sgrna scaffold  (Addgene inc)

Journal: International Journal of Molecular Sciences

Article Title: Role of Endogenous Galectin-3 on Cell Biology of Immortalized Retinal Pigment Epithelial Cells In Vitro †

doi: 10.3390/ijms26157622

Figure Lengend Snippet: Development of galectin-3 knockdown ARPE-19 cells. ( A , B ) Merged phase-contrast and fluorescent imaging for GFP expression of ARPE-19 cells without ( A ) and with pCRISPR-LGALS3 transfection ( B ). ( C , D ) Immunostaining for galectin-3 of ARPE-19 ( C ) and ARPE-19/LGLAS3 +/− cells ( D ) after FACS and single-cell cultivation. ( E , F ) Phase-contrast image of ARPE-19 ( E ) and ARPE-19/LGLAS3 +/− cells ( F ). Magnification bar in ( A , B ) 100 µm; in ( C , D ) 20 µm, in ( E , F ) 100 µm; blue, Hoechst staining. ( G ) Western blot analysis for galectin-3 of ARPE-19 and ARPE-19/LGLAS3 +/− cells. ( H , I ) Galectin-3 concentration of conditioned cell culture medium ( H ) and cell lysate ( I ) from ARPE-19 and ARPE-19/LGLAS3 +/− cells after incubation in unsupplemented cell culture medium for 72 h. Mean ± SD; ** p < 0.01; **** p < 0.0001; n = 10.

Article Snippet: Galectin-3 knockdown ARPE-19 cells were generated using the all-in-one sgRNA pCRISPR plasmid against human LGALS3 (pCRISPR-LGALS3; HCP301784-CG04-3-Bc, Genecopoeia, Rockville, MA, USA).

Techniques: Knockdown, Imaging, Expressing, Transfection, Immunostaining, Staining, Western Blot, Concentration Assay, Cell Culture, Incubation

Journal: International Journal of Molecular Sciences

Article Title: Role of Endogenous Galectin-3 on Cell Biology of Immortalized Retinal Pigment Epithelial Cells In Vitro †

doi: 10.3390/ijms26157622

Figure Lengend Snippet: Reduced expression of galectin-3 decreases viability of immortalized RPE cells in vitro. WST-1 assay of ARPE-19, ARPE-19/FACS, and ARPE-19/LGALS3 +/− cells following incubation in cell culture medium without supplementation and various concentrations of hr-galectin-3 (hr-Gal3) for 72 h. Mean ± SD; **** p < 0.0001; n ≥ 28 of at least 4 independent experiments.

Article Snippet: Galectin-3 knockdown ARPE-19 cells were generated using the all-in-one sgRNA pCRISPR plasmid against human LGALS3 (pCRISPR-LGALS3; HCP301784-CG04-3-Bc, Genecopoeia, Rockville, MA, USA).

Techniques: Expressing, In Vitro, WST-1 Assay, Incubation, Cell Culture

Journal: International Journal of Molecular Sciences

Article Title: Role of Endogenous Galectin-3 on Cell Biology of Immortalized Retinal Pigment Epithelial Cells In Vitro †

doi: 10.3390/ijms26157622

Figure Lengend Snippet: Decreased expression of galectin-3 declines proliferation of immortalized RPE cells in vitro. BrdU ELISA of ARPE-19, ARPE-19/FACS, and ARPE-19/LGALS3 +/− cells following incubation in cell culture medium without supplementation for 72 h with and without various concentrations of hr-galectin-3 ( A ) and/or an additional treatment with 100mM lactose ( B ). Mean ± SD; * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001; n ≥ 20 of at least 4 independent experiments.

Article Snippet: Galectin-3 knockdown ARPE-19 cells were generated using the all-in-one sgRNA pCRISPR plasmid against human LGALS3 (pCRISPR-LGALS3; HCP301784-CG04-3-Bc, Genecopoeia, Rockville, MA, USA).

Techniques: Expressing, In Vitro, Enzyme-linked Immunosorbent Assay, Incubation, Cell Culture

Journal: International Journal of Molecular Sciences

Article Title: Role of Endogenous Galectin-3 on Cell Biology of Immortalized Retinal Pigment Epithelial Cells In Vitro †

doi: 10.3390/ijms26157622

Figure Lengend Snippet: Decreased galectin-3 expression reduces migration of ARPE-19 cells in vitro. Quantification ( A ) and representative images ( B ) of scratch migration assay of native ARPE-19, ARPE-19/FACS, and ARPE-19/LGALS3 +/− cells following incubation with and without hr-galectin-3 in cell culture medium without supplements for 24 h. ( A ) For quantification, the recolonized area after 24 h was calculated and plotted as the relative recolonized area. ( B ) Representative images of native ARPE-19 (left panel) and ARPE-19/LGALS3 +/− cells (right panel) immediately after scratching (0 h, upper row) and after incubation for 24 h (lower panel). Mean ± SD; *** p < 0.001; **** p < 0.0001; n ≥ 10 of 3 independent experiments.

Article Snippet: Galectin-3 knockdown ARPE-19 cells were generated using the all-in-one sgRNA pCRISPR plasmid against human LGALS3 (pCRISPR-LGALS3; HCP301784-CG04-3-Bc, Genecopoeia, Rockville, MA, USA).

Techniques: Expressing, Migration, In Vitro, Incubation, Cell Culture

Journal: International Journal of Molecular Sciences

Article Title: Role of Endogenous Galectin-3 on Cell Biology of Immortalized Retinal Pigment Epithelial Cells In Vitro †

doi: 10.3390/ijms26157622

Figure Lengend Snippet: Reduced expression of endogenous galectin-3 enhances cell attachment of immortalized RPE cells in vitro. ( A ) Cell adhesion of ARPE-19, ARPE-19/FACS, and ARPE-19/LGALS3 +/− cells was quantified 30, 60, 90, 120, 150, and 180 min after seeding and plotted as the relative number of adherent cells. Mean ± SD; n = 12 for each group of 3 independent experiments; * comparison of ARPE-19 versus ARPE-19/LGALS3 +/− cells; # comparison of ARPE-19/FACS versus ARPE-19/LGALS3 +/− cells; **** p < 0.001; #### p < 0.001. ( B ) Relative number of adherent ARPE-19, ARPE-19/FACS, and ARPE-19/LGALS3 +/− cells 90 min after seeding and incubation with various concentrations of hr-galectin-3, which was added immediately before seeding. Mean ± SD; n = 15 for each group of 3 independent experiments; ** p < 0.01; **** p < 0.0001.

Article Snippet: Galectin-3 knockdown ARPE-19 cells were generated using the all-in-one sgRNA pCRISPR plasmid against human LGALS3 (pCRISPR-LGALS3; HCP301784-CG04-3-Bc, Genecopoeia, Rockville, MA, USA).

Techniques: Expressing, Cell Attachment Assay, In Vitro, Comparison, Incubation

Journal: International Journal of Molecular Sciences

Article Title: Role of Endogenous Galectin-3 on Cell Biology of Immortalized Retinal Pigment Epithelial Cells In Vitro †

doi: 10.3390/ijms26157622

Figure Lengend Snippet: Lack of endogenous galectin-3 promotes epithelial-to-mesenchymal transition of immortalized RPE. ( A – F ) Phaloidin staining (( A , B ) orange) as well as immunofluorescent staining for sm-α-actin (( C , D ) orange) and N-cadherin ( E , F ) green) of ARPE-19 ( A , C , E ) and ARPE-19/LGALS3 +/− cells ( B , D , F ). Scale bar, 20 µm; blue, DAPI staining. ( G ) Real-time rt-PCR for sm-α-actin, E-cadherin, and N-cadherin mRNA expression of ARPE-19, ARPE-19/FACS, and ARPE-19/LGALS3 +/− cells. Mean ± SD; n ≥ 7 of 5 independent experiments; * p < 0.05; ** p < 0.01; **** p < 0.0001.

Article Snippet: Galectin-3 knockdown ARPE-19 cells were generated using the all-in-one sgRNA pCRISPR plasmid against human LGALS3 (pCRISPR-LGALS3; HCP301784-CG04-3-Bc, Genecopoeia, Rockville, MA, USA).

Techniques: Staining, Quantitative RT-PCR, Expressing

Journal: International Journal of Molecular Sciences

Article Title: Role of Endogenous Galectin-3 on Cell Biology of Immortalized Retinal Pigment Epithelial Cells In Vitro †

doi: 10.3390/ijms26157622

Figure Lengend Snippet: Endogenous galectin-3 expression maintains basal pAKT, pERK, and β-catenin signaling in immortalized RPE cells. Western blot analysis ( A ) and densitometry for pAKT ( B ), pERK ( C ), and β-catenin ( D ) expression of ARPE-19, ARPE-19/FACS, and ARPE-19/LGALS3 +/− cells. Mean ± SD; n ≥ 6 of 6 independent experiments; * p < 0.05; ** p < 0.01; *** p < 0.001; ns, not significant.

Article Snippet: Galectin-3 knockdown ARPE-19 cells were generated using the all-in-one sgRNA pCRISPR plasmid against human LGALS3 (pCRISPR-LGALS3; HCP301784-CG04-3-Bc, Genecopoeia, Rockville, MA, USA).

Techniques: Expressing, Western Blot