Journal: PLoS ONE

Article Title: Type VI collagen promotes lung epithelial cell spreading and wound-closure

doi: 10.1371/journal.pone.0209095

Figure Lengend Snippet: Quantification of 10hr 16HBE wound-width relative to 0hr controls on COL6, COL1, Matrigel coated wells, and uncoated tissue-culture wells after treatment with inhibitors of PI3K (LY294002, 5 μM), CDC42 (ZCL 278, 55 μM), RHOA (CCG 1423, 3 μM), FAK (PF 573228, 40 nM), RAC (EHT 1864, 600 nM), and ERK (FR 180204, 3 μM). (a) Heat map indicating the effect of inhibitor relative to control for cells on each matrix. A lighter color represents more wound closure relative to 0hr. A yellow circle represents p<0.05. Plots present wound-healing rate of cells on COL6, Matrigel, COL1, and plastic after treatment with (b) PI3K inhibitor, (c) CDC42 inhibitor, and (d) RAC1 inhibitor. ** p<0.01, *** p<0.001. n = 6.

Article Snippet: Plasmids containing GFPmPA-GFP-N1 (a gift from Michael Davidson, Addgene plasmid # 54712), and constitutively active FAK (pGFP FAK Y397F, a gift from Kenneth Yamada, Addgene plasmid # 50516), CDC42 (a gift from Joan Brugge, Addgene plasmid #14568), and RAC1 (pcDNA3-EGFP-RAC1-Q61L, a gift from Gary Bokoch, Addgene plasmid #12981) all contain GFP and were transfected individually [ , ].

Techniques: Control

Journal: PLoS ONE

Article Title: Type VI collagen promotes lung epithelial cell spreading and wound-closure

doi: 10.1371/journal.pone.0209095

Figure Lengend Snippet: (a) Representative images of GFP-only, Rac1, Cdc42 and Pi3k-overexpressing cell spreading on Matrigel. Quantification of GFP-positive 16HBE spreading 3 hours after plating on (b) Matrigel, (c) COL1, (d) uncoated tissue-culture wells, and (e) COL6 after transfection with constitutively active signaling constructs for FAK, RAC1, ERK, RHOA, CDC42, or PI3K. Black lines represent spreading of GFP-transfected cells on each matrix, respectively. Grey lines represent spreading of GFP transfected cells on COL6. N = 6. * p<0.05, ** p<0.01, *** p<0.001.

Article Snippet: Plasmids containing GFPmPA-GFP-N1 (a gift from Michael Davidson, Addgene plasmid # 54712), and constitutively active FAK (pGFP FAK Y397F, a gift from Kenneth Yamada, Addgene plasmid # 50516), CDC42 (a gift from Joan Brugge, Addgene plasmid #14568), and RAC1 (pcDNA3-EGFP-RAC1-Q61L, a gift from Gary Bokoch, Addgene plasmid #12981) all contain GFP and were transfected individually [ , ].

Techniques: Transfection, Construct

Journal: Biochemical and biophysical research communications

Article Title: Cdc42-dependent modulation of rigidity sensing and cell spreading in tumor repopulating cells.

doi: 10.1016/j.bbrc.2018.04.085

Figure Lengend Snippet: Fig. 2. Single-mRNA-transcript statistics revealed a dissimilarity in RhoA and Cdc42 expression in TRCs leading to suppression in cell spreading. a, Representative images showing mRNA-transcript statistics of RhoA, Rac1, and Cdc42 in single control cells and TRCs. b, Correlation analysis between RhoA and Cdc42 transcripts (top) and RhoA and Rac1 transcripts (bottom) is shown here. RhoA and Cdc42 expression in control cells are tightly correlated while TRCs tend to exhibit a heterogeneous expression pattern. Each dot represents a single cell (r, Pearson correlation coefficient). c, RhoA to Cdc42 ratio and RhoA to Rac1 ratio in control cells and TRCs are significantly different (p < 1.35 1058 and 5.86 1013 for RhoA to Cdc42 ratio and RhoA to Rac1 ratio respectively).

Article Snippet: Cdc42 (ID # 12975), Rac1 (ID # 12980), and RhoA (ID #23224) plasmids were obtained from Addgene.

Techniques: Expressing, Control

Journal: Biochemical and biophysical research communications

Article Title: Cdc42-dependent modulation of rigidity sensing and cell spreading in tumor repopulating cells.

doi: 10.1016/j.bbrc.2018.04.085

Figure Lengend Snippet: Fig. 3. TRCs' inability to spread corresponds to very few focal adhesions but can be restored by Cdc42 overexpression. a, Quantification of TIRF microscopy images show very dissimilar FA formation of control cells and TRCs expressing mCherry-vinculin. Color bar represents FA intensity relative to maximum intensity. b-c, Histogram plot of FA area in control cells and TRCs is shown here after 1 h (n ¼ 10 for both control cells and TRCs) and 4 h (n ¼ 10 and 15 for control cells and TRCs respectively) of cell plating. The number of FAs with area 1.5 mm2 per cell in control cells was significantly different between 1 h and 4 h (p < 0.02), but not in TRCs (p > 0.44) (insets). d, Representative images of TRCs with overexpression of Rac1 and Cdc42 show that Cdc42 alone, but not Rac1, can facilitate cell spreading in TRCs with filopodia-like extensions (inset). A TRC not transfected with Cdc42 did not spread (arrowhead, DIC image; cell boundary, fluorescence image). e, Single mRNA-transcripts were quantified after transient overexpression of Cdc42 in TRCs. A repre- sentative image of Cdc42-overexpressed-TRC is shown (left). A plot of CSI vs. Cdc42 density is shown in blue representing overexpressed Cdc42 (n ¼ 46, Pearson's correlation coefficient r ¼ 0.62) and red representing endogenous Cdc42 transcripts in TRCs (n ¼ 168, r ¼ 0.07). A clear threshold of ~25 Cdc42 transcripts per projected cell area is observed for TRC spreading initiation. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: Cdc42 (ID # 12975), Rac1 (ID # 12980), and RhoA (ID #23224) plasmids were obtained from Addgene.

Techniques: Over Expression, Microscopy, Control, Expressing, Transfection

Journal: Biochemical and biophysical research communications

Article Title: Cdc42-dependent modulation of rigidity sensing and cell spreading in tumor repopulating cells.

doi: 10.1016/j.bbrc.2018.04.085

Figure Lengend Snippet: Fig. 4. Overexpression of Cdc42 in TRCs causes increasing cell spreading capability with increasing Ttol. a, TRCs transfected with Cdc42-GFP can spread more with increasing Ttol. b, Summarized data of Cdc42 overexpressed TRC spreading on 43 pN, 56 pN, and >100 pN surfaces (n ¼ 22, 29, 22 for 43 pN, 56 pN, and >100 pN surfaces respectively). Significant differences in projected cell area were observed between 43 pN and 56 pN, and 56 pN and >100 pN (p < 2.89 108 and 5.43 106 respec- tively). Similarly, significant differences in CSI between 43 pN and 56 pN, and 56 pN and >100 pN were observed (p < 0.04 and 9.33 104 respectively).

Article Snippet: Cdc42 (ID # 12975), Rac1 (ID # 12980), and RhoA (ID #23224) plasmids were obtained from Addgene.

Techniques: Over Expression, Transfection