Journal: bioRxiv

Article Title: p21-activated kinase regulates Rab3a vesicles to repair plasma membrane damage caused by Amyloid-β oligomers

doi: 10.1101/2025.02.11.637764

Figure Lengend Snippet: TEM images of A) oAβ 1-40 and B) oAβ 1-42 . C, D) Fluorescently (TMR, red) labelled C) oAβ 1-40 and D) oAβ 1-42 end up to lysotracker positive vesicles (green). E) Percentage of colocalization of TMR-labelled oAβ peptides with lysotracker positive vesicles after 2 hours of exposure, and F) Size of oAβ accumulated lysotracker positive endolysosomes. G) Internalization of oAβ 1-40 -TMR and oAβ 1- 42 -TMR into the EEA1 positive early endosomal vesicles. H) Graphical representation of oAβ peptides colocalization with EEA1. I) WBs of EEA1 Aβ 1-42 treated cells with and without IPA-3. J) Quantification of EEA1 expression. K,L) Lamp1in K) Aβ 1-40 and L) Aβ 1-42 treated cells, with and without IPA-3, a specific inhibitor of pPAK1. M) Quantification of Lamp1 levels. Data are expressed as mean ± SD, *** p ≤ 0.001. Statistical significance was validated using the student-t test for E and F, and one-way ANOVA for I and K. n=3.

Article Snippet: Differential immunostainings were performed to gain insights into actin modulation following membrane damage and the distribution of Rab3a for PM repair ( , ), using pPAK1[phospho-PAK1] (Thr423)/PAK2 (Thr402) antibody (Cell Signaling Technology; CST #2601); f-actin binding phallotoxin phalloidin (A30106, Invitrogen); EEA1 [E4156, Sigmaaldrich]; Lamp1 (PE Conjugated, 2293862, Invitrogen) and Rab3a (PA1-770, Invitrogen).

Techniques: Expressing

Journal: bioRxiv

Article Title: p21-activated kinase regulates Rab3a vesicles to repair plasma membrane damage caused by Amyloid-β oligomers

doi: 10.1101/2025.02.11.637764

Figure Lengend Snippet: WBs of A, B) pPAK1 for Aβ 1-40 and Aβ 1-42 treated cells with and without IPA3 treatment. Quantification of C) pPAK1 levels. WBs of D, E) Rab3a for Aβ 1-40 and Aβ 1-42 treated cells with and without IPA3 treatment. Quantification of F) Rab3a levels. The data are presented as mean ± SD, with *** denoting significance at p ≤ 0.001. The statistical analysis was performed using one-way ANOVA.

Article Snippet: Differential immunostainings were performed to gain insights into actin modulation following membrane damage and the distribution of Rab3a for PM repair ( , ), using pPAK1[phospho-PAK1] (Thr423)/PAK2 (Thr402) antibody (Cell Signaling Technology; CST #2601); f-actin binding phallotoxin phalloidin (A30106, Invitrogen); EEA1 [E4156, Sigmaaldrich]; Lamp1 (PE Conjugated, 2293862, Invitrogen) and Rab3a (PA1-770, Invitrogen).

Techniques:

Journal: bioRxiv

Article Title: p21-activated kinase regulates Rab3a vesicles to repair plasma membrane damage caused by Amyloid-β oligomers

doi: 10.1101/2025.02.11.637764

Figure Lengend Snippet: A) Knockdown efficiency was assessed through the WB experiment conducted on pLKO.1 lentiviral vector-transfected cells as a control and cells with Rab3a knockdown (shRab3A). B) WB of total-PAK1 and pPAK1 on pLKO.1 lentiviral vector-transfected cells as a control and cells with Rab3a knockdown (shRab3A). The quantifications are presented as mean ± SD, with *** indicating p ≤ 0.001. Statistical analysis employed a t-test, and n=3. C-D) Dual nuclear staining using Propidium iodide and Hoechst was performed on C) pLKO.1 and D) shRab3a transfected cells, treated with Aβ 1-42 for 30 minutes and 1 hour. E) Graphical representation of PI-positive cells per unit area percentage in both pLKO.1 and shRab3a transfected cells. F) Cell viability by MTT assay was performed in pLKO.1 and shRab3a transfected cells, treated with and without Aβ 1-42 for 3 and 6 hours. The data is expressed as mean ± SD, with *** indicating p ≤ 0.001. One-way ANOVA was used for statistical analysis. Statistical analysis involved ordinary two-way ANOVA, and the sample size was 9 from three independent experiments (n=3).

Article Snippet: Differential immunostainings were performed to gain insights into actin modulation following membrane damage and the distribution of Rab3a for PM repair ( , ), using pPAK1[phospho-PAK1] (Thr423)/PAK2 (Thr402) antibody (Cell Signaling Technology; CST #2601); f-actin binding phallotoxin phalloidin (A30106, Invitrogen); EEA1 [E4156, Sigmaaldrich]; Lamp1 (PE Conjugated, 2293862, Invitrogen) and Rab3a (PA1-770, Invitrogen).

Techniques: Knockdown, Plasmid Preparation, Transfection, Control, Staining, MTT Assay

Journal: bioRxiv

Article Title: p21-activated kinase regulates Rab3a vesicles to repair plasma membrane damage caused by Amyloid-β oligomers

doi: 10.1101/2025.02.11.637764

Figure Lengend Snippet: The extracellular oAβ induces PM damage: oAβ 1-40 causes less damage, while oAβ 1-42 causes higher damage. The PM repair occurs via the Rab3-dependent recycling of Lamp1-positive lysosomal vesicles coupled with pPAK1-mediated massive endocytosis of the oAβ peptides. PM repair, in response to oAβ1-42 induced damage, occurs at a much faster rate. pPAK1 regulates actin-membrane stress via controlling CIE and by forming long-stretched fibrillary actin conduits or TNT-like structures during rapid PM repair.

Article Snippet: Differential immunostainings were performed to gain insights into actin modulation following membrane damage and the distribution of Rab3a for PM repair ( , ), using pPAK1[phospho-PAK1] (Thr423)/PAK2 (Thr402) antibody (Cell Signaling Technology; CST #2601); f-actin binding phallotoxin phalloidin (A30106, Invitrogen); EEA1 [E4156, Sigmaaldrich]; Lamp1 (PE Conjugated, 2293862, Invitrogen) and Rab3a (PA1-770, Invitrogen).

Techniques: Membrane

Journal: Clinical and Translational Medicine

Article Title: The metastatic promoter DEPDC1B induces epithelial‐mesenchymal transition and promotes prostate cancer cell proliferation via Rac1‐PAK1 signaling

doi: 10.1002/ctm2.191

Figure Lengend Snippet: DEPDC1B regulates the Rho signaling pathway and binds to Rac1. (A) Representative image of silver‐stained SDS‐PAGE gels showing separated proteins that were pulled down using Flag‐labeled DEPDC1B. Anti‐IgG was used as the negative control. (B) The bar graph of top 10 nonredundant enrichment clusters of KEGG using the Metascape website. (C) PPI network visualization in String website showing the proteins that related to DEPDC1B and Rac1. (D) The mass spectrum of a representative peptide fragment of Rac1. (E and F) Western blot analysis determined that DEPDC1B is correlated with Rac1 after performing the pull‐down assay with Flag‐labeled DEPDC1B (E) and an anti‐Rac1 (F) immunoprecipitation antibody. Anti‐IgG was used as the negative control protein in the pull‐down assay. (G and H) Representative image of the Western blotting analysis of active Rac1, total Rac1, phosphorylated PAK1, total PAK1 protein levels after DEPDC1B‐knockdown (G) or ‐overexpression (H) in DU145 and PC3 cells

Article Snippet: Primary antibodies specific to DEPDC1B (1:500, ab124182, Abcam), Rac1 (1:1000, ab33186, Abcam), pPAK1 (2605S), N‐cadherin (13116S), E‐cadherin (3195S), snail (3879S), slug (9585S), claudin1 (13255), β‐catenin (9562, 1:1000, Cell Signaling Technology), PAK (A0809, ABclonal Biotechnology, Wuhan, China), and actin (AA128, Beyotime, Shanghai, China) were used.

Techniques: Staining, SDS Page, Labeling, Negative Control, Western Blot, Pull Down Assay, Immunoprecipitation, Knockdown, Over Expression

Journal: Clinical and Translational Medicine

Article Title: The metastatic promoter DEPDC1B induces epithelial‐mesenchymal transition and promotes prostate cancer cell proliferation via Rac1‐PAK1 signaling

doi: 10.1002/ctm2.191

Figure Lengend Snippet: Inhibitor of active Rac1 (NSC23766) reverses the function in DEPDC1B‐overexpressing cells. (A) The levels of active Rac1, total Rac1, phosphorylated PAK1, and total PAK1 protein were detected by Western blotting in DEPDC1B‐overexpressing cells combined with NSC23766. (B and C) Representative images of cell migration (B) and invasion (C) were analyzed using DEPDC1B‐overexpressing or control cells combined with NSC23766 (left panels) and a quantification analysis of migrated or invaded cell counts (right panels). (D) Representative images of wound‐healing assays using DEPDC1B‐overexpressing or control cells combined with NSC23766 in DU145 (left panels) and PC3 (right panels) cells, showing reversing cell motility after using NSC23766 in DEPDC1B‐overexpressing cells. (E) A quantification analysis of cell migration index is shown. (F) Representative images of three‐dimensional (3D) cell culture using DEPDC1B‐overexpressing or control cells combined with NSC23766, showing reversing cell motility after using NSC23766 in DEPDC1B‐overexpressing cells. (G) Cell viability was reversed by using NSC23766 in DEPDC1B‐overexpressing or control cells. (H) Colony formation assays were constructed in DEPDC1B‐overexpressing or control cells combined with NSC23766. (I) A quantification analysis of colony formation number was shown. NSC23766 was used at 30 µM for 48 h. Unpaired t ‐test was used to analyze two groups of data; * P < .05, ** P < .01, and *** P <.001. Two‐way ANOVA was performed to analyze factorial designed data, # P < .05, ## P < .01, and ### P <.001. Scale bars: 25 µm

Article Snippet: Primary antibodies specific to DEPDC1B (1:500, ab124182, Abcam), Rac1 (1:1000, ab33186, Abcam), pPAK1 (2605S), N‐cadherin (13116S), E‐cadherin (3195S), snail (3879S), slug (9585S), claudin1 (13255), β‐catenin (9562, 1:1000, Cell Signaling Technology), PAK (A0809, ABclonal Biotechnology, Wuhan, China), and actin (AA128, Beyotime, Shanghai, China) were used.

Techniques: Western Blot, Migration, Control, Cell Culture, Construct

Journal: Clinical and Translational Medicine

Article Title: The metastatic promoter DEPDC1B induces epithelial‐mesenchymal transition and promotes prostate cancer cell proliferation via Rac1‐PAK1 signaling

doi: 10.1002/ctm2.191

Figure Lengend Snippet: Mutated active Rac1 plasmid (Q61L) recuses the function in DEPDC1B‐knockdown cells. (A) The levels of active Rac1, total Rac1, phosphorylated PAK1, and total PAK1 protein were detected by Western blotting in DEPDC1B‐knockdown cells combined with Q61L. (B and C) Representative images of cell migration (B) and invasion (C) were analyzed using DEPDC1B‐knockdown or control cells combined with Q61L (left panels), and a quantification analysis of the migrated or invaded cell counts (right panels) is shown. (D) Representative images of wound‐healing assays using DEPDC1B‐knockdown or control cells combined with Q61L in DU145 (left panels) and PC3 (right panels) cells, showing reversing cell motility after using Q61L in DEPDC1B‐overexpressing cells. (E) A quantification analysis of cell migration index is shown. (F) Representative images of three‐dimensional (3D) cell culture using DEPDC1B‐knockdown or control cells combined with Q61L showing rescued cell motility after using Q61L in DEPDC1B‐knockdown cells. (G) Cell viability was rescued by using Q61L in DEPDC1B‐knockdown or control cells. (H) Colony formation assays were constructed in DEPDC1B‐knockdown or control cells combined with NSC23766. (I) A quantification analysis of the colony formation number was shown. Unpaired t ‐test was used to analyze two groups of data; * P < .05, ** P < .01, and *** P <.001. Two‐way ANOVA was performed to analyze factorial designed data, # P < .05, ## P < .01, and ### P <.001. Scale bars: 25 µm

Article Snippet: Primary antibodies specific to DEPDC1B (1:500, ab124182, Abcam), Rac1 (1:1000, ab33186, Abcam), pPAK1 (2605S), N‐cadherin (13116S), E‐cadherin (3195S), snail (3879S), slug (9585S), claudin1 (13255), β‐catenin (9562, 1:1000, Cell Signaling Technology), PAK (A0809, ABclonal Biotechnology, Wuhan, China), and actin (AA128, Beyotime, Shanghai, China) were used.

Techniques: Plasmid Preparation, Knockdown, Western Blot, Migration, Control, Cell Culture, Construct

Journal: eLife

Article Title: An RNAi screen unravels the complexities of Rho GTPase networks in skin morphogenesis

doi: 10.7554/eLife.50226

Figure Lengend Snippet: ( A ) BioID2 constructs used to generate stably transduced lines from primary Krt14-rtTA mouse keratinocytes. TRE, tetracycline regulatory element used with a minimal promoter to drive expression of MYC-BioID2-GFP or MYC-BioID2-RHOU proteins. PGK, constitutively active promoter, used to drive expression of H2B-RFP to mark transduced cells. ( B ) Immunoblot of lysates from Krt14rtTA + keratinocytes transduced with Tre-Myc-BioID2-GFP-pgk-H2B-RFP or Tre-Myc-BioID2-Rhou-pgk-H2B-RFP lentiviruses and then treated for 2 days with doxycycline. Immunoblots were probed with MYC, GFP and RHOU antibodies. ( C ) Cloud analysis representing RHOU’s proximity interactome. Font size represents frequency of most abundant proteins in the interactome. ( D ) Gene ontology (GO) analysis reveals a significant enrichment for ‘cell-cell adherens junction’ and ‘focal adhesion’ proteins in RHOU’s proximity interactome. ( E ) Of the three PAK family members, PAK2 is the most highly expressed at both protein (left) and transcript (right) levels in primary keratinocytes and epidermis. (Left): Immunoblot of lysates from Krt14rtTA + mouse keratinocytes showing the higher expression of PAK2. Immunoblots were probed with a PAN PAK1/2/3, a specific PAK1 and a specific PAK2 antibody. (Right): Transcriptome profiling from E14.5 transduced epidermal cells revealed Pak2 as the most highly expressed Pak1/2/3 gene. ( F ) Overexpression of RHOU promotes PAK2 activation and decreases phosphorylation of MLC2. (Left): Primary keratinocytes were transfected with either empty vector or Myc-Rhou . Immunoblot of lysates were probe with MYC, pPAK1 Ser144 /pPAK2 Ser141 , PAK2, pMLC2, MLC2 and TUBULIN antibodies. (Right): Quantification of protein lysates. Data are represented as SEM from n = 3 experiments. ( G ) Depletion of RHOU reduces the activation of PAK2 and promotes the phosphorylation of MLC2. Primary keratinocytes were transfected with shScr , shRhou-504 or shRhou-505 . Immunoblots of lysates were probed with RHOU, pPAK1 Ser144 /pPAK2 Ser141 , PAK2, pMLC2 and TUBULIN antibodies. (Right): Quantification of protein lysates. Data are represented as SEM from n = 3 experiments for PAK2 level of phosphorylation and n = 4 for pMLC2 level of phosphorylation. ( H ) ARHGEF7 co-immunoprecipitates (co-IP) with MYC-RHOU in primary keratinocytes. Immunoblot of lysates and co-IP transfected with either an empty vector or Myc-Rhou . Immunoblots were probed with MYC and ARHGEF7 antibodies. ( I ) Immunofluorescence showing the co-localization of MYC-BioID2-RHOU with ARHGEF7 and PAK2 at focal adhesions in primary keratinocytes. Scale bars, 10 μm ( J–L ) Similarities between RHOU, PAK2 and ARHGEF7 deficiency phenotypes in skin. ( J ) Planar views from whole-mount immunofluorescence of transduced E15.5 headskin epidermis. P-CADHERIN marks adherens junctions; Phalloidin marks F-ACTIN; RFP verifies transduction. Shown are representative images from the midplanes of the basal cell layers. Representation from n = 3 embryos. Scale bars, 10 μm. ( K ) Quantifications of the numbers of placodes, germs and pegs from the different staggered HF waves in shScr , shPak2-209 and shArhgef7-966 transduced E16.5 head skin. Error bars represent SEM from shScr for Pak2 n = 5, shPak2-209 n = 5, shScr for Arhgef7 n = 4 and shArhgef7 n = 4 embryos. Normal distribution of the data was tested using the Shapiro-Wilk test. Parametric independent unpaired two-tailed t -test was used except for the comparison of shScr germ vs shArhgef7 germ and shScr peg vs shArgef7 peg for which a Mann-Whitney test was used. ( L ) Planar view of P-CADHERIN immunofluoresence of transduced hair peg imaged at the midplane and showing the loss of planar polarized distribution in the absence of RHOU, PAK2 and ARHGEF7. Representation from n = 3 embryos. Scale bars, 10 μm. 10.7554/eLife.50226.021 Figure 6—source data 1. Source data related to .

Article Snippet: The following antibodies were used: α-TUBULIN (mouse, 1:10,000, Sigma T5168), RHOU (rabbit, 1:1000, OriGene TA344077), MYC (mouse, 1:1000, Cell Signaling 2278), GFP (chicken, 1:10 000, Abcam ab13870), pPAK1 Ser144 /pPAK2 Ser141 (rabbit, 1:1000, Cell Signaling 2606), PAK1 (rabbit, 1:1000, Cell signaling 2602), PAK2 (rabbit, 1:1000, Cell signaling 2608), PAK1/2/3 (rabbit, 1:1000, Cell signaling 2604) pMLC2 Thr18/Ser19 (rabbit, 1:1000, Cell Signaling 3674) and MLC2 (rabbit, 1:1000, Cell Signaling 3672), ARHGEF7 (rabbit, 1:1000, Millipore 07–2101).

Techniques: Construct, Stable Transfection, Expressing, Western Blot, Transduction, Over Expression, Activation Assay, Phospho-proteomics, Transfection, Plasmid Preparation, Co-Immunoprecipitation Assay, Immunofluorescence, Two Tailed Test, Comparison, MANN-WHITNEY