C3h mice

All animal studies were approved by the animal care and use committee at our institution, and experiments were performed according to the National Research Council’s Guide for the Care and Use of Laboratory²⁶ . Female C3H mice (Charles River Laboratories, Wilmington, MA) and B6;129S-Ptgs2^tm1Jed/J (COX-2^−/− mice; Jackson Laboratory, Bar Harbor, ME) were aged 10–14 weeks and allowed free access to food and water during studies.

The project (2015-0071) was approved
by the Nijmegen Medical Center animal ethics committee (RUDEC) and
the Dutch animal ethics committee (CCD) of the Radboud University
and performed according to the Institute of Laboratory Animal Research
Guidelines. C3H mice were purchased from Charles River Laboratories
(L’Arbresle, France) and BALB/cRJ nu mice from Janvier (Saint-Berthevin,
France). Mice were housed in a pathogen-free environment in Mouse
IVC Blueline cages (5 mice per cage), had ad libitum access to water
and chow, and were allowed to adapt to laboratory conditions for at
least 1 week before the start of the experiments.

For hair pigmentation experiment, 8-week-old male C3H mice purchased from Charles River Laboratories, Japan Inc. (Kanagawa, Japan), were used. All the mice were allowed to acclimate for 7 days to the laboratory conditions under controlled settings of temperature (21–23°C) and light (light:dark 12:12) with access to food and water, at the Gene Research Center of the University of Tsukuba. Then, C3H mice were randomly divided into two groups (n = 5): TCQA-treated group and control group treated with Milli-Q water (vehicle). The hair pigmentation is tightly linked to the anagen phase of the hair cycle, and at this age (8 weeks old), the HFs of C3H mice are at the telogen phase, so in order to induce the transition from the telogen to anagen phase, the mice were first anesthetized using isoflurane (Wako Pure Chemical Industries, Tokyo, Japan), and then the dorsal part was shaved using a hair clipper. The vehicle or 1% TCQA was applied topically at the shaved area for 1 month, and during this period, the mice were observed daily. The mice were then scarified by cervical spine dislocation, the hair from the treated area at the dorsal part was plucked, and the treated skin was collected and kept in −80°C. All animal procedures were approved by the Animal Study Committee of the University of Tsukuba and were handled according to the guidelines for the Care and Use of Animals.

Eight-week old male C3H mice were supplied by Charles River (Sulzfeld, Germany) and kept in the local central animal facility of the University Hospital Bonn. The mice were housed under standard conditions and had free access to water and food. Animal procedures were performed in accordance with approved protocols and followed recommendations for proper care and use of laboratory animals.
The murine endothelial cell line SVEC4-10 (ATCC CRL-2181) was obtained from LGC Promochem (Wesel, Germany) and cultured in DMEM supplemented with 10% FBS, 200 mM glutamine. HUVE-cells (HUVEC, pooled) were obtained from PromoCell (Heidelberg, Germany) and cultured in EGM with supplements according to the manufacturer's instructions.
Hepa129 cells (Hepatoma 129, obtained from NCI-Frederick Cancer Research and Development Centre (DCT Tumour Repository)) were maintained in RPMI1640 supplemented with 10% FBS, 200 mM glutamine.

Nulliparous female C3H mice (Charles River, Wilmington, MA) were housed 5/cage and acclimated to the vivarium for 1–2 weeks. Females were used to avoid non-experimental wounding from fighting that occurs in cohabitating males. Mice were housed using a 14:10 light:dark cycle with lights on at 06:00 h in a facility controlled for temperature (21 ± 1°C). Rodent chow (Harlan 7912) and water were available ad libitum throughout the study and shredded paper was available for nest building. All animal experiments were approved by the University of Illinois at Chicago or Ohio State University Institutional Animal Care and Use Committee and carried out in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (Institute for Laboratory Animal Resources, 1996). All efforts were made to minimize animal suffering and to reduce the number of mice used.

Knock-in of a dystrophin mutation preventing cleavage by protease 2A was generated as previously described [9 (link)]. Mice were genotyped to identify the knock-in construct using a DysKI-S primer (5′-TCTCTAGGAGAGGTCTCTC) and a DysKI-AS primer (5′-ACCCCACAATCTTGCACATG). Dys^KI mice were backcrossed for 3 to 11 generations with C3H mice (Charles River Laboratories) for viral infection experiments. Control mice were wild type C3H mice from the same litters, Dys^WT.