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. 2020 Dec 12;16(7):1308–1316. doi: 10.4103/1673-5374.301024

Figure 4.

Figure 4

Generation and characterization of the muscle phenotype in HSA-rtTA,tetO-Cre;Tfr1F/F inducible mutant mice.

(A) Schematic of the generation of adult skeletal muscle inducible Tfr1 conditional knockout mice. DOX combines with and activates the rtTA promoter to induce HSA-Cre recombinase to generate the adult skeletal muscle-specific Tfr1 knockout. DOX (10 mg/kg) was injected intraperitoneally once per day to induce HSA-Cre transgene expression for 30 consecutive days. (B) Representative western blots showing the expression of TfR1 in four types of skeletal muscles, including TA, EDL, gastrocnemius and soleus, in the indicated mice at P60. (C) Quantitative analysis of western blots showing significantly decreased expression of TfR1 in all types of skeletal muscles from DOX-treated HSA-rtTA,tetO-Cre;Tfr1F/F mutants compared with saline-treated mutants and Tfr1F/F control littermates. (D) Gross morphology of four types of skeletal muscles, including TA, EDL, gastrocnemius and soleus muscles, dissected from Tfr1F/F control littermates, and saline-treated and DOX-treated HSA-rtTA,tetO-Cre;Tfr1F/F mutants at P60. Significantly reduced muscle volume was found in TA, EDL and gastrocnemius muscles, but not in soleus muscles. Scale bar: 10 mm. (E) Hematoxylin-eosin staining of the cross-sections of four types of skeletal muscles of the indicated genotypes at P60. Scale bar: 100 μm. The cross-sectional area of TA, EDL and gastrocnemius muscles, but not soleus muscles, was markedly reduced in HSA-rtTA,tetO-Cre;Tfr1F/F mutants compared with controls. (F) Quantitative analysis of the average cross-sectional perimeter of single muscle fibers in four types of skeletal muscles of the indicated genotypes at P60. Data are expressed as the mean ± SEM (n = 6). *P < 0.05, **P < 0.01 (one-way analysis of variance followed by Tukey’s post hoc test). DOX: Doxycycline; EDL: extensor digitorum longus; HSA: skeletal muscle actin; i.p.: intraperitoneal injection; n.s.: not significant; P60: postnatal day 60; rtTA: reverse tetracycline-controlled transactivator; TA: tibialis anterior; Tfr1: transferrin receptor 1.