Pshuttle cmv vector

For wild type subfragment-1 (S1) of the myosin construct, human skeletal muscle myosin IIa cDNA (Kazusa Product ID FXC25901, Kisarazu, Japan) was truncated at Ala849. This fragment included the motor domain, essential light chains (ELC) binding domain and regulatory light chains (RLC) binding domain. For oligonucleotide labeling and protein purification, SNAP-tag (New England Biolabs Inc.), FLAG-tag, and 6 × His-tag were attached at the C-terminal via linkers (3 a.a., GGL). Two amino acids (Leu-Glu) corresponding to the restriction endonuclease recognition site (XhoI: CTCGAG) were kept between SNAP-tag and FLAG-tag. For the light chain null construct (lever-arm-less myosin S1), ELC, and RLC binding sites (Lys786-Leu846) were deleted from the S1 construct. These myosin fragments were introduced downstream of the multi cloning site of the pShuttle-CMV vector (Agilent Technologies, Santa Clara, CA, USA). For the double lever-arm length construct (double lever-arm length Myosin S1), the ELC and RLC binding domains were inserted just after Ala849 of WT S1.

The full-length mouse AIF-1 cDNA was PCR-amplified and inserted into the KpnI-XhoI sites of the pShuttle-CMV vector (Agilent Technologies). Recombinant AIF-1-expressing adenovirus AdAIF-1 was produced by homologous recombination of pShuttle-CMV-AIF-1 with the pAdEasy-1 adenoviral vector (Agilent Technologies) in E.coli strain BJ5183 according to the manufacturer's instructions. The AdAIF-1 was amplified in 293T cell line (ATCC). The titer of virus stock was determined by a plaque assay described by Tollefson et al. (49 (link)).

A Flag-tagged FoxO1 expression construct was purchased from Addgene. Mitochondria-GCN5L1 was generated by fusing the GCN5L1 encoding sequence with the COX 8 mitochondrial targeting sequence (36 amino acid at N terminal) and 3×-flag sequence. ER-targeted GCN5L1 was generated by fusing GCN5L1 encoding sequence with the N terminal 17 amino acids of calreticulin at the N terminal and KDEL and 3×-flag at the C terminal. GCN5L1 DNA, mitochondria-GCN5L1 and ER-GCN5L1 were cloned into pShuttle-CMV vector (Agilent), or an empty vector as a negative control. Adenoviruses were produced using Adeasy Adenoviral System (Agilent). After amplification with Ad-293 packaging cell line, virus was purified using ceasium chloride gradient ultracentrifugation and dialyzed into PBS with desalting columns (GE). Adenovirus of flag tagged FoxO1 was purchased from Vector Biolabs. Primary hepatocytes were infected with adenovirus overexpressing either empty vector (control), GCN5L1, Mt-GCN5L1, ER-GCN5L1 or Flag-FoxO1 at a dose of 20 p.f.u per cell. Thirty-six hours after transduction, cells were used for experiments.

All adenoviral vectors were constructed by Cellid, Inc. (Seoul, Korea). To construct the adenoviral vector that expressed the HPV antigen E6/E7 gene in the E1 region of adenovirus, we first constructed a pShuttle-CMV vector (Agilent Technologies, CA, USA) that expressed the HPV antigen E6/E7 protein. The newly generated pShuttle-CMV-E6E7 vector was co-transformed with the pAdeasy-1 adenovirus vector (Agilent Technologies, CA, USA) in which a portion of the Ad type 5 fibre was replaced by a portion of the Ad type 35 fibre (Adk35), yielding the plasmid pAdk35-E6E7. This recombinant plasmid was transfected into human embryonic kidney 293 cells to generate the Adk35-E6E7 adenovirus.

The expression cassettes were cloned into pShuttle-CMV vector (# 240007, Agilent Technologies, USA) between SalI and HindIII sites flanked by CMV promoter and SV40 poly(A) signal. Top 10 E. coli cells (Life technologies, USA) were transformed with the recombinant pShuttle vectors (pSH-CMV/P1-2AB3BC^wt and pSH-CMV/P1-2AB3BC^m). The transformed Top 10 cells were spread-plated on Luria Burtony agar (LB powder, Affymetrix, USA) containing kanamycin (50 µg/ml) and incubated at 37°C for 18-20 h. The Top 10 cells containing the recombinant pShuttle vectors are kanamycin resistant and grow on LB agar containing the antibiotic while non-recombinant Top 10 cells die out. The potential recombinant colonies were tested for GOI by colony PCR. One of the positive colonies was inoculated in LB broth containing kanamycin and incubated at 37°C in a shaker incubator (Thermo Scientific, USA) to amplify the plasmid. The plasmids were isolated by Plasmid Miniprep kit (# K0502, Fermentas, USA) as per the manufacturer’s instruction. The integrity of GOI was checked by PCR and RE digestion, and confirmed by sequencing. The sequencing of the target genes was carried out in an automated DNA Sequencer (Perkin Elmer, USA).

For subfragment-1 (S1) of the myosin construct, human skeletal muscle myosin IIa cDNA (Kazusa Product ID FXC25901) was truncated at Ala849. This fragment included the motor domain, essential light chains (ELC) binding domain and regulatory light chains (RLC) binding domain. For oligonucleotide labeling and protein purification, SNAP-tag (New England Biolabs Inc.), FLAG-tag and 6 × His-tag were attached at the C-terminal. Two amino acids (Leu-Glu) corresponding to the restriction endonuclease recognition site (XhoI: CTCGAG) were kept between SNAP-tag and FLAG-tag. For the light chain null construct (lever-arm-less S1), ELC and RLC binding sites (Lys786-Leu846) were deleted from the S1 construct. These myosin fragments were introduced downstream of the multi cloning site of the pShuttle-CMV vector (Agilent Technologies). For the actin binding domain of the α-actinin construct, human α-actinin 1 cDNA (Addgene; pEGFP-N1 alpha-actin1) was truncated at the actin binding domain (Asp1-Ala247). For oligonucleotide labeling and protein purification, SNAP-tag and 6 × His-tag were attached at the C-terminal via linkers (3 a.a., GGL). This α-actinin1-SNAP-His fragment was introduced downstream of the T7 promoter of pET T7-7 plasmid (Addgene).