Journal: Cell

Article Title: A Flat BAR Protein Promotes Actin Polymerization at the Base of Clathrin-Coated Pits

doi: 10.1016/j.cell.2018.05.020

Figure Lengend Snippet: FCHSD2 Is Not Directly Recruited to CCPs by PI3KC2α or Its Kinase Activity, Related to Figure 2 (A) Top: Transferrin uptake assay by flow cytometry comparing wild-type and FCHSD2 KO cells silenced for PI3KC2α. Uptake of Alexa488 labeled transferrin normalized by the amount of surface transferrin receptor for each condition and against uptake for the wild-type cells in each experiment. Each value represents median fluorescence from at least 5000 cells (n = 12, mean ± SD). ∗∗∗ p > 0.001, ns = non-significant. One-way ANOVA with Tukey’s post hoc analysis. Bottom: Immunoblots showing PI3KC2α knockdown in wild-type and FCHSD2 KO cells. (B) Kymographs of HeLa FCHSD2-Venus stables silenced for PI3KC2α and control cells. Cells were transfected with mCherry-ClathrinLC 24 hs before imaging. Kymographs generated from 120 s movies at 1Hz. Note the elongated CCP lifetimes in PI3KC2α knockdown cells as described in Posor et al. (2013) . (C) Autoinhibition of FCHSD2. Representative images of a center slice from cells expressing different FCHSD2 truncation constructs and co-stained with phalloidin (Actin). The bar graph (upper right) shows the quantification of cellular protrusions/μm for each construct. The non-inhibited BAR domain produces many protrusions. Numbers inside bars represent number of cells measured. The line graph (bottom right) shows the fluorescence profile of sum intensity projections for cells expressing each construct. Due to the natural thinning of cells from their centers to the edge, a gradually decaying line indicates that the fluorescent protein is primarily cytosolic while a flat line with an abrupt fall on the cell edge indicates that the fluorescent protein is primarily bound to the membrane. While the presence of SH3-1 significantly reduces the generation of cellular protrusions generated by the FCHSD2 F-BAR, a significant fraction of the protein remains bound to the membrane (green line). Only the combined presence of SH3-1 and SH3-2 is capable to avoid promiscuous binding of the BAR domain to the membrane. Data is shown as mean ± SD in bar graph and as ± SEM in fluorescence profiles. ∗∗∗ p > 0.001, ns = non-significant. One-way ANOVA with Tukey’s post hoc analysis. (D) Immunoblots for intersectin knockdown in FCHSD2-Venus HeLa cells.

Article Snippet: PI3KC2α siRNA pool , Origene , Cat# SR303516.

Techniques: Activity Assay, Flow Cytometry, Labeling, Fluorescence, Western Blot, Knockdown, Control, Transfection, Imaging, Generated, Expressing, Construct, Staining, Membrane, Binding Assay

Journal: Cell

Article Title: A Flat BAR Protein Promotes Actin Polymerization at the Base of Clathrin-Coated Pits

doi: 10.1016/j.cell.2018.05.020

Figure Lengend Snippet:

Article Snippet: PI3KC2α siRNA pool , Origene , Cat# SR303516.

Techniques: Virus, Recombinant, Labeling, Cloning, Stable Transfection, Expressing, esiRNA, Plasmid Preparation, Software

Journal: Cancer cell

Article Title: Mitotic Spindle Assembly and Genomic Stability in Breast Cancer Require PI3K-C2α Scaffolding Function.

doi: 10.1016/j.ccell.2017.09.002

Figure Lengend Snippet: Figure 2. PI3K-C2a Haploinsufficiency Affects Breast Cancer Growth (A) Kaplan-Meier curve for tumor incidence in Pik3c2a+/+ (WT), Pik3c2a+/ (Het), and NeuT female mice carrying WT (WT/NeuT) or heterozygous deletion of PI3K-C2a (Het/NeuT). n = 20 mice for WT/NeuT and n = 25 mice for Het/NeuT. (B) Growth curve of WT/NeuT and Het/NeuT tumors in the fourth pair of mammary glands. The red shaded background indicates the early stage of tumors while the gray one the late phase. Results are shown as mean ± SEM (n = 20 mice for WT/NeuT and n = 25 mice for Het/NeuT). (C) PCNA staining of WT/NeuT and Het/NeuT tumors at early (E) or late (L) stages of development (n = 6). Scale bars, 100 mm. (D and E) Proliferation curves of WT/NeuT and Het/NeuT MMET derived from E (D) or L (E) stage. FC, fold change. Results are shown as mean ± SEM (n = 8). (F–H) Analysis of metaphase plate width and aberrant metaphases. IF staining (F), metaphase plate width (G), and aberrant metaphases (H) quantification of E and L WT/NeuT and Het/NeuT MMET. Results are shown as mean ± SD (n = 100 cells). Yellow arrows indicate lagging chromosome in metaphase. Yellow dashed lines indicate the metaphase plate width. Scale bars, 5 mm. (I) FACS analysis of E and L WT/NeuT and Het/NeuT MMET to score DNA content (propidium iodide staining). M2 gate represents the percentage of aneuploid cells. Results are shown as mean ± SEM (n = 7). *p < 0.05, **p < 0.01, ***p < 0.001 by ANOVA followed by Bonferroni’s post hoc test. See also Figure S2, and Table S3.

Article Snippet: Three micron thick sections of formalin fixed paraffin embedded (FFPE) samples were stained for haematoxylin and eosin (H&E) and immunohistochemistry with antibodies against PI3K-C2a (clone OTI15H1, formerly 15H1, diluted 1:1000, Origene).

Techniques: Staining, Derivative Assay

Journal: Cancer cell

Article Title: Mitotic Spindle Assembly and Genomic Stability in Breast Cancer Require PI3K-C2α Scaffolding Function.

doi: 10.1016/j.ccell.2017.09.002

Figure Lengend Snippet: Figure 3. PI3K-C2a Localizes on Metaphase Spindle where It Interacts with TACC3 and CHC (A) IF staining of PI3K-C2a in HeLa cells. Co-localization of exogenous PI3K-C2a (upper panel, HeLa transfected with GFP-PI3K-C2a) and endogenous PI3K-C2a (middle panel) with a-tubulin (red) on metaphase spindle. HeLa cells stained for PI3K-C2a expression after its siRNA-mediated knockdown (lower panel). (B) Western blot (WB) analysis of spindle/cytoplasmic fractionation in metaphase-arrested HeLa cells. Whole-cell lysates (WCL) were immunoblotted with indicated antibodies (n = 3). (C and D) PI3K-C2a was immunoprecipitated (IP) from the cell lysate of metaphase-arrested HeLa (C) or MMET (D) with anti-PI3K-C2a antibody (IP) or control immunoglobulin G. Bound proteins were detected by immunoblotting with anti-TACC3, anti-clathrin heavy chain (CHC), or anti-PI3K-C2a antibody (WB, n = 6).

Article Snippet: Three micron thick sections of formalin fixed paraffin embedded (FFPE) samples were stained for haematoxylin and eosin (H&E) and immunohistochemistry with antibodies against PI3K-C2a (clone OTI15H1, formerly 15H1, diluted 1:1000, Origene).

Techniques: Staining, Transfection, Expressing, Knockdown, Western Blot, Fractionation, Immunoprecipitation, Control

Journal: Cancer cell

Article Title: Mitotic Spindle Assembly and Genomic Stability in Breast Cancer Require PI3K-C2α Scaffolding Function.

doi: 10.1016/j.ccell.2017.09.002

Figure Lengend Snippet: Figure 4. PI3K-C2a Organizes the Mitotic Spindle by Stabilizing the TACC3-CHC Complex (A–C) Analysis of TACC3 localization. IF staining (A), relative quantification (B), and WB analysis (C) of TACC3 localization on metaphase spindle and cytosolic fraction in control and PI3K-C2a-silenced HeLa cells. Results are shown as mean ± SEM (IF, n = 200 cells; WB, n = 5). (D–F) Analysis of CHC localization. IF staining (D), relative quantification (E), and WB analysis (F) of CHC localization on metaphase spindle and cytosolic fraction of WT and PI3K-C2a-silenced HeLa cells. Results are shown as mean ± SEM (IF, n = 200 cells; WB, n = 5). (legend continued on next page)

Article Snippet: Three micron thick sections of formalin fixed paraffin embedded (FFPE) samples were stained for haematoxylin and eosin (H&E) and immunohistochemistry with antibodies against PI3K-C2a (clone OTI15H1, formerly 15H1, diluted 1:1000, Origene).

Techniques: Staining, Control

Journal: Cancer cell

Article Title: Mitotic Spindle Assembly and Genomic Stability in Breast Cancer Require PI3K-C2α Scaffolding Function.

doi: 10.1016/j.ccell.2017.09.002

Figure Lengend Snippet: Figure 5. Candidate Genes of the SAC Bypass Metaphase Defect in PI3K-C2a-Deficient Cells (A and B) Time required to progress from cell roundup to anaphase in WT, early, and fast-growing adapted Pik3c2a/ MEFs (A) and MMET cells (B). Results are shown as mean ± SD (n = 100 cells). (C) IF staining of WT and fast-growing adapted Pik3c2a/ MEFs metaphases (n = 5). Scale bar, 5 mm. (D) Real-time qPCR analysis of expression of SAC-related genes in MMET cells. Results are shown as mean ± SD (n = 5). (E) Representative images of late, fast-growing WT and Het/NeuT MMET cells treated with 5 mM nocodazole for 12 hr. Scale bars, 60 mm. (F and G) Effect of spindle assembly checkpoint kinase MPS1 (MPSi) inhibitor on the time required to progress from cell roundup to anaphase on WT and Pik3c2a/ MEFs (F) and MMET (G). Results are shown as mean ± SEM (n = 200 cells). (H) Effect of spindle assembly checkpoint kinase MPS1 (MPSi) inhibitor on MCF10A after PIK3C2A CRISPR/Cas9-targeted deletion. Data are normalized on untreated empty MCF10A cells after 48 hr. Results are shown as mean ± SEM (n = 5). *p < 0.05, ***p < 0.001 by ANOVA followed by Bonferroni’s post hoc test. See also Table S4.

Article Snippet: Three micron thick sections of formalin fixed paraffin embedded (FFPE) samples were stained for haematoxylin and eosin (H&E) and immunohistochemistry with antibodies against PI3K-C2a (clone OTI15H1, formerly 15H1, diluted 1:1000, Origene).

Techniques: Staining, Expressing, CRISPR

Journal: Cancer cell

Article Title: Mitotic Spindle Assembly and Genomic Stability in Breast Cancer Require PI3K-C2α Scaffolding Function.

doi: 10.1016/j.ccell.2017.09.002

Figure Lengend Snippet: Figure 6. PI3K-C2a Is a Synthetic Lethality Partner of Taxane-Based Treatment (A) Effect of paclitaxel (PTX) anti-proliferative activity, measured as percent cell survival after treatment, on WT/NeuT and Het/NeuT MMET (plain bars) and rescue experiments with WT or KD PI3K-C2a (dashed bars). Results are shown as mean ± SD (n = 5). (B) Effect of doxorubicin (DOXO) anti-proliferative activity, measured as percent cell survival after treatment, on WT/NeuT and Het/NeuT MMET (plain bars) and rescue experiments with WT or KD PI3K-C2a (dashed bars) to restore resistance to DOXO treatment. Results are shown as mean ± SD (n = 5). (C) Effect of PTX, DOXO, or combination of the two drugs (PTX + DOXO) on orthotopic MMET tumors engrafted in syngeneic mice. The treatment was started after 20 days from tumor inoculation (start PTX) and was stopped 15 days later (end PTX). Tumor volume is shown as mean ± SEM (n = 6 mice). (D) IF analysis of mitotic spindles in WT/NeuT L and Het/NeuT L treated with 10 nM PTX or vehicle only. Spindles where subdivided into four categories (normal, abnormal chromosome alignment, multipolar, and monopolar) and percentage of each category reported in the graph (NT, no treatment). Yellow dashed lines indicate the metaphase plate width. Results are shown as mean ± SD (n = 80). Scale bars, 5 mm. (E) Time-lapse analysis of late WT/NeuT and Het/NeuT treated with 10 nM or 100 nM of PTX or vehicle only. Percentage of cells exiting mitosis with no division is reported in the graph. Results are shown as mean ± SD (n = 100). (F) FACS analysis of late WT/NeuT and Het/NeuT MMET to score DNA content (propidium iodide staining) after treatment with PTX (indicated dosage, 24 hr). NT, no treatment. Percentage of cells with aneuploidy is shown as mean ± SEM (n = 5). *p < 0.05, ***p < 0.001 by ANOVA followed by Bonferroni’s post hoc test. See also Figure S7 and Movie S2.

Article Snippet: Three micron thick sections of formalin fixed paraffin embedded (FFPE) samples were stained for haematoxylin and eosin (H&E) and immunohistochemistry with antibodies against PI3K-C2a (clone OTI15H1, formerly 15H1, diluted 1:1000, Origene).

Techniques: Activity Assay, Staining

Journal: Cancer cell

Article Title: Mitotic Spindle Assembly and Genomic Stability in Breast Cancer Require PI3K-C2α Scaffolding Function.

doi: 10.1016/j.ccell.2017.09.002

Figure Lengend Snippet: Figure 7. Role of PI3K-C2a in Human Breast Cancer (A) Propidium iodide staining and FACS analysis of shRNA-PI3K-C2a MCF7 (MCF7 sh), shRNA-PI3K-C2a SKBR3 (SKBR3 sh), shRNA-PI3K-C2a MCF7 derived from explanted tumors (MCF7 sh T) and scramble MCF7 derived from explanted tumors (MCF7 Scr T) treated with 100 ng/mL nocodazole for 16 hr. Red arrows indicates G2/M peak. (B) Tumor growth curve of scramble (Scr) and shRNA-PI3K-C2a MCF7 xenografts in NSG mice. Upper graph: tumor volume quantification of scramble (n = 9) versus shRNA (n = 10) MCF7. Lower graph: tumor volume quantification of scramble (circles, n = 9) and shRNA-treated MCF7cells (growth arrested, squares, n = 6; or growing, triangles, n = 4). The red shaded background indicates the early stage of tumors and the gray one the late phase. Results are shown as mean ± SEM. (C) PI3K-C2a expression was assessed by WB analysis in indicated cell lines and primary tumors. WCL lysates were immunoblotted with indicated antibodies (n = 3). (D) Cell survival analyses of scramble (Scr), shRNA-PI3K-C2a, sh + PI3K-C2a WT, and sh + PI3K-C2a KD breast cancer cell lines (BT474, MCF7, MDA231, and SKBR3) after incubating with PTX, DOXO, or a combination of the two drugs (PTX + DOXO) for 24 hr. Cell survival was determined by MTT assays as ratio of live cells after treatment compared with live cells treated with vehicle. Results are shown as mean ± SEM (n = 3).

Article Snippet: Three micron thick sections of formalin fixed paraffin embedded (FFPE) samples were stained for haematoxylin and eosin (H&E) and immunohistochemistry with antibodies against PI3K-C2a (clone OTI15H1, formerly 15H1, diluted 1:1000, Origene).

Techniques: Staining, shRNA, Derivative Assay, Expressing