Oral intake of rice overexpressing ubiquitin ligase inhibitory pentapeptide prevents atrophy in denervated skeletal muscle

Determination of effects of transgenic Cblin peptide-enriched

rice on mice with sciatic denervation

Six-week-old male Jcl:ICR mice (Japan SLC Inc., Shizuoka, Japan) were

housed with access to a standard diet and water ad libitum under lights

on from 8 a.m. to 8.0 p.m. The experimental diet comprised powdered

Cblin rice grains (CbR) and non-transgenic rice (non-Tg) mixed with the

standard diet (50% w/w). Supplementary Table 2 shows the composition

of the diets. The mice were then fed with the diet containing either CbR or

non-Tg for 7 days, then their sciatic nerves were unilaterally transected

under anesthesia. The contralateral innervated (sham-operated) muscles

of the same animals served as controls. Seven days later, the mice were

sacrificed and the Sol and Ga muscles were dissected, weighed, and

frozen in liquid nitrogen.

All animal experiments proceeded according to the guidelines for

animal experiments at Tokushima University and Kyushu University. The

Ethics Review Committee for Animal Experimentation at these institutions

approved all the experimental protocols described herein (Permission Nos.

T27–113 and A30-015-4).

Real-time reverse transcription-polymerase chain reaction

(RT-PCR)

Total RNA was extracted using ISOGEN (Nippon Gene, Osaka, Japan).

Single-stranded cDNA was synthesized using M-MLV Reverse Transcriptase

(Promega). Real-time RT-PCR proceeded using SYBR-Green Master Mix

(Thermo Fisher Scientific Inc., Waltham, MA, USA) and a Real-time PCR

system (Thermo Fisher Scientific Inc.). Supplementary Table 3 shows the

primer sequences. Amounts of target mRNA were normalized relative to

that of Actb.

Immunoblotting

Proteins were extracted from mouse muscles into Tris-HCl buffer, pH 7.5,

containing 150 mM NaCl, 5 mM EDTA, 1% TritonX-100, 10 mM NaF, 2 mM

Na3VO4, 10 μM MG132, and protease inhibitor tablets (Roche Diagnostics,

Basel, Switzerland). Total protein extracts were resolved by SDS-PAGE and

transferred to PVDF membranes. PBS-Tween containing 4% Block Ace

Powder (DS Pharma Biomedical Co. Ltd., Osaka, Japan) was used to block

non-specific binding. Then, the membranes were incubated with primary

antibodies at 4 °C overnight. Chemiluminescent blot was detected using

ImmunoStar LD (Wako Pure Chemical Industries, Osaka, Japan) and signals

were quantified by densitometry. All blots or gels were derived from the

same experiment and were processed in parallel. The primary antibodies

were anti-IRS1 (Merck Millipore Burlington, MA, USA) and anti-ACTIN

(ABclonal Technology, Woburn, MA, USA).

Statistical analysis

Values were statistically evaluated by Student’s t tests using Excel-Toukei

2015 software (Social Survey Research Information Co. Ltd., Osaka, Japan),

and are expressed as means ± SEM. Statistical significance among Papp of

Asp-Gly-pTyr-Met-Pro, Asp-Gly-Tyr-Met-Pro, and Gly-Sar-Sar-Sar-Sar was

assessed by one-way analyses of variance (ANOVA) followed by Tukey post

hoc tests. Data from mice fed with non-Tg or CbR were statistically

evaluated by two-way ANOVA followed by Tukey–Kramer multiple

comparisons for post hoc analysis using Excel-Toukei 2015 software. Values

with P < 0.05 were considered significantly different.

Reporting summary

Further information on research design is available in the Nature Research

Reporting Summary linked to this article.

DATA AVAILABILITY

The authors declare that all data supporting the findings of this study are available

within the paper and supplementary information.

Published in partnership with Beijing Technology and Business University

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Published in partnership with Beijing Technology and Business University

ACKNOWLEDGEMENTS

This study was supported by JSPS Research Fellowship for Young Scientists to W.S.

(No. 19J20948), JSPS KAKENHI to T.N. (JP18H04981 and JP19H04054) from the

Ministry of Education, Culture, Sports, Science, and Technology (MEXT) of Japan, and

AMED-CREST to T.N. (JP19gm0810009h0104). The authors thank Arisa Ochi, Hina

Ishikawa, and Yuki Nomura (Tokushima University) for technical assistance.

AUTHOR CONTRIBUTIONS

R.N., W.S., Y.Sh., K.K., K.O., A.O., T.U., M.T., K.A., T.M., and T.N. designed the study

protocol. R.N., W.S., Y.Sh., K.K., Y.Sa., A.U., K.O., M.N., M.T., K.A., and T.M. conducted

experiments. R.N., W.S., Y.Sh., K.K., Y.Sa., K.O., A.O., M.T., K.A, T.M., and T.N. analyzed

data. R.N., W.S., Y.Sa., M.T., K.A, T.M., and T.N. contributed to writing the manuscript.

All authors read and approved the final version of the manuscript.

COMPETING INTERESTS

The authors declare no competing interests.

ADDITIONAL INFORMATION

Supplementary information The online version contains supplementary material

available at https://doi.org/10.1038/s41538-021-00108-0.

Correspondence and requests for materials should be addressed to T.N.

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