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. 2020 May 11;11(5):357. doi: 10.1038/s41419-020-2570-5

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© The Author(s) 2020

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 2 — a RT-qPCRs to examine MyolncR4 knockdown efficiency. The bars show RNA levels relative to GAPDH. Error bars, standard deviations (n = 3). Statistical analysis was performed using Student’s t test. ***P < 0.001. b (Top): IF with the MHC antibody to examine differentiation of C2C12 transfected with Cntl and MyolncR4 siRNAs. DAPI staining to mark the nuclei. MHC: green; DAPI: red. Scale bar, 100 μm. (Bottom): Quantitation of the number of nuclei present in myotubes. Error bars, standard deviations (n = 3). Statistical analysis was performed using Student’s t test. **P < 0.01, *P < 0.05, ns: not significant. c RT-qPCRs to examine MHC mRNA levels in MyolncR4 knockdown cells. The bars show RNA levels relative to GAPDH. Error bars, standard deviations (n = 3). Statistical analysis was performed using Student’s t test. **P < 0.01. d Illustration of the LEMP KO strategy. e Western blot to examine LEMP expression in Cntl or LEMP KO cells. GAPDH was used as a loading control. f RT-qPCRs to examine MyolncR4 RNA levels in Cntl and LEMP KO cells. The bars show RNA levels relative to GAPDH. Error bars, standard deviations (n = 3). Statistical analysis was performed using Student’s t test. Ns: not significant. g (Left): IF with the MHC antibody to examine differentiation of Cntl or LEMP KO cells. DAPI staining to mark the nuclei. MHC: green; DAPI: red. Scale bar, 100 μm. (Right): Quantitation of the number of nuclei present in myotubes. Error bars, standard deviations (n = 3). Statistical analysis was performed using Student’s t test. ***P < 0.001, **P < 0.01, *P < 0.05, ns: not significant. h RT-qPCRs to examine MHC mRNA levels in Cntl and LEMP KO cells. The bars show RNA levels relative to GAPDH. Error bars, standard deviations (n = 3). Statistical analysis was performed using Student′s t test. **P < 0.01. i Schematic of the LEMPΔATG construct. The start codon of LEMP was deleted. j Western blot and RT-qPCRs to examine LEMP protein and mRNA levels in C2C12 cells transfected with equal amount of Cntl, LEMP-HA, and LEMPΔATG-HA constructs. k (Top): IF with the MHC antibody to examine differentiation of C2C12 cells transfected with plasmid expressing LEMP, LEMPΔATG at 4 days after differentiation induction. DAPI staining to mark the nuclei. MHC: green; DAPI: red. Scale bar, 100 μm. (Bottom): Quantitation of the number of nuclei per myotubes. Error bars, standard deviations (n = 3). Statistical analysis was performed using Student’s t test. ***P < 0.001, **P < 0.01, *P < 0.05, ns: not significant.