Journal: Frontiers in Cell and Developmental Biology

Article Title: Feedback-Driven Mechanisms Between Phosphorylated Caveolin-1 and Contractile Actin Assemblies Instruct Persistent Cell Migration

doi: 10.3389/fcell.2021.665919

Figure Lengend Snippet: Actin protrusive network is upregulated by AMPK-Rac1-PAK1-Cofilin signaling cascade in cells lacking CAV-1. (A) Immunofluorescence microscopy analysis demonstrating that more pronounced endogenous ARPC2 colocalizes with F-actin (visualized by Alexa 568 phalloidin) on the lamellipodia protrusions in CAV-1 deficient cells. Magnified regions of cell edges on the right show the distribution of ARPC2 in WT and CAV-1 KO cells. Bars, 10 μm (in cell images) and 5 μm (in the magnified box). Quantification of width of lamellipodial protrusions are shown on the right. n = 16 regions from 16 cells for each group. (B) Representative images of membrane ruffling by time-lapse microscope. 1-pixel-wide areas were cut out to generate a 300 frame 2 s interval kymograph. Yellow dashed lines indicate the track of cell movement. An enlarged region is displayed on the right, vertical dashed lines show the membrane protrusion distance, while horizontal dashed lines mark the duration of protrusion. Bars, 10 μm (in cell images) and 2 μm (in the magnified box). Quantification of protrusion rate are shown on the right. n = 16 regions from 16 cells for each group. (C) P-AMPK (Thr172) and total AMPK were detected from the lysates of each group by western blotting. Please note that CAV-1(Y14F)-mEGFP can’t be detected by using phospho-CAV-1(Tyr14) antibody. Asterisk denotes the non-specific band. Quantification of P-AMPK (Thr172) levels (compared to total AMPK) from each group was shown on the right panel. n = 3. (D) Pull-down assays were performed for WT, CAV-1 KO, and CAV-1 KO; CAV-1-mEGFP re-expressed cells. Proteins bound to GST-PAK binding domain were analyzed by western blots and further quantified (compared to total Rac1) based on the band’s intensity. n = 3. (E) Western blot analysis and quantification (compared to total PAK1 and Cofilin) of the levels of phosphorylated PAK1 (Thr423) and Cofilin (Ser3) in WT and CAV-1 KO cell lysates. n = 3. (F) Immunostaining and quantification of endogenous P-Cofilin (Ser3) and F-actin distribution in WT and CAV-1 KO cells. A 16 μm length line was used to generate a line profile to illustrate the co-localization of P-Cofilin (Ser3) and F-actin. The lamellipodia region was enlarged on the right, and 2 μm width region was chosen to analyze the mean intensity of P-Cofilin (Ser3) on the leading edge. Bars, 10 μm (in cell images) and 5 μm (in the magnified box). n = 18 regions from 18 cells for each group. (G) Immunostaining and quantification of endogenous P-cofilin (Ser3) distribution upon compound C treatment in WT and CAV-1 KO cells. Magnified regions represent the lamellipodia region. Bars, 10 μm (in cell images) and 5 μm (in the magnified box). n = 18 regions from 18 cells for each group. (H) Western blot analysis and quantification of the phosphorylated levels of AMPK (Thr172), PAK1 (Thr423), Cofilin (Ser3) and activity of Rac1 and RhoA upon compound C treatment. n = 3. In panel (C–E,H) , the obtained intensity value from wild-type cells was set to 1. All the data are presented as mean ± SD. In (C,G,H) , *** P < 0.001; * P < 0.05; N.A., not significant (one-way ANOVA). In (D–F) , *** P < 0.001; ** P < 0.01 (unpaired t -test). All the data are from three independent experiments.

Article Snippet: The following antibodies were used in this study: CAV-1 (D46G3) rabbit antibody (1:1,000 dilution for WB, 1:200 for IF; #3267, Cell Signaling, Beverly, MO, United States); Phospho-CAV-1 (Tyr14) rabbit antibody (dilution 1:1,000 for WB; #3251, Cell signaling); AMPK rabbit antibody (dilution 1:500 for WB; #SAB4502329, Sigma, St. Louis, MO, United States); P-AMPK (Thr172) rabbit antibody (dilution 1:500 for WB, 1:100 for IF; #2531S, Cell Signaling); Cofilin (E-8) mouse antibody (dilution 1:1,000 for WB; #sc-376476, Santa Cruz, Dallas, TX, United States); Phospho-Cofilin (Ser3) rabbit antibody (dilution 1:1,000 for WB, 1:200 for IF; #3313, Cell signaling); p190RhoGAP rabbit antibody (dilution 1:2,000 for WB; #26789, Proteintech, Rosemont, IL, United States); FAK rabbit antibody (dilution 1:1,000 for WB; #3285, Cell Signaling); Phospho-FAK (Tyr397) rabbit antibody (dilution 1:1,000 for WB; #3283, Cell Signaling); PAK1 rabbit antibody (dilution 1:1,000; #2602, Cell Signaling); Phospho-PAK1 (Thr423)/PAK2 (Thr402) rabbit antibody (dilution 1:1,000 for WB, 1:200 for IF; #2601, Cell Signaling); Tpm4.2 (LC24) mouse antibody (dilution 1:500 for WB and IF; a kind gift from Peter W. Gunning, UNSW Australia); Phospho-myosin light chain 2 (Thr18/Ser19) rabbit antibody (dilution 1:500 for WB, 1:200 for IF; #3674, Cell Signaling); Myosin light chain mouse antibody (dilution 1:1,000 for WB; #M4401, Sigma); Vinculin mouse antibody (dilution 1:100 for IF; #V9131, Sigma); ARPC2 rabbit antibody (dilution 1:1,000 for WB and IF; #15058, Proteintech, Rosemont, IL, United States); Myosin-18B rabbit antibody (dilution 1:500 for WB; #HPA000953, Sigma); Rab8 rabbit antibody (dilution 1:100 for IF; #R5530, Sigma); and GAPDH mouse polyclonal antibody (dilution 1:1,000 for WB; #G8795, Sigma).

Techniques: Immunofluorescence, Microscopy, Membrane, Western Blot, Binding Assay, Immunostaining, Activity Assay

Journal: Advanced Science

Article Title: Microfilament‐Myosin II Regulates the Differentiation of Multinucleated Cysts into Oocytes and Influences Oocyte Developmental Potential in Mice

doi: 10.1002/advs.202500358

Figure Lengend Snippet: Arp2/3 complex participated in the oocyte differentiation process. A) The Arp2 protein expression levels during oocyte differentiation process. B,C) Arpc4 and Arp2 knockdown were achieved by using Arpc4 siRNA ( n = 24 in each group). D) Arpc4 and F‐actin fluorescence intensity significantly decreased by using Arpc4 siRNA. E) Oocyte morphology and volume in NC‐siRNA and Arpc4‐siRNA group, scale bar = 50 µm. F) The DDX4 staining of oocytes in NC‐siRNA and Arpc4‐siRNA group, scale bar = 20 µm. G) The polarity degree of oocytes in NC‐siRNA and Arpc4‐siRNA group. H) The rate of oocytes with double nuclei in NC‐siRNA and Arpc4‐siRNA group. I) The proportion of oocyte with a ring Golgi apparatus in NC‐siRNA and Arpc4‐siRNA group, scale bar = 20 µm. J) The relative ATP5a fluorescence intensity in oocytes of NC‐siRNA and Arpc4‐siRNA group, scale bar = 20 µm. K) The relative calreticulin fluorescence intensity in oocytes of NC‐siRNA and Arpc4‐siRNA group, scale bar = 10 µm. L) The Arpc4 and Arp2 protein expression levels in NC‐siRNA, Arpc4‐siRNA and Rescue group. M,N) Arpc4 and F‐actin fluorescence intensity in NC‐siRNA, Arpc4‐siRNA and Rescue group. O) The rate of double nuclei, polarity degree and volume of oocytes in NC‐siRNA, Arpc4‐siRNA, and the Rescue group. P) The proportion of oocyte with a ring Golgi apparatus in NC‐siRNA, Arpc4‐siRNA, and the Rescue group, scale bar = 20 µm. Q) The relative ATP5a fluorescence intensity in oocytes of NC‐siRNA, Arpc4‐siRNA, and the Rescue group, scale bar = 20 µm. * meant p < 0.05, ** meant p < 0.01, *** meant p < 0.001, and **** meant p < 0.0001.

Article Snippet: MYH9 Polyclonal antibody (11128‐1‐AP), MYH10‐Specific Polyclonal antibody (19673‐1‐AP), MYH14 Polyclonal antibody (20716‐1‐AP), MYO7A Polyclonal antibody (20720‐1‐AP), TEX14 Polyclonal antibody (18351‐1‐AP), ARP2 Polyclonal antibody (10922‐1‐AP), ARPC4 Polyclonal antibody (10930‐1‐AP), ROCK1 Polyclonal antibody (21850‐1‐AP), ROCK2 Monoclonal antibody (66633‐1‐Ig) and RHOA Polyclonal antibody (10749‐1‐AP) were purchased from Proteintech (Wuhan, China).

Techniques: Expressing, Knockdown, Fluorescence, Staining