Journal: eLife

Article Title: Deciphering caveolar functions by syndapin III KO-mediated impairment of caveolar invagination

doi: 10.7554/eLife.29854

Figure Lengend Snippet:

Article Snippet: Antibody , goat anti-CAVIN-2 (polyclonal) , R & D Systems (Minneapolis, Minnesota) , AF5759 AB_2269901 , 1:200 (western blot).

Techniques: Transfection, Construct, Western Blot, Recombinant, Plasmid Preparation, Software, Staining

Journal: Cancer Science

Article Title: Serum deprivation‐response protein regulates aldehyde dehydrogenase 1 through integrin‐linked kinase signaling in endometrioid carcinoma cells

doi: 10.1111/cas.14007

Figure Lengend Snippet: Immunohistochemistry of serum deprivation‐response protein ( SDPR ) in clinical endometrioid carcinoma samples. A, Representative immunohistochemically stained images of SDPR and the proportion of positive cases according to histological grade (G1, n = 54; G2, n = 38; G3, n = 34). B, Proportion of positive cases with (n = 30) or without (n = 96) lymphatic invasion. C, Representative immunohistochemically stained image of SDPR with the microcystic, elongated, and fragmented ( MELF ) pattern and the proportion of positive G1 cases with (n = 11) or without (n = 43) the MELF pattern. Scale bar = 50 μm (A) and 200 μm (C). Student's t test: * P < .05, ** P < .01

Article Snippet: Similarly, we cotransfected a control CRISPR/Cas9 plasmid (sc‐418922; Santa Cruz Biotechnology) and SDPR HDR plasmid into HEC‐1B and HEC‐108 cells and selected stably transfected cells using puromycin to generate a stable control cell line (EV).

Techniques: Immunohistochemistry, Staining

Journal: Cancer Science

Article Title: Serum deprivation‐response protein regulates aldehyde dehydrogenase 1 through integrin‐linked kinase signaling in endometrioid carcinoma cells

doi: 10.1111/cas.14007

Figure Lengend Snippet: Immunoblotting of serum deprivation‐response protein ( SDPR ) in endometrioid carcinoma cells. A, SDPR protein levels in HEC 1B, HEC 108, HEC 116, and SNG ‐M cells. B, HEC ‐1B and HEC ‐108, ALDH ‐hi cells showed higher levels of SDPR than that of ALDH ‐lo cells. Equal protein loading was confirmed by quantifying β ‐ actin levels (input control). Data are representative of 3 independent experiments. We quantified the results using ImageJ. SDPR /β ‐ actin quotient of ALDH ‐hi cells is expressed as 1. The relative quotient of ALDH ‐lo cells is presented as the ratio to that of ALDH ‐hi cells. Results are shown as the mean ± SE. Student's t test: * P < .05, ** P < .01

Article Snippet: Similarly, we cotransfected a control CRISPR/Cas9 plasmid (sc‐418922; Santa Cruz Biotechnology) and SDPR HDR plasmid into HEC‐1B and HEC‐108 cells and selected stably transfected cells using puromycin to generate a stable control cell line (EV).

Techniques: Western Blot, Control

Journal: Cancer Science

Article Title: Serum deprivation‐response protein regulates aldehyde dehydrogenase 1 through integrin‐linked kinase signaling in endometrioid carcinoma cells

doi: 10.1111/cas.14007

Figure Lengend Snippet: Generation of serum deprivation‐response protein ( SDPR )‐knockout HEC ‐1B and HEC ‐108 cells using the CRISPR /Cas9 system and functional analysis of SDPR . A, Confirmation of loss of SDPR expression in SDPR ‐knockout cells ( KO 1 and KO 2) by immunoblotting. Equal protein loading was confirmed by quantifying β ‐ actin (input control). EV , control cells. B, Cell proliferation on days 2 and 4. C, Matrigel invasion assay. Representative images of invading KO 1, KO 2, and EV cells are shown. Invasive cells were counted in 5 random fields per well. D, Wound‐healing assay. KO 1, KO 2, and EV cell layers were wounded with a pipette tip, and migration toward the wounded area was monitored. The distance of migration was calculated by subtracting the width of the wound at 24 h from that at 0 h. The distance of migration of EV cells is expressed as 1. The relative migration distance of KO 1 and KO 2 cells is presented as the ratio to that of EV cells. E, Chemotaxis assay. Representative images of KO 1, KO 2, and EV cells; proportion of cells in which F‐actin aggregated in the direction of transforming growth factor‐β1 ( TGF ‐β1) in 10 random fields. F, Shapes of KO 1, KO 2, and EV cells. G, Immunoblotting of N‐cadherin and vimentin in the cell lysate and snail in the nuclear extracts from KO 1, KO 2, and EV cells. Equal protein loading was confirmed by quantifying β‐actin in cell lysates and lamin A/C in nuclear extracts. H, Colony formation assay. Representative images of colonies of KO 1, KO 2, and EV cells. Colonies were counted in 5 random fields per well. Data are representative of 3 independent experiments and are shown as means ± SE (B–F, H). Scale bar = 100 μm (C), 200 μm (D), 20 μm (E), 100 μm (F), and 1 mm (H). Student's t test: * P < .05, ** P < .01

Article Snippet: Similarly, we cotransfected a control CRISPR/Cas9 plasmid (sc‐418922; Santa Cruz Biotechnology) and SDPR HDR plasmid into HEC‐1B and HEC‐108 cells and selected stably transfected cells using puromycin to generate a stable control cell line (EV).

Techniques: Knock-Out, CRISPR, Functional Assay, Expressing, Western Blot, Control, Invasion Assay, Wound Healing Assay, Transferring, Migration, Chemotaxis Assay, Colony Assay

Journal: Cancer Science

Article Title: Serum deprivation‐response protein regulates aldehyde dehydrogenase 1 through integrin‐linked kinase signaling in endometrioid carcinoma cells

doi: 10.1111/cas.14007

Figure Lengend Snippet: Serum deprivation‐response protein ( SDPR ) regulates aldehyde dehydrogenase 1 ( ALDH 1) through integrin‐linked kinase ( ILK ) signaling. A, Proportion of ALDH ‐hi cells according to Aldefluor staining (left and middle) and the ALDH 1A1 protein level (right) in SDPR‐knockout KO 1, KO 2, and empty vector ( EV ) HEC ‐1B and HEC ‐108 cells. Equal protein loading was confirmed by quantifying β‐actin (input control). B, Effect of OSU ‐T315 on the proportion of ALDH ‐hi cells (left and middle) and the ALDH 1A1 and SDPR protein level in EV HEC ‐108 cells (right). Equal protein loading was confirmed by quantifying β‐actin (input control). C, Conformation of ILK knockdown in si ILK #1, #2, and #3 EV HEC ‐108 cells and effect of ILK knockdown on the ALDH 1A1 and SDPR protein level (left). Equal protein loading was confirmed by quantifying β‐actin (input control). Proportion of ALDH ‐hi cells in si ILK #1, #2, #3, and siControl cells according to Aldefluor staining (middle and right). Dot‐blot of Aldefluor assay without inhibitor (left panels), and that with inhibitor (right panels). D, Immunoblotting of AKT and phospho‐ AKT protein levels in KO 1, KO 2, and EV HEC ‐108 cell lysates. Equal protein loading was confirmed by quantifying β‐actin (input control). E, Representative images of KO 1, KO 2, and EV HEC ‐108 cells in a chamber slide. Cells were fixed and stained with an anti‐ ILK 1 Ab (left 3 pictures). Scale bar = 20 μm. The plot profile along the lines passing through the center of the cell (left 3 graphs). The relative quotient was defined as the ratio of the pixel intensity of the cell cortex (α) to that of the perinuclear region (β) (middle). ILK 1 protein levels in KO 1, KO 2, and EV HEC ‐108 cell lysates (right) were measured by immunoblotting. Equal protein loading was confirmed by quantifying β‐actin (input control). Data are representative of 3 independent experiments and are shown as means ± SE (A,C,E). Student's t test: * P < .05, ** P < .01

Article Snippet: Similarly, we cotransfected a control CRISPR/Cas9 plasmid (sc‐418922; Santa Cruz Biotechnology) and SDPR HDR plasmid into HEC‐1B and HEC‐108 cells and selected stably transfected cells using puromycin to generate a stable control cell line (EV).

Techniques: Staining, Knock-Out, Plasmid Preparation, Control, Knockdown, Dot Blot, Western Blot

Journal: JCI Insight

Article Title: Key molecular alterations in endothelial cells in human glioblastoma uncovered through single-cell RNA sequencing

doi: 10.1172/jci.insight.150861

Figure Lengend Snippet: ( A ) Plot of transcription factor activity score estimated by SCENIC to fold change of their expression between ECs in periphery and tumor core. Red/blue dot corresponds to transcription factor activated in ECs in the tumor core or tumor periphery, respectively. ( B ) Venn diagram illustrating the overlaps of 374 EC-enriched genes with differentially expressed genes between ECs in tumor core and tumor periphery. ( C ) Heatmap showing differentially expressed EC-enriched genes between ECs in the tumor core and peripheral brain tissue. ( D ) Bar plots of CAVIN2 , HSPG2 , and MYO1B among different EC subclusters. ( E ) IHC staining and quantification of CAVIN2, HSPG2, and MYO1B in human GBM tumor core and paired peripheral brain tissue (CAVIN2, n = 13; HSPG2, n = 13; MYO1B, n = 14). Staining was scored semiquantitatively on scale from 0 to 2 based on proportional of vessels stained (Wilcoxon test, ** P < 0.01, *** P < 0.001). Scale bar: 50 μm.

Article Snippet: Then, the sections incubated with primary antibody toward CD31 (AF806, R&D Systems), CAVIN2 (NBP1-44090, Novus), HSPG2 (AF2364, R&D Systems), MYO1B (ab194356, Abcam), ABCG2 (ab24115, Abcam), SLC2A1 (HPA058494, Sigma-Aldrich), ABCB1 (HPA002199, Sigma-Aldrich), TJP1 (HPA001637, Sigma-Aldrich), CLDN5 (341600, Invitrogen), and PLVAP (NBP1-83911, Novus).

Techniques: Activity Assay, Expressing, Immunohistochemistry, Staining

Journal: Current Biology

Article Title: The Hippo Pathway Regulates Caveolae Expression and Mediates Flow Response via Caveolae

doi: 10.1016/j.cub.2018.11.066

Figure Lengend Snippet:

Article Snippet: Anti-CAVIN2 , Atlas Antibodies , Cat# HPA039325, RRID: AB_10805473.

Techniques: Virus, Recombinant, Transfection, Western Blot, Reverse Transcription, SYBR Green Assay, Luciferase, Mutagenesis, Software

Journal: Cancer Growth and Metastasis

Article Title: A Role for the Cavin-3/Matrix Metalloproteinase-9 Signaling Axis in the Regulation of PMA-Activated Human HT1080 Fibrosarcoma Cell Neoplastic Phenotype

doi: 10.4137/CGM.S18581

Figure Lengend Snippet: PMA treatment triggers MMP-9 secretion and cavin-3 expression, but reduces the HT-1080 cell migration index. ( A ) Human HT-1080 fibrosarcoma cells were treated with varying concentrations of PMA in serum-free medium for 18 hours. Cells were then collected and analyzed for their migration capacity as described in the Methods section. ( B ) Total RNA was extracted from the remaining of the cells and qRT-PCR was performed to assess expression of cavin-1 , cavin-2 , cavin-3 and MMP-9 transcripts. Values were normalized over the housekeeping genes GAPDH and PPIγ and are the mean ± S.E.M of triplicate values from one out of three representative experiments. ( C ) Conditioned media was harvested to assess the extent of MMP-2 and MMP-9 gelatinolytic activity by zymography. ( D ) PMA-induced changes in endogenous cavin-3 protein expression were analyzed by Western blotting.

Article Snippet: C-terminal turboGFP-tagged full length cDNAs encoding human cavin-1, cavin-2, and cavin-3 were purchased from OriGene Technologies.

Techniques: Expressing, Migration, Quantitative RT-PCR, Activity Assay, Zymography, Western Blot

Journal: Cancer Growth and Metastasis

Article Title: A Role for the Cavin-3/Matrix Metalloproteinase-9 Signaling Axis in the Regulation of PMA-Activated Human HT1080 Fibrosarcoma Cell Neoplastic Phenotype

doi: 10.4137/CGM.S18581

Figure Lengend Snippet: Overexpression of recombinant cavin-3 specifically decreases cell migration and abrogates PMA-induced MMP-9 expression. ( A ) Human HT-1080 fibrosarcoma cells were transiently transfected with 2 μg of cDNA plasmids and fluorescent confocal microscopy was used to visualize the encoded recombinant GFP-tagged cavin-1, cavin-2 or cavin-3 proteins 24–48 hours post transfection. pEGFP cDNA plasmid was used as control. ( B ) Cells were then harvested and cell migration assayed as described in the Methods section. ( C ) HT-1080 cells were transiently transfected with plasmids as described above. Twenty-four hours post transfection, cells were treated with vehicle (white bars) or 100 nM PMA (Black bars) in a serum-free medium for 18 hours. Total RNA was extracted and qRT-PCR was performed to measure the levels of MMP-9 transcript. Values of PCR are normalized over the expression of housekeeping genes GAPDH and PPI γ and are the mean ± S.E.M of triplicate values from one representative experiment.

Article Snippet: C-terminal turboGFP-tagged full length cDNAs encoding human cavin-1, cavin-2, and cavin-3 were purchased from OriGene Technologies.

Techniques: Over Expression, Recombinant, Migration, Expressing, Transfection, Confocal Microscopy, Plasmid Preparation, Quantitative RT-PCR