Journal: Scientific Reports

Article Title: Tumor suppressor DEAR1 regulates mammary epithelial cell fate and predicts early onset and metastasis in triple negative breast cancer

doi: 10.1038/s41598-022-22417-4

Figure Lengend Snippet: Stable DEAR1 -KD in HMECs results in increased numbers of mammosphere independent of SMAD3 expression. ( a ) Representative bar plots showing mean ± SD counts of primary mammospheres (> 50um) from control vector (CshR) and DEAR1 -KD (DshR) MCF10A cells across three trials. Each cell line was grown in at least 8 wells of 96-well plate in mammosphere growing conditions for 17 days for each individual trial. P-values were calculated using a two-tailed Student t-test between groups. **** p < 0.0001. ( b ) Representative images of mammospheres from ( a ) taken using a Zeiss microscope at ×10 magnification (Scale bars equal 100 µm). ( c ) Bar graphs representing the mean ± SD number of mammospheres (> 50 µm) formed from two control vector clones (CshR) and three DEAR1- KD clones (DshR) from 76N-E6 cells grown in mammosphere-growing conditions for 14 days across three trials. P-values were calculated using Wilcoxon Rank Sum tests. **p < 0.01 and ****p < 0.0001. ( d ) Representative images at ×10 magnification of CshR1 (left) and DshR3 (right) mammospheres quantified in panel ( c ) in the absence (top) and presence (bottom) of TGF-β (Scale bars equal 100 µm). ( e ) Bar plots showing the mean ± SD number of mammospheres formed from CshR, DshR, DshR/shC, and DshR/shSM3 cells without (left) and with (right) TGF-β treatment across 3 trials. p-values calculated using Wilcoxon Rank Sum tests. *p < 0.05–0.1 and ****p < 0.0001. ( f ) Bar graphs represent the mean ± SD number of mammospheres (> 50 µm) formed from two control vector clones (CshR) and three DEAR1- KD clones (DshR) from 76N-E6 cells treated with TGF-β and grown in mammosphere growing conditions for 14 days across three trials. P-values were calculated using Wilcoxon Rank Sum tests. *p < 0.05–0.1 and ****p < 0.0001. The barplots in ( c ), ( e ) and ( f ) were created using RStudio (Version 1.0.143), https://www.rstudio.com .

Article Snippet: The barplots in ( c ), ( e ) and ( f ) were created using RStudio (Version 1.0.143), https://www.rstudio.com .

Techniques: Expressing, Control, Plasmid Preparation, Two Tailed Test, Microscopy, Clone Assay

Journal: Scientific Reports

Article Title: Tumor suppressor DEAR1 regulates mammary epithelial cell fate and predicts early onset and metastasis in triple negative breast cancer

doi: 10.1038/s41598-022-22417-4

Figure Lengend Snippet: The regulation of SNAI2 by DEAR1. ( a ) Bar plots of qPCR results (mean ± SD) of SNAI2 mRNA levels in control (CshR), DEAR1 -KD (DshR), DEAR1 -KD/shRCt clone (DshR/shC), and DEAR1-SMAD3 double KD (DshR-shSM3) across 2 runs. P-values were calculated using Wilcoxon Rank Sum tests. *p < 0.05–0.1, **p < 0.01, ***p < 0.001. The barplots were created using RStudio (Version 1.0.143), https://www.rstudio.com . ( b ) Western blot showing SNAI2 protein expression in control (CshR) and DEAR1 -KD clones (DshR) with and without TGF-β treatment. Full length gels for Western blots are shown in Supplementary Fig. . ( c ) Co-immunoprecipitation (Co-IP) to determine possible interaction between DEAR1 and SNAI2. HEK293T cells were co-transfected with an HA - DEAR1 -expressing plasmid and/or a Myc-SNAI2 expression plasmid. After 24 h post-transfection, cell lysates were immunoprecipitated with anti-HA (rabbit) antibody. SNAI2 proteins were blotted with anti-Myc (mouse) antibody. Full length gels for Western blots are shown in Supplementary Fig. . ( d ) Co-IP assay to detect ubiquitination of SNAI2 by DEAR1. HEK293T cells were co-transfected with Myc-SNAI2 , HA - ubiquitin , and DEAR1 . Cells were lysed 48 h post-transfection. Lysates were denatured and immunoprecipitated with anti-Myc (mouse) antibody. Ubiquitin was blotted with anti-HA (Rat) antibody. Full length gels for Western blots are shown in Supplementary Fig. .

Article Snippet: The barplots in ( c ), ( e ) and ( f ) were created using RStudio (Version 1.0.143), https://www.rstudio.com .

Techniques: Control, Western Blot, Expressing, Clone Assay, Immunoprecipitation, Co-Immunoprecipitation Assay, Transfection, Plasmid Preparation, Ubiquitin Proteomics

Journal: bioRxiv

Article Title: Planar polarization of cilia in the zebrafish floor-plate involves Par3-mediated posterior localization of highly motile basal bodies

doi: 10.1101/702282

Figure Lengend Snippet: a) BB movements in wt (left column) and vangl2 m209 (right column) embryos at early (first line) and late (second line) developmental stages. Each dot represents the endpoint of a single BB movement, the starting point being the center of the circle; thus, the angles outside the circle represent BB movements orientation relative to the embryo anteroposterior axis, and the distance relative to the center of the circle represents its length (each circle radius corresponds to 7 µm). The color of the dots indicates whether a movements leads to a membrane contact and, if so, the nature of the contact (anterior, posterior or lateral). (early stages: wt 8 embryos, 20 cells, 238 movements; vangl2 m209 : 9 embryos, 20 cells, 220 movements. Late stages: wt 4 embryos, 19 cells, 255 movements; vangl2 m209 : 4 embryos, 42 cells, 708 movements). b, c) Orientation of BB movements in wt and vangl2 m209 embryos at early (b) or late (c) stages. d, e) BB movements speed, nature of membrane contacts and total time spent in contact with membranes at early (d) and late (e) stages. Statistical tests: Wilcoxon test for comparison of BB speeds; Fisher test for comparison of BB movements orientation and BB/membrane contacts.

Article Snippet: BB/membrane contacts were half less frequent than at early stages, both in wt and vangl2 m209/m209 (around 7% of total BB movements), but we could still detect a significant difference in their position between wt and vangl2 m209/m209 , with more lateral and anterior contacts in vangl2 m209/m209 ( , middle barplot).

Techniques: Membrane, Comparison

Journal: bioRxiv

Article Title: Planar polarization of cilia in the zebrafish floor-plate involves Par3-mediated posterior localization of highly motile basal bodies

doi: 10.1101/702282

Figure Lengend Snippet: a) Polarization index of vangl2 m209/m209 determined from immunostaining data wt: 2 embryos, 49 cells; vangl2 m209/+ : 3 embryos, 66 cells; vangl2 m209/m209 : 5 embryos, 57 cells. b) Immunostaining of phoshorylated Par3 (BazP1085 antibody) in vangl2 +/+ (wt) and vangl2 m209/m209 embryo FP at 18 s. In each case ZO1 staining was removed in the right image to reveal Par3 patches (yellow arrows). c) Quantification of the number of Par3 patches per cell on transverse membranes from immunostaining data as shown in b. d) Prominence of Par3 patches (BazP1085 antibody) in wt and vangl2 m209/m209 mutant embryo FP at 18 s In a-d, vangl2 +/+ : 7 embryos, 186 cells; vangl2 m209/+ : 5 embryos, 112 cells; vangl2 m209/m209 : 7 embryos, 129 cells. e) Percentage of cells displaying a lateral Par3-RFP patch in live-imaging experiments (such as the one described in f). vangl2 +/+ and vangl2 m209/+ : 16 embryos, 45 cells; vangl2 m209/m209 : 7 embryos, 17 cells. f) Images from movies of 5s vangl2 m209/m209 embryos mosaically injected with Par3-RFP, Centrin-GFP and Membrane-GFP mRNA at the 16-32 cell stage. Yellow arrows point at contact events between lateral Par3 patches and BBs. Statistical tests: Wilcoxon test for comparison of p.i. and prominence; Fisher test for comparison of patch number and percentage of cells with lateral patches.

Article Snippet: BB/membrane contacts were half less frequent than at early stages, both in wt and vangl2 m209/m209 (around 7% of total BB movements), but we could still detect a significant difference in their position between wt and vangl2 m209/m209 , with more lateral and anterior contacts in vangl2 m209/m209 ( , middle barplot).

Techniques: Immunostaining, Staining, Mutagenesis, Imaging, Injection, Membrane, Comparison

Journal: bioRxiv

Article Title: Planar polarization of cilia in the zebrafish floor-plate involves Par3-mediated posterior localization of highly motile basal bodies

doi: 10.1101/702282

Figure Lengend Snippet: a) Scheme showing BB behavior at early and late stages in the FP of wt and vangl2 mutants. In wt at early stages of polarization, the BB makes back and forth movements (dotted lines) between anterior and posterior Par3 patches (magenta) but very few lateral movements. In contrast, in vangl2 mutants, BBs make more lateral movements and contacts and these contacts always occur at ectopic lateral Par3 patches. At later stages, the BB displays low motility. In wt, BBs are either docked to posterior Par3 patch or move in the posterior side of the cell, close to the patch, whereas in vangl2 mutants BBs move around the center of the apical surface, which is probably due to earlier polarization defects and the still abnormal dispersion of Par3 patches around the apical junctions. Note that in both wt and vangl2 embryos, at all stages studied, cilia are present and do not seem to impair BB movements. b) Hypothetical microtubule-based mechanism for BB polarization. Within the apical surface, dynamic microtubules emanate in all direction from the BB but can be captured at Par3 patches, at which their depolymerization is triggered (in this case at the posterior patch). The resulting mechanical force pulls on the membrane and the BB, which can result in digitation formation and/or BB movement towards the patch. The posterior Par3 enrichment makes it more likely for posterior pulling to happen (compared to anterior pulling) which ultimately results in a stably docked posterior BB at later stages.

Article Snippet: BB/membrane contacts were half less frequent than at early stages, both in wt and vangl2 m209/m209 (around 7% of total BB movements), but we could still detect a significant difference in their position between wt and vangl2 m209/m209 , with more lateral and anterior contacts in vangl2 m209/m209 ( , middle barplot).

Techniques: Dispersion, Membrane, Stable Transfection