Journal: eLife

Article Title: On demand expression control of endogenous genes with DExCon, DExogron and LUXon reveals differential dynamics of Rab11 family members

doi: 10.7554/eLife.76651

Figure Lengend Snippet: ( A ) Schematic of triple knock-in A2780 cells (mNeonGreen-Rab11a, mCherry-Rab11b, DExCon mTagBFP2-Rab25) created with BioRender.com . ( B–D ) mNeonGreen-Rab11a/mCherry-Rab11b knock-in A2708 cells were further modified with a DExCon-mTag-BFP2 module at the Rab25 locus (Tet-On transactivator introduced by lentivirus with hygromycin selection). Airyscan confocal fluorescence images of triple knock-in cells treated by doxycycline (dox) (>94 hr) trafficking Alexa-647 labeled transferrin (TFN-647, 15–60 min). Colors represent Rab11s as indicated and line profile corresponds to yellow dashed line. ( B ) Scale bar=5µm. ( C ) Maximum intensity Z-projections: top (2D, FN-coated), bottom (3D cell-derived matrix [CDM]). Scale bar=20µm; see also – . ( D–E ) dox induced (>94 hr) triple knock-in A2780 cells recycling TFN-647 were imaged and mNeonGreen/mCherry/mTagBFP2/TFN-647 positive vesicles tracked using Comdet plugin. ( D ) Bar graph representing the percentage of colocalizing vesicles (100 cells, 46,000 vesicles [Rab11s]; 23,000 vesicles [TFN]) and the contribution of individual channels (25- Rab25; TFN- transferrin; a- Rab11a; b- Rab11b). One-way ANOVA Tukey post hoc test used for statistical analysis. ( E ) Venn diagram with the percentage of colocalization for every channel as 100% total; black numbers for Rab11s; yellow for TFN-647. ( F ) Lattice light-sheet imaging of dox induced (>94 hr) triple knock-in A2780 cells recycling TFN-647 are shown in gray as individual channels (one focal plane) or as maximum intensity Z-projection for merged channels; scale bar=10 µm. Line profiles (normalized to maximal intensity, background subtracted) correspond to orange dashed lines, which connects the same vesicles (1. and 3. dot) in the 3D cell volume. See also ,E and and .

Article Snippet: Lentiviral pCDH-antiGFPnanobody-mCherry-Rab11a (overexpressing control) was cloned using pCDH-EF1-tagBFP-T2A-mycBirA*-Rab11a (previously prepared in our lab) via SpeI/SalI and XbaI/XhoI complementary cleavage sites. pMito-mCherry-FRB , a gift from Stephen Royle (Addgene plasmid #59352), was used as a template for generation of pMito-iRFP670-FRB (used with GFP-FKBP for knocksideways). pSH-EFIRES-P-AtAFB2 (Addgene plasmid #129715) , pCDH-EF1-tagBFP-T2A-mycBirA*-Rab11a, and mTagBFP2 were used to generate lentiviral pCDH-AtAFB2-mTagBFP2.

Techniques: Knock-In, Modification, Selection, Fluorescence, Labeling, Derivative Assay, Imaging

Journal: eLife

Article Title: On demand expression control of endogenous genes with DExCon, DExogron and LUXon reveals differential dynamics of Rab11 family members

doi: 10.7554/eLife.76651

Figure Lengend Snippet: ( A–E ) Triple knock-in A2780 cells (mNeonGreen-Rab11a, mCherry-Rab11b, mTagBFP2-Rab25 DExCon) treated by doxycycline (dox) (>94 hr); ( B–E ) recycling Alexa-647 labeled transferrin (TFN-647, 30 min). ( A ) Immunoblots. Fluorescent antibodies: anti-Rab11 targeting both Rab11a/b (Rab11), anti-Rab25 (R25) shown as black and white; Tubulin (Tub), loading control. wt=unmodified A2780 or OVCAR3; a=Rab11a; b=Rab11b (heterozygote); R25=Rab25±fluorophore knock-in. Sorted (1 or 2×S). ( B ) AiryScan fluorescence images (LSM880, 63×; scale bar=20 µm). ( C ) Images obtained by Incucyte S3 system (scale bar=20 µm). ( D ) Lattice LightSheet images (3i, scale bar=10 µm; live cells); one focal plane or maximum intensity Z-projection for merged channels with increased contrast. See also and . ( E ) Lattice LightSheet images (3i, scale bar=5 µm) of fixed cells (3D projection using Imaris Cell Imaging Software); See also .

Article Snippet: Lentiviral pCDH-antiGFPnanobody-mCherry-Rab11a (overexpressing control) was cloned using pCDH-EF1-tagBFP-T2A-mycBirA*-Rab11a (previously prepared in our lab) via SpeI/SalI and XbaI/XhoI complementary cleavage sites. pMito-mCherry-FRB , a gift from Stephen Royle (Addgene plasmid #59352), was used as a template for generation of pMito-iRFP670-FRB (used with GFP-FKBP for knocksideways). pSH-EFIRES-P-AtAFB2 (Addgene plasmid #129715) , pCDH-EF1-tagBFP-T2A-mycBirA*-Rab11a, and mTagBFP2 were used to generate lentiviral pCDH-AtAFB2-mTagBFP2.

Techniques: Knock-In, Labeling, Western Blot, Fluorescence, Imaging, Software

Journal: eLife

Article Title: On demand expression control of endogenous genes with DExCon, DExogron and LUXon reveals differential dynamics of Rab11 family members

doi: 10.7554/eLife.76651

Figure Lengend Snippet:

Article Snippet: Lentiviral pCDH-antiGFPnanobody-mCherry-Rab11a (overexpressing control) was cloned using pCDH-EF1-tagBFP-T2A-mycBirA*-Rab11a (previously prepared in our lab) via SpeI/SalI and XbaI/XhoI complementary cleavage sites. pMito-mCherry-FRB , a gift from Stephen Royle (Addgene plasmid #59352), was used as a template for generation of pMito-iRFP670-FRB (used with GFP-FKBP for knocksideways). pSH-EFIRES-P-AtAFB2 (Addgene plasmid #129715) , pCDH-EF1-tagBFP-T2A-mycBirA*-Rab11a, and mTagBFP2 were used to generate lentiviral pCDH-AtAFB2-mTagBFP2.

Techniques: Modification, Transfection, Construct, shRNA, Recombinant, Expressing, Plasmid Preparation, CRISPR, Knock-In, Labeling, Sequencing, Software, Invasion Assay