Cyclophilin A

An aliquot of 0.5 μg total RNA was treated with 1 unit DNAse (Fermentas, St. Leon-Rot, Germany) 30 min at 37°C. Reverse transcription of RNA (0.5 μg) was performed with oligo (dT)12–18 primer and 200 units of SUPERSCRIPT II (Invitrogen, Karlsruhe, Germany) and 24 units of Ribo LockTM RNAse inhibitor (Fermentas) for 1 h at 42°C. The cDNA was used for PCR analysis. All cDNA probes were analyzed for: ACTA1, (NM_001100), amplicon length 85 bp; MYOG, (NM_002479), amplicon length 113 bp; MYH3, (NM_002470), amplicon length 84 bp and the RG: ACTB (NM_001101), amplicon length 104 bp; B2M, (NM_004048), amplicon length 98 bp; GAPDH, (NM_002046), amplicon length 119 bp; cyclophilin A/PPIA, (NM_203430), amplicon length 121 bp; RPLPO, (NM_001002), amplicon length 170 bp; TBP, (NM_003194), amplicon length 132 bp. The QuantiTect/PrimerAssays were purchased from QIAGEN GmbH (Hilden, Germany). cDNAs were amplified with Brilliant^® II SYBR^® Green QRT-PCR Master Mix (Stratagene-Agilent Technologies, Waldbronn, Germany). The thermal profile consisted of 1 cycle at 50°C for 2 minutes followed by 1 cycle at 95°C (2 min), 45 cycles at 95°C (15 sec), 60°C (1 min). Amplification was performed using the Mx3005P™ QPCR System (Stratagene). For relative quantification, a standard curve was generated in every individual run. Shortly, total RNA was pooled from muscle biopsies of healthy human volunteers, reverse transcription was performed and a serial dilution of the cDNA was used to perform the calibration curve. The data were analyzed using the relative standard curve method. For each unknown sample, the relative amount is calculated using linear regression analysis from their respective standard curves. Data were analyzed using the Mx3005P analysis software (Stratagene-Agilent Technologies, Waldbronn, Germany). The efficiencies of all GOI and RG were calculated in every individual run (Table 1).

Fluorescently labeled pseudoviruses were produced and characterized, as described previously [11 (link),68 (link),73 (link)]. Briefly, HEK293T/17 cells grown in a 100 mm dish were transfected with the plasmids encoding for the HIV-1 backbone pR9ΔEnv (8 μg), along with VSV-G (1 μg), Vpr-IN-sfGFP (4 μg) and CypA-fluorescent protein plasmids (4 μg) using the JetPrime Transfection reagent (VWR, Radnor, PA). Control viruses were produced without CypA-DsRed. For infectivity assays, pseudoviruses were produced as above but without the Vpr-IN-sfGFP plasmid. Alternatively, NL4-3-based pseudoviruses were produced by co-transfecting VSV-G (1 μg), pNL4.3R-E-Luc (8 μg), Nef-HA (4 μg) and CypA-DsRed (4 μg). Where noted, saquinavir was used at a final concentration of 500 nM to generate immature particles. Twelve hours after transfection, the medium was replaced with 7 ml of fresh DMEM/10% FBS without phenol red, and the sample incubated for additional 36 h at 37°C, 5% CO₂. Viral supernatant was collected, pooled, filtered through a 0.45 μm filter and quantified for p24 content using AlphaLISA immunoassay kit (PerkinElmer, Waltham, MA) or for the RT activity using the PERT protocol [74 (link)]. Viruses were aliquoted and stored at -80°C.
For Western blotting, the viruses were pelleted through a 20% sucrose cushion by centrifuging at 100,000×g for 2 h at 4°C, using the SW41 swinging bucket rotor (Beckman, Indianapolis IN), re-suspended in PBS and lysed with 0.5% Triton X-100 for 30 min at room temperature. Equal amounts of p24 were loaded onto a 12% polyacrylamide gel (Bio-Rad, Hercules, CA). Proteins were transferred onto a nitrocellulose membrane, blocked with PBS/0.1% Tween20/10% Blotting-grade Blocker (Bio-Rad) for 1 h at room temperature and incubated with HIV IG (1∶3000 dilution), rabbit anti-Cyclophilin A antibody (Millipore) (1∶500 dilution), or anti-α-tubulin (Sigma) (1∶3000 dilution) in PBS/0.1% Tween20/5% Blocking-grade Blocker overnight at 4°C. Horseradish peroxidase-conjugated (HRP) goat anti-rabbit antibody (1∶500 dilution, Santa Cruz, Dallas, Texas), HRP-Protein G (1∶2000, Bio-Rad) or HRP-rabbit anti-mouse (Millipore) were employed for protein detection using a chemiluminescence reagent from GE Healthcare. The resulting signal was visualized on the Chem-Doc Imager (Bio-Rad). PrecisionPlus Protein Standards (Kaleidoscope Bio-Rad) were used for molecular weight markers.

Total RNA was isolated from tissues and cell lines with the RNeasy Kit (Qiagen) following reverse transcription (Applied Biosciences). 1 µg RNA was used for generation of 50 µl cDNA. qPCR was performed with the StepOnePlus real time PCR system (Applied Biosystems) by using the StepOne Software v2.3. Power SYBR Green PCR Master Mix was used in a 25 µl mixture containing 100 nM of each primer. Only primers with an amplification efficiency between 1.8 and 2.2 were applied. qPCR primers are given in Table 2. mRNA expression was analysed on 5 µl of 1:5 diluted cDNA in either duplicate or triplicate. All expression values were normalized to the housekeeping gene Cyclophilin A (CypA) or GAPDH. A melt curve was performed after each run to check for unwanted primer dimerization. Data analysis was carried out using Excel version 16.65 (Microsoft Corporation) according to 2^-ΔΔCt method.Table 2

qPCR primers for testing mRNA expression level

Gene	Name of primer	Sequence (5’ – 3’)
CDH1 human	hCDH1-forward hCDH1-reverse	CCGAGAGCTACACGTTC TCTTCAAAATTCACTCTGCC
Cdh1 mouse	mCdh1-forward mCdh1-reverse	GAGCGTGCCCCAGTATCG CGTAATCGAACACCAACAGAGAGT
p16Ink4a	mp16-forward mp16-reverse	CCCAACGCCCCGAACT GTGAACGTTGCCCATCATCA
p19Arf	mp19-forward mp19-reverse	TCGCAGGTTCTTGGTCACTGT GAACTTCACCAAGAAAACCCTCTCT
Ccna1	mCcnA1-forward mCcnA1-reverse	GCTGTCTCTTTACCCGGAGCA ACGTTCACTGGCTTGTCTTCTA
Ccna2	mCcnA2-forward mCcnA2-reverse	CACTGACACCTCTTGACTATCC CGTTCACTGGCTTGTCTTCT
Ccnb1	mCcnB1-forward mCcnB1-reverse	TTGTGTGCCCAAGAAGATGCT GTACATCTCCTCATATTTGCTTGCA
Ccnb2	mCcnB2-forward mCcnB2-reverse	TGAAGTCCTGGAAGTCATGC GAGGCCAGGTCTTTGATGAT
SNAIL human	hSNAIL-forward hSNAIL-reverse	CTCTAATCCAGAGTTTACCTTC GACAGAGTCCCAGATGAG
SNAIL mouse	mSNAIL-forward mSNAIL-reverse	GCCGGAAGCCCAACTATAGC GGTCGTAGGGCTGCTGGAA
KRAS human	hKRAS-forward hKRAS-reverse	GACTGAATATAAACTTGTGGTAGTTGGA CATATTCGTCCACAAAATGATTCTG
Cyclophilin A (CypA)	CypA-forward CypA-reverse	ATGGTCAACCCCACCGTGT TTCTTGCTGTCTTTGGAACTTTGTC
GAPDH human	hGAPDH-forward hGAPDH-reverse	AATCCCATCACCATCTTCCA TGGACTCCACGACGTACTCA

Statistical analyses were performed with Microsoft Excel (version 16.61). For the experiments in which we compared two different conditions, Student’s two‐tailed t test or Mann-Whitney test were applied. For the experiments in which multiple conditions were compared, we used the one-way ANOVA test. All the data were obtained by performing at least 3 independent experiments and are expressed as means ± Standard Deviation (SD) of 3 independent experiments. The reproducibility, sample sizes and, where appropriate, statistical analyses are described in the figure legends. Relative mRNA levels (in which the control sample was arbitrarily set as 1) are reported as results of Real-Time PCR, in which the expression of Cyclophilin-A (CyA) served as housekeeping gene. A p-value <0.05 was considered statistically significant (Confidence level: 95%).

Messenger RNAs were extracted with TRIzol reagent (Life Technologies Ltd, Carlsbad, CA, USA, cod. 15596018). Complementary DNAs (cDNAs) were prepared with SuperScript™ VILO™ MasterMix (Life Technologies Ltd, Carlsbad, CA, USA, cod. 11755-050) as indicated by the manufacturer. The cDNAs were amplified by PCR in a CFX Connect Real-Time PCR Detection System (BioRad, Hercules, California, USA, cod. 1855201) with the fluorescent double-stranded DNA-binding dye SYBR Green (BioRad, Hercules, California, USA, cod. 1708882). Specific primers for each gene were designed to work under the same cycling conditions (95 °C for 10 min followed by 40 cycles at 95 °C for 15 sec and 60 °C for 1 min), thereby generating products of comparable sizes (about 200–300 bp for each amplification). Primer combinations were positioned whenever possible to span an exon-exon junction and the RNAs were digested with DNAse to avoid genomic DNA interference. Primer sequences are indicated in Supplementary Data 2. For each reaction, standard curves for reference genes were constructed based on six four-fold serial dilutions of cDNA. All samples were run in triplicate. The template concentration was calculated from the cycle number when the amount of PCR product passed a threshold established in the exponential phase of the PCR. The relative amounts of gene expression were calculated with Cyclophilin-A (CyA) expression as an internal standard (calibrator). The results, expressed as N-fold differences in target gene expression, were determined as follows: N*target = 2^{(ΔCt sample-ΔCt calibrator)}.