Journal: OncoTargets and therapy

Article Title: Integrated RNA Sequencing and Single-Cell Mass Cytometry Reveal a Novel Role of LncRNA HOXA-AS2 in Tumorigenesis and Stemness of Hepatocellular Carcinoma

doi: 10.2147/OTT.S272717

Figure Lengend Snippet: Purified Antibodies About the Stem-Like Cells Centric Panel

Article Snippet: Vimentin , 156Gd , RV202 , Fluidigm , 3156023A.

Techniques: Purification

Journal: Circulation Research

Article Title: Induction of Cardiomyocyte-Like Cells in Infarct Hearts by Gene Transfer of Gata4, Mef2c, and Tbx5

doi: 10.1161/circresaha.112.271148

Figure Lengend Snippet: Figure 1. Retrovirus infection in mouse hearts. A, Section of mouse heart 7 days after MI (Azan staining). B, Retroviral GFP expression was localized to the infarct-border region. The infarcted area is delineated by dots. C and D, GFP retrovirus was injected into the infarct heart. GFP+ cells were immunopositive for vimentin and collagen 1 (C) but not for α-actinin (D). E, Ratios of α-actinin+ cardiomyoctes (CM), vimentin+ CF, and others to GFP+ cells (n=4). F and G, Dose dependency of retrovirus infection in mouse hearts, with quantitative analyses shown in G (n=4). H, Genomic DNA PCR analyses for GFP and GAPDH in cardiac tissues infected by 1- to 100-fold concentrated retrovirus solution. I, FACS analyses for Thy1+/GFP+ cells from mouse hearts after 1 week of GFP retrovirus infection into MI hearts. J, One hundred–fold concentrated GFP retrovirus was injected into the infarcted heart. GFP+ cells were immunopositive for vimentin. K, Z-stack image of the GFP+/vimentin+ cells in J. Representative data are shown in each panel. Inset boxes in the first panels are enlarged in the second to fourth panels (C, D, and J). All data are presented as mean±SEM. **P<0.01 versus relevant control. Scale bars, 1 mm (A and B); 50 μm (C, D, F, J, and K).

Article Snippet: Cells were fixed in 4% paraformaldehyde for 15 minutes at room temperature, blocked, and then incubated with primary antibodies against α-actinin, GFP, cTnT, vimentin, CD31, SMA (Sigma), collagen 1, Mef2c (Aviva Systems Biology), Gata4 (Santa Cruz Biotechnology), ANP (Chemicon), Nkx2.5 (Santa Cruz), or SM- MHC (Biomedical Technologies), with secondary antibodies conjugated to Alexa 488 or 546.

Techniques: Infection, Staining, Retroviral, Expressing, Injection, Control

Journal: Circulation Research

Article Title: Induction of Cardiomyocyte-Like Cells in Infarct Hearts by Gene Transfer of Gata4, Mef2c, and Tbx5

doi: 10.1161/circresaha.112.271148

Figure Lengend Snippet: Figure 5. Induction of cardiomyocyte-like cells by a polycistronic vector. A, Schematic representation of the 3F2A retrovirus containing GMT for polycistronic gene expression. B, Western blot analysis of 293 cells transfected with 3F2A for 2 days. C, Immunocytochemistry for Gata4 and vimentin in mouse fibroblasts transduced with 3F2A. D, Gata4 and Mef2c were expressed in the same cells after transduction of 3F2A. E, 3F2A retrovirus induced α-actinin expression and cross striations. The cell, indicated by arrows in the first and second panels, is enlarged in the third to fifth panels. White boxes are enlarged in the insets. F, Z-stack image of the GFP+/ α-actinin+ cell in E. G and H, Quantitative analyses of the α-actinin+ to GFP+ cell ratios (G) and the proportions of α-actinin+/GFP+ cells showing cross striations (H) (120 sections from 5 mice in control, 140 section from 5 mice in GMT, and 110 sections from 5 mice in 3F2A). I, The 3F2A-expressing cells also showed cTnT expression. White boxes in the first panels are enlarged in the second to fourth panels. J, Schematic representation to analyze mRNA expression in GFP- or 3F2A/GFP-infected cells. GFP+ cells were sorted by FACS after 1 week. K, 3F2A/GFP-transduced cells expressed significantly higher cardiac genes compared with controls determined by qPCR (n=3). Representative data are shown in each panel. All data are presented as mean±SEM. *P<0.05;**P<0.01 versus control. Scale bars, 50 μm.

Article Snippet: Cells were fixed in 4% paraformaldehyde for 15 minutes at room temperature, blocked, and then incubated with primary antibodies against α-actinin, GFP, cTnT, vimentin, CD31, SMA (Sigma), collagen 1, Mef2c (Aviva Systems Biology), Gata4 (Santa Cruz Biotechnology), ANP (Chemicon), Nkx2.5 (Santa Cruz), or SM- MHC (Biomedical Technologies), with secondary antibodies conjugated to Alexa 488 or 546.

Techniques: Plasmid Preparation, Gene Expression, Western Blot, Transfection, Immunocytochemistry, Transduction, Expressing, Control, Infection

Journal: Journal of cell science

Article Title: Cytoplasmic intermediate filaments mediate actin-driven positioning of the nucleus.

doi: 10.1242/jcs.076356

Figure Lengend Snippet: Fig. 1. Cytoplasmic IFs surround the nucleus and accumulate on the side of the nucleus facing the free edge of the cell. Astrocytes were plated onto 100-m- diameter circular fibronectin-printed micropatterns and fixed 7 hours later. When indicated, the culture medium (+Ca) was changed to Ca2+-free medium 2 hours after plating (–Ca). (A,C)Hoechst (blue), phalloidin (red), vimentin (green), nestin (yellow) stainings and (B) perinuclear density (see supplementary material Fig. S3 for details) of actin, nestin and vimentin. Data are given as means+s.d. for three independent experiments comprising at least 180 cells. *P<0.05; **P<0.005; N.S., not significant. The gray horizontal line indicates the theoretical value (13%) of the perinuclear density in the case of an entirely homogenous staining inside the cell. The right-hand panels in C show segmented images obtained after the image treatment described in supplementary material Fig. S3. ‘’ (°) is the angle between the axis defined by the nucleus center and the center of the IF or actin area (green line) and the micropattern radius passing through the nucleus center (black line). (D)Distribution of values. The median angle and statistical differences are indicated in black. **P<0.005. (E)Hoechst (white), phalloidin (red), nestin (green) stainings. The left-hand panels show three-dimensional reconstructions of the 20 planes acquired with a step of 0.2m in the z-direction. The right- hand panels show orthogonal sections of the same three-dimensional reconstructions. Scale bars: 20m (A, left-hand panels E); 2m (right-hand panels E).

Article Snippet: We used primary antibodies against the following proteins: pan-cadherin CH-19 (Sigma), GFAP, vimentin, lamin A (Santa Cruz Biotechnology), nestin (Chemicon), pericentrin, nesprin-1, myosin IIA, myosin IIB (Covance), plectin, lamin B1 (Abcam) and -tubulin (AbD Serotec).

Techniques: Staining

Journal: Journal of cell science

Article Title: Cytoplasmic intermediate filaments mediate actin-driven positioning of the nucleus.

doi: 10.1242/jcs.076356

Figure Lengend Snippet: Fig. 2. The actin cytoskeleton controls cytoplasmic IF organization. (A,B)Primary rat astrocytes were plated onto large fibronectin-printed micropatterns. Cells were treated with 1M cytochalasin D (CytoD) 4 hours after plating and fixed 7 hours after plating. (A)Hoechst (blue), phalloidin (red) and vimentin (green) stainings. The middle and right-hand panels show higher magnification of the stainings in the free edge of the cell (1) or in the perinuclear zone (2). (B)Perinuclear density of nestin (Nest) and vimentin (Vim). Data are given as means+s.d. for three independent experiments comprising at least 180 cells. *P<0.05; **P<0.005. The gray horizontal line indicates the theoretical value (13%) of the perinuclear density in the case of an entirely homogenous staining inside the cell. (C–E) Astrocytes transiently coexpressing EGFP–vimentin and LifeAct–Cherry were left to adhere onto adhesive micropatterns for 2 hours in Ca2+-containing medium, which was then exchanged for Ca2+-free medium for a further 2 hours. Ca2+ was then added back into the medium, to promote adherens junction formation and nuclear movement. The cell was imaged every 15 minutes from 2 hours after the Ca2+ re-addition (t0). (C)Selected images, at the indicated times, from supplementary material Movie 2. The right-hand panels show a higher magnification of the GFP image in the perinuclear region. The nucleus shape is highlighted by a continuous white line. At t495 minutes, the initial position of the nucleus (broken line) is also shown. White arrows point to the perinuclear IF accumulation. (D)High-magnification image of the free edge of the cell upon Ca2+ addition, showing vimentin filaments (white arrowheads) moving together with actin fibers (yellow arrowheads). (E)Velocities of actin, vimentin filaments and the nucleus measured in six cells during the Ca2+ switch experiment. *P<0.05; N.S., not significant. Scale bars: 20m.

Article Snippet: We used primary antibodies against the following proteins: pan-cadherin CH-19 (Sigma), GFAP, vimentin, lamin A (Santa Cruz Biotechnology), nestin (Chemicon), pericentrin, nesprin-1, myosin IIA, myosin IIB (Covance), plectin, lamin B1 (Abcam) and -tubulin (AbD Serotec).

Techniques: Staining, Adhesive

Journal: Journal of cell science

Article Title: Cytoplasmic intermediate filaments mediate actin-driven positioning of the nucleus.

doi: 10.1242/jcs.076356

Figure Lengend Snippet: Fig. 3. IFs control nucleus position in several cell types. Three days following nucleofection of the indicated siRNAs, astrocytes [or for panel J, SW13 cells expressing (Vim +) or not expressing (Vim –) vimentin, and wild-type MEFs or MEFs from vimentin-knockout mice (Vim–/–)] were plated onto 100-m-diameter circular micropatterns (A,B) or crossbow-shaped micropatterns (F–J). Astrocytes were incubated for 2 hours in the presence of Ca2+. Cells were either incubated in Ca2+-free medium (–Ca) for 7 hours or incubated for 2 hours in Ca2+-free medium followed by 7 hours in Ca2+-containing medium (+Ca). When indicated, cells were treated, 4 h after plating, with 1M cytochalasin D; cytochalasin-D-treated cells were fixed after 3 hours (CytoD), or further incubated for 5 hours in normal medium (CytoD washout). Alternatively, cells were micro-injected 2 hours after plating with dextran, or with dextran and a dominant-negative GFAP construct (GFAP-DN) and incubated for 5 hours. ‘si triple IF’ indicates the triple depletion of vimentin, GFAP and nestin by siRNA. (A)Cells were fixed and stained for pan- cadherin (green) and with Hoechst (blue). The lower-left-hand panels show phalloidin staining and the lower-right-hand panels a higher magnification of a typical cell with the cell center indicated (yellow cross). (B)Nucleus off-centering, defined as the distance between the nucleus center and the cell centroid divided by the square root of the cell area. The green horizontal line indicates the off-centering value (0.12) in single cells plated onto a symmetrical micropattern. Data are given as means+s.d. of three independent experiments totalizing at least 180 cells; statistical differences between the gray or white bars and the corresponding control black bar on their left (Ctl) are indicated. **P<0.005. (C–E) IF-depleted astrocytes (si triple IF) transiently expressing LifeAct–Cherry were left to adhere onto adhesive micropatterns for 2 hours in the presence of Ca2+, the medium was then exchanged for Ca2+-free medium for 2 hours. Ca2+ was then added back to the medium and, after a further 2 hours (t0), the cell was imaged every 15 minutes. (C)Selected image, at t0, from supplementary material Movie 3. The nucleus shape is highlighted by a white line. (D)Higher magnification of the perinuclear region upon Ca2+ addition showing actin fibers (red, orange and yellow) moving in various directions above or under the nucleus. (E)Velocities of actin filaments and the nucleus measured in six IF-depleted cells during the Ca2+ switch experiment. **P<0.005. (F,I)Dextran fluorescence (green) and Hoechst staining (blue) showing the micropattern and the nucleus. (G)Schematic defining the ‘y nucleus’ distance (m) between the nucleus center (blue dot) and the cell center (green cross) along the symmetry axis (Y axis) of the pattern. (H,J)y nucleus position (gray spots) measured on at least 78 cells of the indicated cell types. The blue horizontal line and vertical bar indicate means±s.d. Statistical differences are indicated. **P<0.005. Scale bars: 20m (A,C); 10m (F,I).

Article Snippet: We used primary antibodies against the following proteins: pan-cadherin CH-19 (Sigma), GFAP, vimentin, lamin A (Santa Cruz Biotechnology), nestin (Chemicon), pericentrin, nesprin-1, myosin IIA, myosin IIB (Covance), plectin, lamin B1 (Abcam) and -tubulin (AbD Serotec).

Techniques: Control, Expressing, Knock-Out, Incubation, Injection, Dominant Negative Mutation, Construct, Staining, Adhesive, Fluorescence

Journal: Cancer Cell

Article Title: Single-cell analysis defines a pancreatic fibroblast lineage that supports anti-tumor immunity

doi: 10.1016/j.ccell.2021.06.017

Figure Lengend Snippet:

Article Snippet: Anti-mouse VIM 154Sm clone D21H3 , Fluidigm , 3154014A.

Techniques: Blocking Assay, Virus, Recombinant, Plasmid Preparation, Saline, Lysis, Staining, Reverse Transcription, In Vivo, Electroporation, Mass Cytometry, Conjugation Assay, Illumina Sequencing, Library Quantification, In Vitro, Quantitative RT-PCR, Software, Real-time Polymerase Chain Reaction, Cytometry, Microscopy, Imaging, Spectrophotometry

Journal: Translational Oncology

Article Title: Uncovering Differently Expressed Markers and Heterogeneity on Human Pancreatic Cancer

doi: 10.1016/j.tranon.2020.100749

Figure Lengend Snippet: Workflow of M-IMC. (A) After tissue staining with metal tagged antibodies on TMA, first step was to create images. After single-cell segmentation of tissue images, higher-order analysis was performed to visualize results such as t-SNE maps. (B) The element of each metal tagged antibody is listed. Five EMT markers: α-SMA, vimentin, cytokeratin 7, pan-keratin, and collagen I. One tumor proliferation marker: Ki-67. One immune cell marker: HLA-DR. Two nuclear counterstainings. (C, D, and E) The expression of each target was generated by heat map; heat map of tumor grade (C), TNM stage (D), tumor stage (E).

Article Snippet: Iridium DNA Intercalator (Fluidigm, 201192A) and metal-labeled antibodies pan-keratin (Nd 148, Fluidigm 3148020D), cytokeratin 7 (Dy 164, Fluidigm 3164028D), Ki-67 (Er 168, Fluidigm 3168022D), HLA-DR (Yb 174, Fluidigm 3174023D), α-SMA (Pr 141, Fluidigm 3141017D), vimentin (Nd 143, Fluidigm 3143029D), and collagen I (Tm 169, Fluidigm 3169023D) were purchased from Fluidigm.

Techniques: Staining, Marker, Expressing, Generated

Journal: Translational Oncology

Article Title: Uncovering Differently Expressed Markers and Heterogeneity on Human Pancreatic Cancer

doi: 10.1016/j.tranon.2020.100749

Figure Lengend Snippet: Individual t-SNE map shows different expression of pattern in each marker. (A) The t-SNE was performed on three EMT markers (left). Bar graph was generated to show the level of expression (right): α-SMA, vimentin, and cytokeratin 7. (B) The t-SNE was performed on two EMT makers and one tumor proliferation marker (left). Bar graph was generated to show the level of expression (right); pan-keratin, Collagen I, and Ki-67. (C) The t-SNE was performed on an leukocyte antigen marker (left), Bar graph was generated to show the level of expression (right); HLA-DR. (D) The t-SNE was performed on nuclear staining (left), Bar graph was generated to show the level of expression (right); HLA-DR.

Article Snippet: Iridium DNA Intercalator (Fluidigm, 201192A) and metal-labeled antibodies pan-keratin (Nd 148, Fluidigm 3148020D), cytokeratin 7 (Dy 164, Fluidigm 3164028D), Ki-67 (Er 168, Fluidigm 3168022D), HLA-DR (Yb 174, Fluidigm 3174023D), α-SMA (Pr 141, Fluidigm 3141017D), vimentin (Nd 143, Fluidigm 3143029D), and collagen I (Tm 169, Fluidigm 3169023D) were purchased from Fluidigm.

Techniques: Expressing, Marker, Generated, Staining

Journal: Translational Oncology

Article Title: Uncovering Differently Expressed Markers and Heterogeneity on Human Pancreatic Cancer

doi: 10.1016/j.tranon.2020.100749

Figure Lengend Snippet: The corresponding histoCAT images to H&E staining. (A) Vimentin in purple and α-SMA in blue were shown in top, and corresponding H&E staining is in the bottom. No expression of α-SMA and vimentin was observed on normal region. The high expression on tumor grade I and II and low expression on grade III were observed. (B) The expression of collagen I was dominantly expressed on tumor grades I and II. Collagen I was observed in only connective tissues on normal and adjacent region (AT). (C) The high expression of Ki-67 was observed some of AT and tumor grades I and II. No expression was observed on normal region and AT. (D) The expression of cytokeratin 7 was observed highly on tumor grade I and AT. (E) The high expression of pan-keratin was observed only on tumor grade I and AT. The low expression was observed on tumor grades II and III. (F) The dominant expression of HLA-DR was observed on tumor grades I and III and AT. Low expression of HLA-DR on tumor grade II was observed. Scale bar: 500 μm.

Article Snippet: Iridium DNA Intercalator (Fluidigm, 201192A) and metal-labeled antibodies pan-keratin (Nd 148, Fluidigm 3148020D), cytokeratin 7 (Dy 164, Fluidigm 3164028D), Ki-67 (Er 168, Fluidigm 3168022D), HLA-DR (Yb 174, Fluidigm 3174023D), α-SMA (Pr 141, Fluidigm 3141017D), vimentin (Nd 143, Fluidigm 3143029D), and collagen I (Tm 169, Fluidigm 3169023D) were purchased from Fluidigm.

Techniques: Staining, Expressing