lenti x tet on 3g system  (TaKaRa)

Journal: Brain Sciences

Article Title: Inducible Knockout of 14-3-3β Attenuates Proliferation and Spheroid Formation in a Human Glioblastoma Cell Line U87MG

doi: 10.3390/brainsci13060868

Figure Lengend Snippet: A. Generation of a Tet-inducible CRISPR knockout of 14-3-3β in U87MG cells. ( A ) Relative expression (2 −∆∆Ct ) of Cas9 expression in stably expressing Tet-CAS9 clonal U87MG cells upon doxycycline (1 mg/mL) administration for 48 h. ( B ) Quantification of the western blot analysis. *** denotes p < 0.001 and * denotes p < 0.01 (Holm-Sidak multiple t -test). ( C ) Western blot analysis of 14-3-3β and β-tubulin expression in parental clone1 Tet-Cas9 U87MG cells, as well as Tet-Cas9 YWHAB sgRNA U87MG cells of differing populations (P1, P2, P3, P4). ( D ) Comparison of the Tet-Cas9 YWHAB sgRNA populations with and without the addition of doxycycline (1 mg/mL for 72 h). ( E ) Average 14-3-3β protein expression of the YWHAB sgRNA U87MG cell populations (P1, P2, P3, and P4) with ±1 mg/ml doxycycline. * denotes p < 0.05 (Holm Sidak multiple t -tests).

Article Snippet: The Lenti-X Tet-On 3G CRISPR/Cas9 system was purchased from Takara (Takara, Shiga, Japan) and used to create the doxycycline-inducible knockouts of YWHAB (14-3-3β protein).

Techniques: CRISPR, Knock-Out, Expressing, Stable Transfection, Western Blot, Comparison

Journal: Brain Sciences

Article Title: Inducible Knockout of 14-3-3β Attenuates Proliferation and Spheroid Formation in a Human Glioblastoma Cell Line U87MG

doi: 10.3390/brainsci13060868

Figure Lengend Snippet: Tet-inducible CRISPR knockout of 14-3-3β reduces U87MG cell proliferation and tumor spheroid growth in vitro. ( A ) Wild-type (WT) U87MG cells and Tet-Cas9 YWHAB gRNA U87MG cells were plated in replicates of 12 with and without doxycycline (1 mg/mL), and three experimental replicates were counted at days 2, 4, 6, and 8. * denotes p < 0.05 ** denotes p < 0.01 (Tukey two-way ANOVA). ( B ) Wild-type (WT) U87MG cells and Tet-Cas9 YWHAB gRNA U87MG cells were plated on 96-well ultra-low adhesion round bottom plates in replicates of 12 with and without doxycycline (1 mg/mL). The first group (on the left) shows WT U87MG cells with ±1 mg/mL doxycycline, and the second group (on the right) shows YWHAB gRNA expressing U87MG cells with ±1 mg/ml doxycycline YWHAB gRNA U87MG cells with doxycycline would be the group with knockout for 14-3-3β. The cells were counted on day 7; cells were plated on day 0, and doxycycline (if applicable) was also added on day 0. ** denotes p < 0.01 (Tukey two-way ANOVA).

Article Snippet: The Lenti-X Tet-On 3G CRISPR/Cas9 system was purchased from Takara (Takara, Shiga, Japan) and used to create the doxycycline-inducible knockouts of YWHAB (14-3-3β protein).

Techniques: CRISPR, Knock-Out, In Vitro, Expressing