Journal: BMC Cancer

Article Title: PKCα promotes the mesenchymal to amoeboid transition and increases cancer cell invasiveness

doi: 10.1186/s12885-015-1347-1

Figure Lengend Snippet: Expression of dominant-negative PKCα results in decreased invasiveness of cancer cells. Invasion analysis of A375m2 (A) , K2 (B) and MDA-MB-231 (C) strains overexpressing wt PKCα or expressing dominant-negative PKCα. Cells were transfected with pCMV6 vector expressing either wt PKCα or dominant-negative PKCα and let to invade into 3D collagen for 48 hours. 3D collagen cross-sections were analyzed using NIS Elements software (Nikon) and average invasion depth was calculated and normalized to that of untreated cells. The invasion depth in each individual field of view was assessed as sum of invasion depths of individual cells divided by the total number of cells in the respective field of view (including cells on the top of collagen). Statistical significance (marked by asterisks) was evaluated using ANOVA followed by Tukey’s honest significant difference test.

Article Snippet: Flag-tagged wt PKCα, constitutive active form (with myristoylation sequence from Src) and dominant-negative PKCα (K368R) in pCMV6 vector were all obtained from Addgene Inc. Silencer® Select Validated siRNA against PKCα was obtained from Applied Biosystems and a final concentration of 10 nM was used for transfections.

Techniques: Expressing, Dominant Negative Mutation, Transfection, Plasmid Preparation, Software

Journal: bioRxiv

Article Title: PROTAC molecule-mediated SpCas9 protein degradation for precise genome editing

doi: 10.1101/2025.01.07.631496

Figure Lengend Snippet: A .Optimized CASPROTAC 6 degraded dCas9 proteins. 293T-Tet-On 3G cells with stable integration of dCas9-APEX2 were pretreated with doxycycline (500 ng/mL) for 9 h to the simultaneous expression of Cas9-APEX2 protein complex, 15 hrs after removing doxycycline, CASPROTAC 6 was added for 24 hrs in a dose manner. The protein level of dCas9-APEX2 was monitored by Western blotting. B . Quantification of the intensity of the staining from A. Anti-Flag antibody was used to detect the level of dCas9-APEX-Flag proteins. Anti-β-actin was used as the loading control. C . The 293T-nCas9-PE2 bulk cells were pretreated with doxycycline (500 ng/mL) for 24 h, then removing doxycycline, CASPROTAC 6 was added for 24 hrs. The protein level of nCas9-PE2 was monitored by Western blotting. D . Quantification of the intensity of the staining from C. Anti-Cas9 antibody was used to detect the level of dCas9-APEX-Flag proteins. Anti-β-actin was used as the loading control. E . 293T-nCas9-BE3RA bulk cells were pretreated with doxycycline (500 ng/mL) for 24 h. Then, after removing doxycycline, CASPROTAC 6 was added for another 24 hrs. The protein level of nCas9-BE3RA was monitored by Western blotting. F . Quantification of the intensity of the staining from E. Anti-Cas9 antibody was used to detect the level of dCas9-APEX-Flag proteins. Anti-β-actin was used as the loading control. Bars show mean value□±□s.e.m. and significance was calculated using Student’s t-test (n = 2 or 3). *p < 0.05, **p < 0.005, and ***p < 0.0001 (versus the control). G . CASPROTAC 6 reduced CRISPR editing efficiency. FANCF-gRNA and SpCas9 protein were mixed at a 1:1 molar ratio (20 pmol SpCas9+ 20 pmol FANCF-gRNA for 1 million cells) and incubated at room temperature for 20 min to allow the formation of the CRISPR ribonucleoprotein complexes. 293T cells were electroporated with FANCF-RNP. Six hours after electroporation, cells were incubated with CASPROTAC 6 for 24 hrs. CRISPR editing efficiency was measured by amplicon sequencing. H . The reduction of CRISPR/SpCas9-mediated editing based on the amplicon sequencing results. I . Batch output mode comparing CRISPR editing of control and CASPROTAC 6-treated cells. Each column represents the base composition of a single nucleotide at the FANCF site 2 locus. The percentage of each base or indel at each nucleotide is shown as a proportion of each row (n=1). *p < 0.05, **p < 0.005, and ***p < 0.0001 (versus the control).

Article Snippet: For 293T-nCas9-PE2 bulk cells, the wild-type 293T cell line was grown in 6 well plates (50% confluency) and co-transfected with 1.0 μg Xlone-PE2 (Addgene 136463) plasmid and 1.0 μg PiggyBac Transposase vector 6 µg PEI (Polyscience, 23966) in 50 µl of serum-free medium and selected with 3□μg ml -1 Blasticidin (Biomol) for 2-3 days.

Techniques: Expressing, Western Blot, Staining, Control, CRISPR, Incubation, Electroporation, Amplification, Sequencing