hpgk lifeact egfp wpre sequence  (Addgene inc)

Journal: bioRxiv

Article Title: Spatial confinement induces reciprocating migration of epidermal keratinocytes and forms triphasic epithelia

doi: 10.1101/2024.11.12.623158

Figure Lengend Snippet: (a) Schematic diagram of LifeAct-KCs cultured on a microporous membrane. Observation at the level of cell exit from the micropores. (b) Time-lapse images of LifeAct-KCs at the level of cell exit from 3.0-µm micropores. Representative images are shown at 30-minute time intervals. Time stamp shows hour:minute. Arrows indicate the cells that exit from the pores and expand under the membrane. Arrowheads indicate the cells that show reciprocating movement. Scale bar: 20 µm. (c) Schematic diagram of LifeAct-KCs cultured on a microporous membrane. Observation at the level of cell entry into the micropores. (d) Time-lapse images of LifeAct-KCs at the level of cell entry into 3.0-µm micropores. Representative images are shown at 5-minute intervals. Time stamp shows hour:minute. Arrowheads indicate the cells that show reciprocating movement. Scale bar: 20 µm. (e, f) Time-lapse images and schematic diagrams of LifeAct-KCs at the level of cell entry into 3.0-µm (e) or 8.0-µm (f) micropores. Representative images are shown at 2-minute intervals. Time stamp shows hour:minute. Scale bar: 10 µm. (g–i) Quantification of reciprocal movement of LifeAct-KCs on 3.0-µm or 8.0-µm micropores. (g) LifeAct signals (white) are shown as a spectrogram, where the X-axis represents each pore, and the Y-axis represents time. (h) Graphs comparing the number of pores presenting the LifeAct signals within 3.0-µm or 8.0-µm micropores per time. The left panel shows oscillation time as the X-axis and the number of pores as the Y-axis. The right panel shows unidirectional movement of the cells (no oscillation within 30 minutes) within each micropore. (i, j) E-cad and Vim labeling of LifeAct-KCs at the level of cells above (i) or within (j) 3.0-µm or 8.0-µm micropores. Autofluorescence of micropores is shown in blue ( j ). Scale bar: 10 µm.

Article Snippet: The GFP-LifeAct-expressing vector was generated by inserting the LifeAct sequence (a gift from Dr. Kurisu, Tokushima University) into the pRetroQ- AcGFP1-C1 vector (Takara Bio).

Techniques: Cell Culture, Membrane, Labeling

Journal: bioRxiv

Article Title: Spatial confinement induces reciprocating migration of epidermal keratinocytes and forms triphasic epithelia

doi: 10.1101/2024.11.12.623158

Figure Lengend Snippet: (a,b) Quantification of LifeAct-KCs present within 3.0-µm micropores 6 hours after cell seeding. Cells were treated with TGF-β ligand (a) or TGF-β receptor inhibitors, SB431542, and SB525334 (b) . (c, d) Quantification of LifeAct-KCs present below 3.0-µm micropores 24 hours after cell seeding. Cells were treated with TGF-β ligand (c) or TGF-b receptor inhibitors (d) . (e, f) Quantification of LifeAct-KCs present within 3.0-µm micropores 6 hours after cell seeding. Cells were treated with cytochalasin D (e) or blebbistatin (f) . (g, h) Quantification of LifeAct-KCs present below 3.0-µm micropores 24 hours after cell seeding. Cells were treated with cytochalasin D (g) or blebbistatin (h). Two-tailed Mann–Whitney U tests (a, c, e–h) and Kruskal–Wallis tests followed by Dunn’s multiple comparison test (b, d) were performed. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Article Snippet: The GFP-LifeAct-expressing vector was generated by inserting the LifeAct sequence (a gift from Dr. Kurisu, Tokushima University) into the pRetroQ- AcGFP1-C1 vector (Takara Bio).

Techniques: Two Tailed Test, MANN-WHITNEY, Comparison

Journal: bioRxiv

Article Title: Spatial confinement induces reciprocating migration of epidermal keratinocytes and forms triphasic epithelia

doi: 10.1101/2024.11.12.623158

Figure Lengend Snippet: (a,b) Quantification of ruthenium red–treated LifeAct-KCs present within 3.0 µm micropores at 6 hours (a) and the cells present below 3.0-µm micropores 24 hours after cell seeding (b) . (c, d) Quantification of Piezo1 KO LifeAct-KCs present within 3.0 µm micropores at 6 hours (c) and the cells present below 3.0-µm micropores 24 hours after cell seeding (d) . (e, f) Quantification of Yoda1-treated LifeAct-KCs present within 3.0-µm micropores at 6 hours (e) and the cells present below 3.0-µm micropores 24 hours after cell seeding (f) . (g) Schematic diagram of bulk RNA-seq comparing HaCaT KCs cultured on a 0.4-µm- or 3.0-µm-pored membrane. The magenta dashed rectangles indicate the cells compared in the bulk RNA-seq. (h) Heatmap of differentially expressed genes identified by the bulk RNA-seq. (i) Gene ontology biological process terms enriched in upregulated and downregulated genes. (j) Heatmap of differentially expressed keratin genes in HaCaT KCs cultured on a 0.4-µm- or 3.0 µm-pored membrane. (k) Keratin 6 (KRT6) labeling of normal human epidermal KCs cultured on a 0.4-µm- or 3.0-µm-pored membrane for 14 days. Scale bar: 20 µm. (l, m) Quantification of pan-KRT6 KO LifeAct-KCs present within 3.0-µm micropores at 6 hours (l) and the cells present below 3.0-µm micropores 24 hours after cell seeding (m) . Two-tailed Mann–Whitney U tests (a–f) and Kruskal–Wallis tests followed by Dunn’s multiple comparison test (l, m) were performed. * p < 0.05; ** p < 0.01; **** p < 0.0001.

Article Snippet: The GFP-LifeAct-expressing vector was generated by inserting the LifeAct sequence (a gift from Dr. Kurisu, Tokushima University) into the pRetroQ- AcGFP1-C1 vector (Takara Bio).

Techniques: RNA Sequencing, Cell Culture, Membrane, Labeling, Two Tailed Test, MANN-WHITNEY, Comparison

Journal: The Journal of Reproduction and Development

Article Title: Efficient derivation of embryonic stem cells and primordial germ cell-like cells in cattle

doi: 10.1262/jrd.2023-087

Figure Lengend Snippet: bPGCLC induction. (A) Scheme for the direct induction of bPGCLCs from bES cells. (B) PGCLC induction with a WNT agonist plus WNT antagonist. Shown are bright-field (BF), fluorescence images and FACS analysis of D4 aggregates of bES cells. BT, BLIMP1-tdTomato. TN, TFAP2C-mNeonGreen. Scale bar: 200 µm. (C) Scheme for the induction of bPGCLCs under the culture conditions indicated. (D) bES cells after 24 h of incubation under the conditions indicated. (E) PGCLC induction with BMP4 and a WNT antagonist. Shown are bright-field (BF) and fluorescence images and FACS patterns of D4 aggregates of bES cells after 24 h of pre-incubation. BT, BLIMP1-tdTomato. TN, TFAP2C-mNeonGreen. Scale bar: 200 µm. (F) PGCLC induction from various cell lines. C1-144 yielded the most BTTN-positive cells when the BMP4 concentration was 200 ng/ml, while C11-928 and N5-319 were best when the BMP4 concentration was 50 ng/ml. The concentration of CHIR was 6 µM in the all experiments. FACS analyses were done on day 4 of PGCLC induction. BT, BLIMP1-tdTomato. TN, TFAP2C-mNeonGreen. Scale bar: 200 µm.

Article Snippet: Existing targeting vectors were treated with restriction enzymes, and mNeonGreen sequences were amplified by PCR from LifeAct-mNeonGreen (98877; Addgene) and cloned using an in-fusion HD cloning kit (TaKaRa Bio, Shiga, Japan).

Techniques: Fluorescence, Incubation, Concentration Assay

Journal: The Journal of Reproduction and Development

Article Title: Efficient derivation of embryonic stem cells and primordial germ cell-like cells in cattle

doi: 10.1262/jrd.2023-087

Figure Lengend Snippet: Separation of bPGCLCs by surface markers. (A) FACS analysis of surface marker protein expression in BTTN-positive and -negative cells in D6 aggregate. Red and grey histograms show the cells with antibody and without antibody, respectively. BT, BLIMP1-tdTomato. TN, TFAP2C-mNeonGreen. (B) Separation of induced PGCLCs (C1-144 ES cell) by surface markers KIT/CD117 and CD44. BT, BLIMP1-tdTomato. TN, TFAP2C-mNeonGreen. (C) Separation of PGCLCs derived from non-reporter bES cells by surface markers. (D) Comparison of gene expression in BTTN+ cells and KIT+/CD44– cells. Shown are ΔC t values determined by qPCR analysis.

Article Snippet: Existing targeting vectors were treated with restriction enzymes, and mNeonGreen sequences were amplified by PCR from LifeAct-mNeonGreen (98877; Addgene) and cloned using an in-fusion HD cloning kit (TaKaRa Bio, Shiga, Japan).

Techniques: Marker, Expressing, Derivative Assay, Comparison, Gene Expression