memerald clip170 c 18  (Addgene inc)

Journal: Cell death & disease

Article Title: Tension of plus-end tracking protein Clip170 confers directionality and aggressiveness during breast cancer migration.

doi: 10.1038/s41419-022-05306-6

Figure Lengend Snippet: Fig. 2 TTL expression regulates Clip170 tension and breast cancer invasion and migration. A Left panel: 15-min time-lapse images of FRET analysis in MDA-MB-468 cells expressing the Clip170-cpstFRET probe and transfected with the TTL siRNA or TTL overexpression plasmid after CXCL12 treatment. Calibration bar, 0.2–2.0; scale bar, 10 μm. Right panel: Normalized CFP/FRET signals for the whole cell corresponding to tension versus time, respectively. (Mean ± SEM, n ≥6 cells in each group) (B) Left panel: 15-min time-lapse images of FRET analysis in MCF7 cells expressing the Clip170-cpstFRET probe and transfected with the TTL siRNA or TTL overexpression plasmid after CXCL12 treatment. Calibration bar, 0.2–2.0; scale bar, 10 μm. Right panel: Normalized CFP/FRET signals for the whole cell corresponding to tension versus time, respectively. (Mean ± SEM, n ≥6 cells in each group). C Representative CFP images of MDA-MB-468 cells expressing the Clip170-cpstFRET probe and transfected with the TTL siRNA or TTL overexpression plasmid. D Normalized CFP/FRET signals, comet lengths, comet quantities, and fluorescence intensities for the whole cell in different groups (scale bar, 10 μm, n ≥6 cells in each group, ***P < 0.001 compared with WT- Clip170 alone group). E Representative images of the migration and invasion of MDA-MB-468 cells in the control, TTL siRNA, TTL overexpression and TTL rescue groups. F Quantitative analysis of the migration and invasion cells (Mean ± SEM, n = 3 independent experiments and at least three fields, ***P < 0.001 compared with WT-Clip170 alone group).

Article Snippet: Plasmid construction and small interfering RNA (siRNA) design The mEmerald-CLIP170-C-18 (#54043) was purchased from Addgene (Watertown, MA, USA). pCMV3-TTL (HG23280-UT) and Pcmv3-FlagIQGAP2 plasmids were obtained from Sino Biological (Beijing, China).

Techniques: Expressing, Migration, Transfection, Over Expression, Plasmid Preparation, Control

Journal: Cell death & disease

Article Title: Tension of plus-end tracking protein Clip170 confers directionality and aggressiveness during breast cancer migration.

doi: 10.1038/s41419-022-05306-6

Figure Lengend Snippet: Fig. 6 RSK promotes Clip170 tension and breast cancer aggressiveness. A Left panel: Representative images of FRET analysis of cells treated with the RSK inhibitor (BRD7389) for 0, 1, 3, and 6 h. Calibration bar, 0.2–2.0; scale bar, 10 μm. Right panel: Mean CFP/FRET ratios for the whole cell. (mean ± SEM, n ≥6 cells in each group, **P < 0.01, ***P < 0.001 compared with 0h-treatment group). B Left panel: 15-min time-lapse images of FRET analysis in MDA-MB-468 cells transfected with the Clip170-cpstFRET probe alone or combined with RSK siRNA. Calibration bar, 0.2–2.0; scale bar, 10 μm. Right panel: Normalized CFP/FRET signals for the whole cell corresponding to Clip170 tension versus time (mean ± SEM, n ≥5 cells in each group). C Left panel: Representative images of FRET analysis of MDA-MB-468 cells transfected with the Clip170-cpstFRET, Clip170-S-A-cpstFRET, Clip170-S-D-cpstFRET, Clip170-S311A-cpstFRET, or Clip170-S311D-cpstFRET probes treated with the RSK inhibitor (BRD7389) for 6 h. Calibration bar, 0.2–2.0; scale bar, 10 μm. Right panel: Mean CFP/FRET ratios for the whole cell. (mean ± SEM, n ≥6 cells in each group, ***P < 0.001 compared with the cells without BRD7389 treatment). D Western blotting analysis of RSK-siRNA and RSK inhibitor treatment in MDA-MB-468 cells. E Western blotting analysis of RSK and p-RSK levels in MDA-MB-468, MCF7, and MDA-MB-231 after CXCL12 treatment. F Representative images of the migration and invasion of control, NC, RSK-siRNA, DMSO, and RSK inhibitor (BRD7389) treatment groups. G–H Quantitative analysis of the migration (G) and invasion (H) rates (Mean ± SEM, n = 3 independent experiments and at least three fields, ***P < 0.001 compared with NC or DMSO group). I Western blotting analysis of RSK and p-RSK levels in breast cancer tissue (situ) and adjacent normal tissue (para). J Quantitative analysis of the p-RSK/RSK ratio in breast cancer tissue and matched normal tissues (n = 10).

Article Snippet: Plasmid construction and small interfering RNA (siRNA) design The mEmerald-CLIP170-C-18 (#54043) was purchased from Addgene (Watertown, MA, USA). pCMV3-TTL (HG23280-UT) and Pcmv3-FlagIQGAP2 plasmids were obtained from Sino Biological (Beijing, China).

Techniques: Transfection, Western Blot, Migration, Control

Journal: Cell death & disease

Article Title: Tension of plus-end tracking protein Clip170 confers directionality and aggressiveness during breast cancer migration.

doi: 10.1038/s41419-022-05306-6

Figure Lengend Snippet: Fig. 7 IQGAP 1 location regulates cortactin-associated formation of filopodia and lamellipodia. A Representative images of MF and IQGAP1 or IQGAP2 distribution in the directional migration model or after CXCL12 treatment. (green, FITC-stained MFs; red, TRITC-stained IQGAP1/IQGAP2), scale bar, 10 μm. B Representative images of MF and IQGAP1 or IQGAP2 distribution in the wound healing assay. (green, FITC-stained MFs; red, TRITC-stained IQGAP1/IQGAP2), scale bar, 10 μm. C Western blotting analysis of Cortactin, IQGAP1, and IQGAP2 levels in the NC, IQGAP1-siRNA, IQGAP2-siRNA, and IQGAP2 and IQGAP2 rescue groups. D Western blotting analysis of cortactin levels in the WT, S311A, S311D, S-A, and S-D groups. E Western blotting analysis of cortactin levels in the control, NC, RSK- siRNA, and RSK inhibitor treatment groups. F Representative images of filopodia and lamellipodia structures in the Clip170 mutation, and NC, IQGAP1 and IQGAP2-siRNA groups (scale bar, 10 μm, green, FITC-stained MFs; blue, nucleus; white arrows: filopodia and lamellipodia structures).

Article Snippet: Plasmid construction and small interfering RNA (siRNA) design The mEmerald-CLIP170-C-18 (#54043) was purchased from Addgene (Watertown, MA, USA). pCMV3-TTL (HG23280-UT) and Pcmv3-FlagIQGAP2 plasmids were obtained from Sino Biological (Beijing, China).

Techniques: Migration, Staining, Wound Healing Assay, Western Blot, Control, Mutagenesis

Journal: Cell death & disease

Article Title: Tension of plus-end tracking protein Clip170 confers directionality and aggressiveness during breast cancer migration.

doi: 10.1038/s41419-022-05306-6

Figure Lengend Snippet: Fig. 8 IQGAP1 and 2 are correlated in Clip170 tension magnitude and distribution. A Representative images of MDA-MB-468 expressing the Clip170-cpstFRET probe in the IQGAP1-siRNA, IQGAP2-siRNA, IQGAP2 overexpression, and IQGAP2 rescue groups. B Normalized CFP/FRET signals, comet lengths, comet quantities, and fluorescence intensities for the whole cell in the different groups (scale bar, 10 μm, n ≥6 cells in each group, ***P < 0.001 compared with NC group). C Representative images of MDA-MB-468 cells expressing the Clip170-cpstFRET probe in the IQGAP1-siRNA, IQGAP2-siRNA, IQGAP2 overexpression, and IQGAP2 rescue groups in the directional migration model, scale bar, 10 μm.

Article Snippet: Plasmid construction and small interfering RNA (siRNA) design The mEmerald-CLIP170-C-18 (#54043) was purchased from Addgene (Watertown, MA, USA). pCMV3-TTL (HG23280-UT) and Pcmv3-FlagIQGAP2 plasmids were obtained from Sino Biological (Beijing, China).

Techniques: Expressing, Over Expression, Migration