Journal: The American Journal of Pathology

Article Title: Promotion of β-Catenin/Forkhead Box Protein O Signaling Mediates Epithelial Repair in Kidney Injury

doi: 10.1016/j.ajpath.2021.03.005

Figure Lengend Snippet: Proximity ligation assay (PLA) analysis of β-catenin/forkhead box protein O 1 (Foxo1) and β-catenin/T-cell factor (TCF) interactions in scratch assay using C1.1 cells. A: Representative phase contrast images of WT C1.1 cells using in vitro scratch assay at 0 and 48 hours. The area between broken lines is the scratch wound. The solid lines outline the field are used in immunofluorescence imaging. B: Representative β-catenin/Foxo1 (red spot) and β-catenin/TCF (green spot) interaction in control, recombinant human transforming growth factor-β1 (rhTGF-β1), rhTGF-β1 and ICG-001, rhTGF-β1 and iCRT3, ICG-001 alone, or iCRT3 alone treated C1.1 cells using scratch assay (cells to the right of broken lines are cells migrating after the scratch). Insets: Higher-resolution images of the smaller boxed areas outlined with solid green lines . Nuclei were stained with DAPI. C and D: Quantitation of PLA signals, using one-way analysis of variance, followed by Tukey post hoc test. Results are shown as means ± SEM ( C and D ). n = 4 ( C and D ). ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001. Scale bar = 40 μm ( B ). Original magnification, ×40 ( B ).

Article Snippet: Foxo1 knockout (KO) C1.1 cell line was produced by cotransfection of C1.1 cells with the Foxo1 CRISPR/Cas9 KO plasmid and a Foxo1 Homology directed repair (HDR) plasmid (Santa Cruz Biotechnology, Inc., Dallas, TX), following the manufacturer's instructions.

Techniques: Proximity Ligation Assay, Wound Healing Assay, In Vitro, Immunofluorescence, Imaging, Control, Recombinant, Staining, Quantitation Assay

Journal: The American Journal of Pathology

Article Title: Promotion of β-Catenin/Forkhead Box Protein O Signaling Mediates Epithelial Repair in Kidney Injury

doi: 10.1016/j.ajpath.2021.03.005

Figure Lengend Snippet: In vitro scratch assay in wild-type (WT), Foxo1 knockout (KO), and TCF1 KO C1.1 cells. A: Representative images of the wound closure of WT, Foxo1 KO, and TCF1 KO C1.1 untreated cells (control) or cells treated with recombinant human transforming growth factor-β1 (rhTGF-β1), rhTGF-β1 and ICG-001, rhTGF-β1 and iCRT3, ICG-001 alone, or iCRT3 alone. White dotted lines indicate front edge of cells after scratch at 0 hour. B and C: Quantitation of percentage wound closure ( B ) and number of cells ( C ) at 48 hours in WT (black dots), Foxo1 KO (reds dots), and TCF1 KO (green dots) with various treatments is shown. Results are representative of four independent experiments. Statistical significance was determined by one-way analysis of variance, followed by Tukey post hoc test. Results are shown as means ± SEM ( B and C ). ∗ P < 0.05, ∗∗ P < 0.01, and ∗∗∗ P < 0.001. Scale bar = 100 μm ( A ). Original magnification, ×20 ( A ).

Article Snippet: Foxo1 knockout (KO) C1.1 cell line was produced by cotransfection of C1.1 cells with the Foxo1 CRISPR/Cas9 KO plasmid and a Foxo1 Homology directed repair (HDR) plasmid (Santa Cruz Biotechnology, Inc., Dallas, TX), following the manufacturer's instructions.

Techniques: In Vitro, Wound Healing Assay, Knock-Out, Control, Recombinant, Quantitation Assay

Journal: The American Journal of Pathology

Article Title: Promotion of β-Catenin/Forkhead Box Protein O Signaling Mediates Epithelial Repair in Kidney Injury

doi: 10.1016/j.ajpath.2021.03.005

Figure Lengend Snippet: Sirius red collagen detection assay in wild-type (WT), Foxo1 knockout (KO), and TCF1 KO C1.1 cells. Quantitation of collagen (fold change) at 48 hours in WT (black dots), Foxo1 KO (red dots), and TCF1 KO C1.1 (green dots) with various treatments. F-TrCP-Ecad (βTrCP-E-cadherin chimera construct) were used for targeted degradation of cytosolic β-catein. Results are representative of four independent experiments. Statistical significance was determined by one-way analysis of variance, followed by Tukey post hoc test. ∗ P < 0.05, ∗∗ P < 0.01. rhTGF-β1, recombinant human transforming growth factor-β1.

Article Snippet: Foxo1 knockout (KO) C1.1 cell line was produced by cotransfection of C1.1 cells with the Foxo1 CRISPR/Cas9 KO plasmid and a Foxo1 Homology directed repair (HDR) plasmid (Santa Cruz Biotechnology, Inc., Dallas, TX), following the manufacturer's instructions.

Techniques: Detection Assay, Knock-Out, Quantitation Assay, Construct, Recombinant

Journal: The American Journal of Pathology

Article Title: Promotion of β-Catenin/Forkhead Box Protein O Signaling Mediates Epithelial Repair in Kidney Injury

doi: 10.1016/j.ajpath.2021.03.005

Figure Lengend Snippet: In vitro collagen gel contraction assay in wild-type (WT), Foxo1 knockout (KO), and TCF1 KO C1.1 cells. Top panels: Representative images of a collagen gel in a well from the bottom of plate. White dotted circle lines indicate edge of collagen gel at 0 hour. Bottom panels: Quantitation of percentage gel surface area at 48 hours in WT, Foxo1 KO, and TCF1 KO with various treatments. Results are representative of four independent experiments. Statistical significance was determined by one-way analysis of variance, followed by Tukey post hoc test. Results are shown as means ± SEM ( bottom panels ). ∗ P < 0.05, ∗∗ P < 0.01, and ∗∗∗ P < 0.001. TGF-β, transforming growth factor-β.

Article Snippet: Foxo1 knockout (KO) C1.1 cell line was produced by cotransfection of C1.1 cells with the Foxo1 CRISPR/Cas9 KO plasmid and a Foxo1 Homology directed repair (HDR) plasmid (Santa Cruz Biotechnology, Inc., Dallas, TX), following the manufacturer's instructions.

Techniques: In Vitro, Collagen Gel Contraction Assay, Knock-Out, Quantitation Assay

Journal: The American Journal of Pathology

Article Title: Promotion of β-Catenin/Forkhead Box Protein O Signaling Mediates Epithelial Repair in Kidney Injury

doi: 10.1016/j.ajpath.2021.03.005

Figure Lengend Snippet: Proximity ligation assay (PLA) analysis of β-catenin/forkhead box protein O 1 (Foxo1) interactions in kidney of unilateral ischemia reperfusion (UIR) mice. A: Representative β-catenin/Foxo1 (red spot) interaction in kidney sections of control, untreated UIR, and UIR mice treated with recombinant human transforming growth factor-β1 (rhTGF-β1), rhTGF-β1 and ICG-001 (day 1 to 7), rhTGF-β1 and ICG-001 (day 7 to 14), or ICG-001 alone. B: Representative β-catenin/Foxo1 (red spot) interaction in kidney sections of UIR mice treated with iCRT3 alone, rhTGF-β1 and iCRT3 (day 1 to 7), or rhTGF-β1 and iCRT3 (day 7 to 14). Nuclei were stained with DAPI. Fluorescence images were merged with differential interference contrast images to show the localization of PLA signals in tubular cells (indicated by broken lines ). Quantitation of PLA signals with the various treatments was compared by one-way analysis of variance, followed by Tukey post hoc test. Results are shown as means ± SEM ( A and B ). n = 5 ( A and B ). ∗ P < 0.05, ∗∗ P < 0.01, and ∗∗∗ P < 0.001. Scale bar = 40 μm ( A and B ). Original magnification, ×60 ( A and B ).

Article Snippet: Foxo1 knockout (KO) C1.1 cell line was produced by cotransfection of C1.1 cells with the Foxo1 CRISPR/Cas9 KO plasmid and a Foxo1 Homology directed repair (HDR) plasmid (Santa Cruz Biotechnology, Inc., Dallas, TX), following the manufacturer's instructions.

Techniques: Proximity Ligation Assay, Control, Recombinant, Staining, Fluorescence, Quantitation Assay

Journal: Genome Research

Article Title: Megabase-scale deletion using CRISPR/Cas9 to generate a fully haploid human cell line

doi: 10.1101/gr.177220.114

Figure Lengend Snippet: Combination of gRNAs 1 and 3 causes deletion of the Chromosome 15 fragment. HAP1 cells were transfected with various combinations of gRNAs (as indicated). Around 10 d post transfection, genomic DNA was isolated from pools of transfected cells. ( A ) The regions targeted by individual gRNAs were amplified by PCR using suitable primer pairs. Digestion of these PCR products by T7 endonuclease provides a semiquantitative measure for Cas9 editing efficiency. ( B ) To assess whether the fragment between gRNAs 1 and 3 had been excised following Cas9 cleavage, we performed a deletion PCR using a forward primer (HG6090) that binds to position Chr 15: 61,105,055 and a reverse primer (HG6093) that binds to position Chr 15: 89,889,818. We also included a control PCR (primer pair HG6090/HG6091) to confirm that every sample contained genomic DNA, suitable for PCR.

Article Snippet: HAP1 cells were transiently transfected with expression plasmids for Streptococcus pyogenes Cas9 and suitable gRNAs using TurboFectin (OriGene) according to manufacturer’s instructions.

Techniques: Transfection, Isolation, Amplification