PMC

(A) Representative splicing products obtained by RT-PCR amplification of RNA isolated from 5 days differentiated myotubes (T5) obtained from CTR, DM1, DM2 and T2DM patients. Bands were quantified and proportions of isoform INSR-B (+exon 11) were calculated. (B) Representative western blot analysis of the basal expression of IRβ in myotubes (T5). (C) Histograms representing mean values of IRβ expression and bars represent standard error of the mean (SEM). The number of samples analysed in each group (n) is reported in histogram legend. Density of the bands has been normalized to GAPDH expression.

(A) Representative western blot analysis of the expression and activation of proteins involved in the insulin pathway. Myotubes (T5) were cultured in absence or in presence of 10 nM insulin for 5 to 30 minutes. Quantification of IRS1 activation (B), of Akt/PKB (C), p70S6K (D), GSK3β (E), ERK1 (F) and ERK2 (G). Histograms represents mean values and bars represent standard error of the mean (SEM). The number of samples analysed in each group (n) is reported in histogram legends. The effect of time within groups was assessed by one-way ANOVA (repeated measures). Results from post-test are marked with connecting capped arcs. *Results from Student t-test DM1 or DM2 versus CTR at 0-5-15-30 minutes of insulin stimulation: *p<0.05, **p<0.01.

(A-D) H&E staining of muscle sections obtained from two DM1 patients who performed two successive biopsies at different years of age. In the first biceps brachii biopsy (A) of DM1-1 patient a variation in fiber size, central nuclei (asterisk) and atrophic fibers (black arrow) were present, while the second tibialis anterior biopsy (B) was severely affected with end-stage changes including loss of muscle fibers, fibrosis, marked increase in the number of fibers with internal nuclei (asterisk) and fiber hypertrophy (arrow head) and atrophy (black arrow). In DM1-4 patient, the first biceps brachii biopsy (C) and the second tibialis anterior biopsy (D) showed minimal myopathic changes. (E, F) Fast myosin immunostaining of biceps brachii muscle sections obtained from DM1-2 (E) and from DM2-2 patients (F). Atrophic fibers were prevalently of type 1 in DM1 section (E, black arrow) and of type 2 in the DM2 section (F, black arrow). Nuclear clumps fast myosin positive were also present in DM2 section (F; arrow head). Original magnification 200x.

(A) INSR splicing products obtained by RT-PCR amplification of RNA isolated from biceps brachii biopsies obtained from CTR, DM1 and DM2 patients. Bands were quantified and proportions of isoform INSR-B (+exon 11) were calculated. (B) Representative western blot analysis of the expression of proteins involved in the insulin pathway in CTR, DM1 and DM2 patients. Due to the high number of samples analysed, the figure panel is composed by images of bands originating from different gels. Details on how western blot experiments were performed are reported in Materials and Methods. (C) Quantification of proteins expression normalized to GAPDH. Histograms represent mean values and bars represent standard error of the mean (SEM). The number of samples analysed in each group (n) is reported in histogram legend. Differences between groups have been evaluated by Student t-test. *p<0.05.

(A) Histogram representing insulin action on 2-deoxyglucose uptake in human myotubes (T5). Cells were depleted of serum, incubated in absence or presence of 10 nM insulin for 40 min and then 2-deoxyglucose uptake was measured using a colorimetric assay kit. Histograms represent mean values and bars represent standard error of the mean (SEM). (B) Representative western blot analysis of the expression of GLUT4 during insulin stimulation. Myotubes (T5) were cultured in absence or in presence of 10 nM insulin for 5 to 30 minutes. Due to the high number of samples analysed, the figure panel is composed by images of bands originating from different gels. Details on how western blot experiments were performed are reported in Materials and Methods. (C) Histograms representing mean values of GLUT4 expression analysed by densitometry. Density of the bands has been normalized to GAPDH expression. Bars represent standard error of the mean (SEM). The number of samples analysed in each group (n) is reported in histogram legend. Differences between groups have been evaluated by Student t-test. *p<0.05.

(A) INSR splicing products obtained by RT-PCR amplification of RNA isolated from biceps brachii and tibialis anterior biopsies obtained from CTR and DM1 patients. Bands were quantified and proportions of isoform INSR-B (+exon 11) were calculated. (B) Representative western blot analysis of the expression of proteins involved in the insulin pathway in biceps brachii and tibialis anterior biopsies obtained from CTR and DM1 patients. Due to the high number of samples analysed, the figure panel is composed by images of bands originating from different gels. Details on how western blot experiments were performed are reported in Materials and Methods. (C) Quantification of proteins expression normalized to GAPDH in tibialis anterior biopsies obtained from CTR and DM1 patients. Histograms represent mean values and bars represent standard error of the mean (SEM). (D) Quantification of proteins expression normalized to GAPDH in tibialis anterior and biceps brachii biopsies obtained from two DM1 patients who performed two successive biopsies at different years of age. Data obtained from one BB and one TA sample from DM1-1 and DM1-4 were compared with mean values obtained from 2 CTR BB and 3 CTR TA. Bars represent standard error of the mean (SEM). The number of samples analysed in each group (n) is reported in histogram legends. Differences between groups have been evaluated by Student t-test. *p<0.05; **p<0.01.