokogawa/Olympus CV1000 spinning disk confocal microscope.

Techniques: Mutagenesis, Western Blot, Expressing, Flow Cytometry, Confocal Microscopy, Staining, Clinical Proteomics, Membrane, Microscopy, Imaging, Molecular Weight

Journal: Science Advances

Article Title: Bi-handed assembly chaperones regulate protein complex assembly through an intramolecular handover mechanism

doi: 10.1126/sciadv.adw9158

Figure Lengend Snippet: ( A ) Diagrams of WT and mutant AAGAB proteins. ( B ) Representative immunoblots showing the expression of the indicated proteins in HeLa cells. ( C ) Representative confocal microscopy images showing AP2 puncta (α staining) on the plasma membrane of the indicated HeLa cell lines. Images were captured using a 100× oil immersion objective on a Yokogawa/Olympus CV1000 spinning disk confocal microscope. Scale bars, 10 μm. ( D ) Quantification of AP2 puncta on the plasma membrane. Images were captured as in (C) and analyzed using ImageJ. Each dot represents imaging data from an individual cell. Data of all samples were normalized to those of AAGAB KO cells expressing the WT rescue gene. Error bars indicate SD. *** P < 0.001; n.s. P > 0.05 (compared to AAGAB KO cells, calculated using one-way ANOVA with Holm-Sidak corrections).

Article Snippet: Confocal images were captured using a 100× oil immersion objective on a Yokogawa/Olympus CV1000 spinning disk confocal microscope.

Techniques: Mutagenesis, Western Blot, Expressing, Confocal Microscopy, Staining, Clinical Proteomics, Membrane, Microscopy, Imaging

Journal: Science Advances

Article Title: Bi-handed assembly chaperones regulate protein complex assembly through an intramolecular handover mechanism

doi: 10.1126/sciadv.adw9158

Figure Lengend Snippet: ( A ) Diagrams of WT AAGAB and the flipped AAGAB variant (amino acids 258 to 315, 178 to 257, and 1 to 177). ( B ) Representative immunoblots showing the expression of the indicated proteins in AAGAB KO HeLa cells. Cells grown on a 24-well plate were transfected with the indicated amounts of plasmids (0.1 to 0.3 μg for each well). ( C ) Flow cytometry measurements showing normalized surface levels of TfR in the indicated HeLa cell lines. Data are presented as mean ± SD of three biological replicates. *** P < 0.001; n.s. P > 0.05, calculated using one-way ANOVA with Holm-Sidak corrections. ( D ) Representative confocal microscopy images showing AP2 puncta (α staining) on the plasma membrane of the indicated HeLa cell lines. Images were captured using a 100× oil immersion objective on a Yokogawa/Olympus CV1000 spinning disk confocal microscope. Scale bars, 10 μm. ( E ) Quantification of AP2 puncta on the plasma membrane. Images were captured as in (D) and analyzed using ImageJ. Each dot represents imaging data from an individual cell. Data of all samples were normalized to those of AAGAB KO cells expressing the WT rescue gene. Error bars indicate SD. *** P < 0.001 (compared to AAGAB KO cells); n.s. P > 0.05, calculated using one-way ANOVA with Holm-Sidak corrections.

Article Snippet: Confocal images were captured using a 100× oil immersion objective on a Yokogawa/Olympus CV1000 spinning disk confocal microscope.

Techniques: Variant Assay, Western Blot, Expressing, Transfection, Flow Cytometry, Confocal Microscopy, Staining, Clinical Proteomics, Membrane, Microscopy, Imaging

Journal: Science Advances

Article Title: Bi-handed assembly chaperones regulate protein complex assembly through an intramolecular handover mechanism

doi: 10.1126/sciadv.adw9158

Figure Lengend Snippet: ( A ) Representative confocal microscopy images showing AP2 puncta (α staining) on the plasma membrane of AAGAB KO HeLa cells and KO cells expressing either WT AAGAB or a YY mutant (Y53R/Y54R). Images were captured using a 100× oil immersion objective on a Yokogawa/Olympus CV1000 spinning disk confocal microscope. Scale bars, 10 μm. ( B ) Quantification of AP2 puncta on the plasma membrane. Images were captured as in (A) and analyzed using ImageJ. Each dot represents imaging data from an individual cell. Data from all samples were normalized to those of AAGAB KO cells with a WT rescue gene. Error bars indicate SD. *** P < 0.001; n.s. P > 0.05 (compared to AAGAB KO cells, calculated using one-way ANOVA with Holm-Sidak corrections). ( C ) Representative immunoblots showing the expression of the indicated proteins in HeLa cells. ( D ) Representative immunoblots showing the interaction of the 3xFLAG-tagged AAGAB YY mutant with HA-tagged α and σ2 in HeLa cells. ( E ) Representative Coomassie blue–stained gel showing the transfer of α and σ2 from the CTD to the GD of AAGAB. The AAGAB-CTD:α:σ2 ternary complex was isolated from E. coli using glutathione beads and incubated with lysates of E. coli expressing AAGAB-GD. After incubation for 1 hour at 4° or 37°C, the glutathione beads were washed, and proteins bound to the beads were detected using Coomassie blue staining (top) or immunoblotting (bottom). ( F ) Quantification of proteins bound to glutathione beads. Intensities of proteins were normalized to those of GST-α. Data are presented as mean ± SD of three biological replicates. *** P < 0.001 (calculated using one-way ANOVA with Holm-Sidak corrections).

Article Snippet: Confocal images were captured using a 100× oil immersion objective on a Yokogawa/Olympus CV1000 spinning disk confocal microscope.

Techniques: Confocal Microscopy, Staining, Clinical Proteomics, Membrane, Expressing, Mutagenesis, Microscopy, Imaging, Western Blot, Isolation, Incubation

Journal: bioRxiv

Article Title: Plastin-3 membrane recruitment drives cell-in-cell invasion during entosis

doi: 10.64898/2026.03.17.709257

Figure Lengend Snippet: (A) MCF7 cells were treated with vehicle (DMSO), 1 µM narciclasine, 20 µM Y27632, or pre-treated with 20 µM Y27632 followed by 1 µM narciclasine for 24 h. Entotic structures were visualized by immunofluorescence staining for β-catenin (membrane, green), LAMP1 (lysosomal compartments, red), and DAPI (nuclei, blue), followed by z-stack confocal microscopy. Representative 3D confocal images are shown. White arrows indicate entotic structures. Selected regions (boxed) are magnified, with corresponding orthogonal z-stack views displayed adjacent to each image. (B) Summary of quantification of entotic events in MCF7 cells treated from Figure A. (C–D) Equivalent analyses performed in ZR75-1 (C) and EFM19 (D) cells. Panels show representative entotic structures following treatment with 1 µM narciclasine and the corresponding quantification. (E) Representative immunoblot images of ROCK1, RhoA, and tubulin (loading control) in control or ROCK1-silenced cells, together with densitometric quantification (absolute OD unit) of ROCK1 and RhoA protein levels normalized to tubulin. (F) Quantification of entotic events in control and ROCK1-silenced MCF7 cells treated with vehicle or 1 µM narciclasine. For all confocal microscopy panels, orthogonal z-stack views and magnified insets are provided. Experiments were performed in biological triplicate (n = 3). Data are presented as mean ± SEM. Statistical analysis for (B–D) was performed using one-way ANOVA followed by Bonferroni post-hoc test (***p < 0.001, ****p < 0.0001), whereas panel E was analysed using a paired two-tailed Student’s t-test (****p < 0.0001). Panel F was analyzed using two-way ANOVA followed by Bonferroni post-hoc test (**p < 0.01, ****p < 0.0001).

Article Snippet: Membranes were blocked in 5% non-fat milk in TBS-T or in 5% BSA in TBS-T when detecting phospho-specific antibodies and incubated with primary antibodies against ROCK1 (rabbit monoclonal, 1:1000; Cell Signaling Technology, #28999S), RhoA (rabbit monoclonal, 1:1000; Cell Signaling Technology, #2117S), PLS2 (rabbit monoclonal, 1:1000; Atlas Antibodies, #HPA019493), PLS3 (mouse monoclonal, 1:1000; Invitrogen, #MA5-27772), α-tubulin (rabbit polyclonal, 1:3000; Cell Signaling Technology, #2144S), phospho-MLC2 (Ser19) (rabbit, 1:1000; Cell Signaling Technology, #3671S), and GFP (chicken polyclonal, 1:1000; Rockland Immunochemicals, #600-901-215).

Techniques: Immunofluorescence, Staining, Membrane, Confocal Microscopy, Western Blot, Control, Two Tailed Test

Journal: bioRxiv

Article Title: Plastin-3 membrane recruitment drives cell-in-cell invasion during entosis

doi: 10.64898/2026.03.17.709257

Figure Lengend Snippet: (A) Representative immunoblot images of ROCK1, RhoA, pMCL2 and tubulin (loading control) in MCF7 treated with 1uM narciclasine at different time-points. (B) Densitometric quantification of ROCK1, RhoA and pMCL2 protein levels from immunoblot images. (C) Immunofluorescence staining for pMCL2 (green) and actin (red) in MCF7 cells treated with 1 µM narciclasine for 6 hours. Nuclei were counterstained with DAPI (blue). (D) Summary of quantification of entotic events in MCF7 cells treated with vehicle, 1 µM narciclasine, or 50 µM blebbistatin (1 hour pretreatment) in combination with 1 µM narciclasine for 24 hours. (E) Representative 3D confocal images of entotic structures following 6 hours of treatment with vehicle or 1 µM narciclasine. White arrows indicate entotic events. Selected regions (boxed) are magnified, with corresponding orthogonal z-stack views shown adjacent to each image. (F) Summary of quantification of entotic events from the conditions shown in panel E. For all confocal microscopy panels, orthogonal z-stack views and magnified insets are provided. Experiments were performed in biological triplicate (n = 3). Data are presented as mean ± SEM. Data in panel B are normalized to untreated controls within each experiment (set to 100%). Statistical analysis for panels B and D was performed using one-way ANOVA followed by Bonferroni’s post hoc test (**p < 0.01, ***p < 0.001, *p < 0.0001), whereas panel F was analyzed using a paired two-tailed Student’s t-test (*p < 0.05).

Article Snippet: Membranes were blocked in 5% non-fat milk in TBS-T or in 5% BSA in TBS-T when detecting phospho-specific antibodies and incubated with primary antibodies against ROCK1 (rabbit monoclonal, 1:1000; Cell Signaling Technology, #28999S), RhoA (rabbit monoclonal, 1:1000; Cell Signaling Technology, #2117S), PLS2 (rabbit monoclonal, 1:1000; Atlas Antibodies, #HPA019493), PLS3 (mouse monoclonal, 1:1000; Invitrogen, #MA5-27772), α-tubulin (rabbit polyclonal, 1:3000; Cell Signaling Technology, #2144S), phospho-MLC2 (Ser19) (rabbit, 1:1000; Cell Signaling Technology, #3671S), and GFP (chicken polyclonal, 1:1000; Rockland Immunochemicals, #600-901-215).

Techniques: Western Blot, Control, Immunofluorescence, Staining, Confocal Microscopy, Two Tailed Test

Journal: bioRxiv

Article Title: Plastin-3 membrane recruitment drives cell-in-cell invasion during entosis

doi: 10.64898/2026.03.17.709257

Figure Lengend Snippet: (A) Schematic overview of ROCK1 construct transfection used to assess entosis induction. (B) Representative immunoblots showing ROCK1, phosphorylated MLC2 (pMLC2), and tubulin (loading control) in MCF7 cells transfected with GFP, GFP–ROCK1, or constitutively active GFP–ROCK1 Δ3. Immunoblot analysis of pMLC2 was performed 6 h post-transfection, whereas ROCK1 expression was assessed at 24 h. (C) Representative 3D confocal images of live MCF7 cells expressing GFP–ROCK1 Δ3 and stained with SiR-Actin (red) and Hoechst (blue). White arrows indicate entotic structures. Boxed regions showing representative entotic structures are magnified, with corresponding orthogonal z-stack views shown alongside each image. (D) Quantification of entotic events in MCF7 cells expressing GFP–ROCK1 or GFP–ROCK1 Δ3. (E) Distribution percentages of GFP-positive cells participating in CIC structures as outer cells, inner cells, or both. (F) Representative time-lapse imaging (phase contrast, Hoechst, and GFP) capturing an entotic event in MCF7 cells expressing GFP–ROCK1 Δ3. Yellow dotted lines outline inner cells, and red dotted lines outline host cells. Experiments were performed in biological triplicate (n = 3). Data are presented as mean ± SEM. Statistical analysis in panel D was performed using a paired two-tailed Student’s t-test (**p < 0.01), whereas panel E was analyzed using two-way ANOVA followed by Bonferroni post hoc test (*p < 0.05, **p < 0.01, ****p < 0.0001).

Article Snippet: Membranes were blocked in 5% non-fat milk in TBS-T or in 5% BSA in TBS-T when detecting phospho-specific antibodies and incubated with primary antibodies against ROCK1 (rabbit monoclonal, 1:1000; Cell Signaling Technology, #28999S), RhoA (rabbit monoclonal, 1:1000; Cell Signaling Technology, #2117S), PLS2 (rabbit monoclonal, 1:1000; Atlas Antibodies, #HPA019493), PLS3 (mouse monoclonal, 1:1000; Invitrogen, #MA5-27772), α-tubulin (rabbit polyclonal, 1:3000; Cell Signaling Technology, #2144S), phospho-MLC2 (Ser19) (rabbit, 1:1000; Cell Signaling Technology, #3671S), and GFP (chicken polyclonal, 1:1000; Rockland Immunochemicals, #600-901-215).

Techniques: Construct, Transfection, Western Blot, Control, Expressing, Staining, Imaging, Two Tailed Test

Journal: bioRxiv

Article Title: Plastin-3 membrane recruitment drives cell-in-cell invasion during entosis

doi: 10.64898/2026.03.17.709257

Figure Lengend Snippet: (A) Immunofluorescence staining for PLS3 (red) in MCF7 cells treated with 1 µM narciclasine or vehicle for 24 hours. Nuclei were counterstained with DAPI (blue). (B) Summary of quantification of membrane to cytosol ratio (mean intensity) from immunofluorescence PLS3 images. (C) Representative 3D confocal images of live MCF7 cells expressing PLS3-GFP and stained with SiR-Actin (red) and Hoechst (blue). White arrows indicate entotic structures. Boxed regions showing representative entotic structures are magnified, with corresponding orthogonal z-stack views shown alongside each image. (D) Quantification of entotic events in MCF7 cells expressing GFP or PLS3-GFP. (E) Representative time-lapse images (GFP and Hoechst) showing induction of entosis in MCF7 cells expressing PLS3. (F) Representative immunoblot images of PLS3 and tubulin (loading control) in control or PLS3-silenced cells, together with densitometric quantification of PLS3 normalized to tubulin. (G) Summary of quantification of entotic events in control and PLS3 -silenced MCF7 cells treated with vehicle or 1 µM narciclasine. For all confocal microscopy panels, orthogonal z-stack views and magnified insets are provided. Experiments were performed in biological triplicate (n = 3). Data are presented as median ± IQR for panel B and as mean ± SEM for panels D, F and G. Statistical analysis for panel B was performed using an unpaired two-tailed Student’s t-test (****p < 0.0001), whereas panel D and F by a paired two-tailed Student’s t-test (*p < 0.05). Panel G was assessed by a two-way ANOVA followed by Bonferroni’s post hoc test (*p < 0.05, ****p < 0.0001).

Article Snippet: Membranes were blocked in 5% non-fat milk in TBS-T or in 5% BSA in TBS-T when detecting phospho-specific antibodies and incubated with primary antibodies against ROCK1 (rabbit monoclonal, 1:1000; Cell Signaling Technology, #28999S), RhoA (rabbit monoclonal, 1:1000; Cell Signaling Technology, #2117S), PLS2 (rabbit monoclonal, 1:1000; Atlas Antibodies, #HPA019493), PLS3 (mouse monoclonal, 1:1000; Invitrogen, #MA5-27772), α-tubulin (rabbit polyclonal, 1:3000; Cell Signaling Technology, #2144S), phospho-MLC2 (Ser19) (rabbit, 1:1000; Cell Signaling Technology, #3671S), and GFP (chicken polyclonal, 1:1000; Rockland Immunochemicals, #600-901-215).

Techniques: Immunofluorescence, Staining, Membrane, Expressing, Western Blot, Control, Confocal Microscopy, Two Tailed Test