Journal:

Article Title: FLI1 monoallelic expression combined with its hemizygous loss underlies Paris-Trousseau/Jacobsen thrombopenia

doi: 10.1172/JCI200421197

Figure Lengend Snippet: Restoration of megakaryocytic phenotype in two PTS patients after FLI1 cDNA transfer in vitro. (A) Flow-cytometry analysis of CD41 and CD42 expression in MKs from a control and two patients (P1 and P2) 11 days after transduction of their peripheral blood CD34+ cells with PGK-IRES-eGFP or with PGK-Fli1-IRES-eGFP. Gate R3 and numbers indicated below the gates represent the percentage of mature MKs with high levels of CD41 and CD42 expression. (B) Immunolabeling of FLI1 (red staining) and vWF (green staining) in patient 1 and 3 (P1 and P3) MKs grown from CD34+ cells transduced (T), or not (NT), with PGK-Fli1-IRES-eGFP. Images were captured using epifluorescence microscope (Nikon Eclipse 600) with a ×40 objective. Large, presumably polyploid MKs characterized by a typical polylobulated nucleus (DAPI staining) and by a unique, continuous cytoplasmic membrane (vWF staining) in patient 1 and 3 after FLI1 transduction are indicated by a white arrowhead.

Article Snippet: Images were captured using epifluorescence microscope (Nikon Eclipse 600) with a ×40 objective.

Techniques: In Vitro, Flow Cytometry, Expressing, Transduction, Immunolabeling, Staining, Microscopy

Journal:

Article Title: FLI1 monoallelic expression combined with its hemizygous loss underlies Paris-Trousseau/Jacobsen thrombopenia

doi: 10.1172/JCI200421197

Figure Lengend Snippet: Fli1 expression throughout normal MK differentiation. CD34+ cells were cultured in the presence of TPO and analyzed at day 6. (A) Fli1 expression level determined by real-time RT-PCR. Diploid MKs were sorted according to their differentiation stages. Fli1 and two housekeeping β2-M and β5-tubulin (β5-T) genes (constant throughout MK differentiation; our unpublished data) were amplified in separate wells in five independent experiments (four using β2-M and one using β5-T). The relative expression level of Fli1 in each fraction was normalized to the corresponding values for β2-M or β5-T. A representative experiment is shown with error bars representing the SD of the mean of triplicate wells. (B) Representative picture of TPO-cultured cells showing the distribution of Fli1 nuclear RNA (green) and chromosome 12 (red) visualized by FISH. Cell with a monoallelic expression of Fli1 is on the left, and cell with its biallelic expression is on the right.The number of green spots indicates the number of transcribed Fli1 alleles. The number of Fli1 alleles in the same MK is determined by the number of red spots. Chromatin is counterstained with DAPI (blue). (C) Statistical analysis of monoallelic/biallelic expression of Fli1 in diploid MKs during their differentiation. Cells were sorted as described in A, and RNA-FISH was performed as described in B. One hundred cells of each population were analyzed on an epifluorescence microscope (Nikon Eclipse 600) using a ×60 objective for the presence of Fli1 pre-mRNA. The error bars represent the SD of the mean of three independent experiments.

Article Snippet: Images were captured using epifluorescence microscope (Nikon Eclipse 600) with a ×40 objective.

Techniques: Expressing, Cell Culture, Quantitative RT-PCR, Amplification, Microscopy

Journal: Journal of Cell Science

Article Title: Kindlin-2 interacts with a highly conserved surface of ILK to regulate focal adhesion localization and cell spreading

doi: 10.1242/jcs.221184

Figure Lengend Snippet: I427E mutation of αH in surface 2 of ILK-pKD impairs binding of GFP–ILK to GST–kindlin-2 F2PH. (A) GFP or GFP–ILK co-expressed with FLAG–α-parvin in CHO cells bound to glutathione bead-immobilized GST–kindlin-2 F2PH or GST–kindlin-2 F2PH L357A (L/A) as a negative control detected by immunoblotting. One representative blot for each construct tested is shown. The lane labeled ‘3%’ indicates 3% of input lysate. Bead loading was visualized by Ponceau S staining. (B) Quantification of GFP or GFP–ILK binding to GST–kindlin-2 F2PH or GST–kindlin-2 F2PH L/A (mean±s.e.m.; n=4). (C) Representative immunoblots for pulldown of GFP–ILK mutants co-expressed with FLAG–α-parvin in CHO cell lysates by GST–kindlin-2 F2PH or GST–kindlin-2 F2PH L/A. The lane labeled ‘3%’ indicates 3% of input lysate. Bead loading was visualized by Ponceau S staining. (D) Quantification of binding of GFP–ILK and GFP–ILK mutants to GST–kindlin-2 F2PH or GST–kindlin-2 F2PH L/A (mean±s.e.m.; n≥3); *P<0.005 (Student's t-test). Pulldown quantification graphs are shown as bar charts with individual data points plotted (dots).

Article Snippet: Primary antibodies against GFP (Rockland, catalog #601-101-215; Limerick, PA) (used at 1:1000), ILK (Cell Signaling, catalog #3862; Danvers, MA) (used at 1:1000), FLAG (Sigma, catalog #F1804; St. Louis, MO) (used at 1:1000), vinculin (Sigma, catalog #V9131) (used at 1:10,000), kindlin-2 (Proteintech, catalog #11453-1-AP; Rosemont, IL) (used at 1:1000), α-parvin (Cell Signaling, catalog #8190) (used at 1:1000), PINCH1 (Proteintech, catalog #55336-1-AP) (used at 1:1000), carbonyl reductase (Santa Cruz Biotechnology, catalog #sc-70212; Dallas, Texas) (used at 1:1000) and β-tubulin (Developmental Studies Hybridoma Bank, catalog #E7; Iowa City, Iowa) (used at 1:1000) as well as IRDye-conjugated secondary antibodies (Li-Cor; Lincoln, NE) (used at 1:10,000) were purchased from commercial sources.

Techniques: Mutagenesis, Binding Assay, Negative Control, Western Blot, Construct, Labeling, Staining

Journal: Journal of Cell Science

Article Title: Kindlin-2 interacts with a highly conserved surface of ILK to regulate focal adhesion localization and cell spreading

doi: 10.1242/jcs.221184

Figure Lengend Snippet: Additional residues on αH in the ILK-pKD are implicated in the interaction with kindlin-2. (A) Ribbon diagram of helix-αH and surrounding residues in the ILK-pKD–α-parvin-CH2 co-crystal structure (PDB ID: 3KMW) generated with Chimera software (Pettersen et al., 2004). Residues selected for mutation are labeled and shown as a ball-and-stick representation. Conservation coloring is indicated using the same color scale as shown in Fig. 1A. (B,C) Pulldown of GFP–ILK or GFP–ILK mutants by GST–kindlin-2 F2PH and GST–kindlin-2 F2PH L357A (L/A) from CHO cell lysate co-overexpressing FLAG–α-parvin assessed by representative immunoblots (B) and quantified (C); mean±s.e.m.; n≥3; *P<0.001 (Student's t-test). (D,E) Pulldown of GFP–ILK or GFP–ILK mutants from CHO cell lysate co-overexpressing FLAG–α-parvin using GST–kindlin-2 329-368 or GST–kindlin-2 329-368 L/A were assessed by representative immunoblots (D) and quantified (E); mean±s.e.m.; n≥3; *P≤0.0006. (F,G) Pulldown of GFP–ILK or GFP–ILK mutants from CHO cell lysate co-overexpressing FLAG–α-parvin using GST or GST–kindlin-2 were assessed in representative immunoblots (F) and quantified (G); mean±s.e.m.; n=4; *P≤0.0001 (Student's t-test). Pulldown quantification graphs are shown as bar charts with individual data points plotted (dots). GST- protein loading is indicated by Ponceau S staining.

Article Snippet: Primary antibodies against GFP (Rockland, catalog #601-101-215; Limerick, PA) (used at 1:1000), ILK (Cell Signaling, catalog #3862; Danvers, MA) (used at 1:1000), FLAG (Sigma, catalog #F1804; St. Louis, MO) (used at 1:1000), vinculin (Sigma, catalog #V9131) (used at 1:10,000), kindlin-2 (Proteintech, catalog #11453-1-AP; Rosemont, IL) (used at 1:1000), α-parvin (Cell Signaling, catalog #8190) (used at 1:1000), PINCH1 (Proteintech, catalog #55336-1-AP) (used at 1:1000), carbonyl reductase (Santa Cruz Biotechnology, catalog #sc-70212; Dallas, Texas) (used at 1:1000) and β-tubulin (Developmental Studies Hybridoma Bank, catalog #E7; Iowa City, Iowa) (used at 1:1000) as well as IRDye-conjugated secondary antibodies (Li-Cor; Lincoln, NE) (used at 1:10,000) were purchased from commercial sources.

Techniques: Generated, Software, Mutagenesis, Labeling, Western Blot, Staining

Journal: Journal of Cell Science

Article Title: Kindlin-2 interacts with a highly conserved surface of ILK to regulate focal adhesion localization and cell spreading

doi: 10.1242/jcs.221184

Figure Lengend Snippet: R243G/R334G double mutation of GFP–ILK (GFP–ILK RR/GG) impairs binding of the ILK to α-parvin. (A) Ribbon diagram of selected regions in the ILK KD–α-parvin-CH2 complex co-crystal structure (PDB ID: 3KMW) surrounding I244, F245, and S246, generated with Chimera software (Pettersen et al., 2004). Residues selected for mutagenesis are labeled and shown as a ball-and-stick representation. Conservation coloring is indicated using the same color scale as shown in Fig. 1A. (B,C) Pulldown of GFP–ILK or GFP–ILK RR/GG from CHO cell lysates co-overexpressing FLAG–α-parvin using GST–kindlin-2 F2PH or GST–kindlin-2 F2PH L357A (L/A) assessed by representative immunoblots (B) and quantified (C); mean±s.e.m.; n=3; *P≤0.0001 (Student's t-test). (D) Diagram of the GFP nanotrap experiment. GFP–ILK was purified from lysate of cells co-expressing GFP–ILK and FLAG-α-parvin. The amount of co-purifying FLAG–α-parvin was assessed by immunoblotting and the FLAG:GFP ratio for each construct was calculated. (E,F) GFP-nanotrap co-purification of GFP–ILK constructs co-expressed FLAG–α-parvin in CHO cells was assessed by immunoblot from one experiment (E) and quantified (F) as a raw FLAG:GFP ratio. Dots represent single data points for each construct tested. The lane labeled ‘2%’ indicates the 2% input of lysate. (G) The FLAG:GFP ratio for each GFP or GFP–ILK construct tested is expressed relative to the FLAG:GFP ratio of the GFP–ILK control within each experiment, which is set to 1. The dataset includes the experiment shown in E and F (mean±s.e.m.; n≥4); *P≤0.0015; ns, statistically not significant, P>0.05 (Student's t-test). (H,I) Pulldown of GFP–ILK K220M from CHO cell lysate co-overexpressing FLAG–α-parvin using GST–kindlin-2 F2PH or GST–kindlin-2 F2PH L/A assessed by representative immunoblot (H) and quantified (I); mean±s.e.m.; n=3; *P≤0.0001. Pulldown and co-purification quantification graphs are shown as bar charts with individual data points plotted (dots). GST-protein loading control for pulldown experiments is indicated by Ponceau S staining.

Article Snippet: Primary antibodies against GFP (Rockland, catalog #601-101-215; Limerick, PA) (used at 1:1000), ILK (Cell Signaling, catalog #3862; Danvers, MA) (used at 1:1000), FLAG (Sigma, catalog #F1804; St. Louis, MO) (used at 1:1000), vinculin (Sigma, catalog #V9131) (used at 1:10,000), kindlin-2 (Proteintech, catalog #11453-1-AP; Rosemont, IL) (used at 1:1000), α-parvin (Cell Signaling, catalog #8190) (used at 1:1000), PINCH1 (Proteintech, catalog #55336-1-AP) (used at 1:1000), carbonyl reductase (Santa Cruz Biotechnology, catalog #sc-70212; Dallas, Texas) (used at 1:1000) and β-tubulin (Developmental Studies Hybridoma Bank, catalog #E7; Iowa City, Iowa) (used at 1:1000) as well as IRDye-conjugated secondary antibodies (Li-Cor; Lincoln, NE) (used at 1:10,000) were purchased from commercial sources.

Techniques: Mutagenesis, Binding Assay, Generated, Software, Labeling, Western Blot, Purification, Expressing, Construct, Copurification, Control, Staining

Journal: Journal of Cell Science

Article Title: Kindlin-2 interacts with a highly conserved surface of ILK to regulate focal adhesion localization and cell spreading

doi: 10.1242/jcs.221184

Figure Lengend Snippet: GFP–ILK mutants that are impaired in kindlin-2 binding localize poorly to focal adhesions. CHO cells stably expressing mCherry-paxillin were transiently co-transfected with FLAG–α-parvin and either GFP alone, GFP–ILK or one of the GFP–ILK mutants. Six hours after replating on fibronectin-coated glass-bottom dishes, live cells were imaged by epifluorescence (EPI) and/or TIRF microscopy as indicated. Images in each channel were linearly and uniformly adjusted, and cropped for clarity. Scale bar: 20 µm.

Article Snippet: Primary antibodies against GFP (Rockland, catalog #601-101-215; Limerick, PA) (used at 1:1000), ILK (Cell Signaling, catalog #3862; Danvers, MA) (used at 1:1000), FLAG (Sigma, catalog #F1804; St. Louis, MO) (used at 1:1000), vinculin (Sigma, catalog #V9131) (used at 1:10,000), kindlin-2 (Proteintech, catalog #11453-1-AP; Rosemont, IL) (used at 1:1000), α-parvin (Cell Signaling, catalog #8190) (used at 1:1000), PINCH1 (Proteintech, catalog #55336-1-AP) (used at 1:1000), carbonyl reductase (Santa Cruz Biotechnology, catalog #sc-70212; Dallas, Texas) (used at 1:1000) and β-tubulin (Developmental Studies Hybridoma Bank, catalog #E7; Iowa City, Iowa) (used at 1:1000) as well as IRDye-conjugated secondary antibodies (Li-Cor; Lincoln, NE) (used at 1:10,000) were purchased from commercial sources.

Techniques: Binding Assay, Stable Transfection, Expressing, Transfection, Microscopy

Journal: Journal of Cell Science

Article Title: Kindlin-2 interacts with a highly conserved surface of ILK to regulate focal adhesion localization and cell spreading

doi: 10.1242/jcs.221184

Figure Lengend Snippet: The ILK–kindlin-2 interaction is important for normal cell spreading in HeLa cells. (A) Immunoblotting of shScr and shILK HeLa cells to show protein levels of ILK, kindlin-2, α-parvin, PINCH1 and vinculin. (B) Bar graph showing residual protein levels in shILK cells calculated relative to those in shScr cells (mean±s.e.m.); individual data points are indicated (dots; n≥4). (C) Immunofluorescence staining of endogenous vinculin in fixed shScr or shILK HeLa cells spread on fibronectin-coated glass coverslips and acquired by epifluorescence microscopy. Scale bars: 20 µm. (D) CellProfiler quantification of GFP-positive cell areas pooled across three independent experiments shown as box and whiskers plots indicating 10th and 90th percentile range. n=155 shScr+GFP cells, 169 shILK+GFP cells, 154 shILK+GFP–ILK cells, 162 shILK+I427E cells, 143 shILK+K423D cells, 137 shILK+K426D cells, 125 shILK+I413D cells; *, significantly different from shScr+GFP calculated using one-way ANOVA and Tukey's correction for multiple comparisons (P≤0.01); ns, statistically not significant; P>0.05. (E) Immunoblotting of shScr and shK2 HeLa cells to show protein levels of kindlin-2, ILK, and β-tubulin. (F) Bar graph showing residual kindlin-2 or ILK protein levels in shK2 cells calculated relative to those in shScr cells (mean±s.e.m.); individual data points are indicated (dots; n≥3). (G) Immunofluorescence staining of endogenous vinculin in fixed shScr or shK2 HeLa cells spread on fibronectin-coated glass coverslips and acquired by epifluorescence microscopy. Scale bars: 20 µm. (H) CellProfiler quantification of GFP-positive cells areas pooled across three independent experiments; n=143 shScr+GFP cells, 121 shK2+GFP cells, 145 shK2+GFP-K2 cells, 130 shK2+GFP-K2 LA cells. Data are shown as box and whiskers plot, with whiskers indicating the 10th and 90th percentile range. *, significantly different from shScr+GFP calculated using one-way ANOVA and Tukey's correction for multiple comparisons (P≤0.015); ns, statistically not significant. Microscopy images were linearly and uniformly adjusted for clarity.

Article Snippet: Primary antibodies against GFP (Rockland, catalog #601-101-215; Limerick, PA) (used at 1:1000), ILK (Cell Signaling, catalog #3862; Danvers, MA) (used at 1:1000), FLAG (Sigma, catalog #F1804; St. Louis, MO) (used at 1:1000), vinculin (Sigma, catalog #V9131) (used at 1:10,000), kindlin-2 (Proteintech, catalog #11453-1-AP; Rosemont, IL) (used at 1:1000), α-parvin (Cell Signaling, catalog #8190) (used at 1:1000), PINCH1 (Proteintech, catalog #55336-1-AP) (used at 1:1000), carbonyl reductase (Santa Cruz Biotechnology, catalog #sc-70212; Dallas, Texas) (used at 1:1000) and β-tubulin (Developmental Studies Hybridoma Bank, catalog #E7; Iowa City, Iowa) (used at 1:1000) as well as IRDye-conjugated secondary antibodies (Li-Cor; Lincoln, NE) (used at 1:10,000) were purchased from commercial sources.

Techniques: Western Blot, Immunofluorescence, Staining, Epifluorescence Microscopy, Microscopy