Journal: eLife

Article Title: Septin7 is indispensable for proper skeletal muscle architecture and function

doi: 10.7554/eLife.75863

Figure Lengend Snippet: ( A ) Expression of different septin isoforms at mRNA level in human skeletal muscle ( m. quadriceps femoris ). Related to and . Independent samples from three patients (P1–P3) were examined. Extended experiment of . First and last lines in the ladders correspond to 200 and 1000 bp, respectively. ( B ) mRNA expression of all septin isoforms in differentiating C2C12 myoblast cells. Proliferating cells (p1) and myotubes at different stages of development (d1, d3, and d5) are demonstrated. First and last lines in the ladders correspond to 100 and 900, 100 and 1500, and 200 and 1500 bp, from top to bottom panels, respectively. ( C ) Age-related (age of 4 days [4d], 4 weeks [4w], 4 months [4m]) mRNA expression of Septin7 in different skeletal muscle types of mouse. Gapdh was used as loading control; NB: newborn muscle; TA: m. tibialis anterior ; FDB: m. flexor digitorum brevis; EDL: m. extensor digitorum longus; and Sol: m. soleus were examined. First and last lines in the ladders correspond to 200 and 700, 100 and 1000, 200 and 600, and 200 and 600 bp, from left to right and top to bottom panels, respectively.( D ) Ontogenesis-dependent Septin7 protein expression in differentiating C2C12 cells, newborn and different muscle types of adult (4-month-old) mice; α-actinin was used as control. First and last lines in the ladder correspond to 35 and 130 kDa, respectively. ( E ) Screening for the presence of the HSA-MCM transgene from genomic DNA by PCR. First and last lines in the ladder correspond to 300 and 1500 bp, respectively. ( F ) Altered Septin7 expression in Cre+ mice following a tamoxifen diet compared to Cre- and control BL6 mice. The m. quadriceps femoris (Q) and m. pectoralis (P) were analyzed; α-actinin was used as a normalizing control. First and last lines in the ladder correspond to 55 and 130 kDa, respectively.( G ) Pooled data of the Septin7 protein expression in different muscle types of tamoxifen-fed mice. Eight Cre+ and five Cre- were examined from three independent litters. Data represent mean ± standard error of the mean, *p<0.05 from t-test. Figure 1—figure supplement 1—source data 1. Expression of SEPTIN1-4 isoforms at mRNA level in human skeletal muscle ( m. quadriceps femoris ). Independent samples from three patients (P1–P3) were examined. Relevant parts of the original gel images are presented in . Figure 1—figure supplement 1—source data 2. Expression of SEPTIN5-8 isoforms at mRNA level in human skeletal muscle. Figure 1—figure supplement 1—source data 3. Expression of SEPTIN9,10,11,14 isoforms at mRNA level in human skeletal muscle. Figure 1—figure supplement 1—source data 4. mRNA expression of Septin1-8 isoforms in C2C12 cultured cells. Proliferating cells (p1) and myotubes at different stages of development (d1, d3, and d5) are demonstrated. Relevant parts of the original gel images are presented in . Figure 1—figure supplement 1—source data 5. mRNA expression of Septin9-12 and Septin14 isoforms in C2C12 cultured cells. Figure 1—figure supplement 1—source data 6. Age-related (age of 4 days [4d], 4 weeks [4w], 4 months [4m]) mRNA expression of Gapdh in newborn and in different skeletal muscle types ( m. flexor digitorum brevis, m. extensor digitorum longus , and m. soleus ) of adultmouse. Gapdh was used as loading control; NB: newborn muscle; TA: m. tibialis anterior; FDB: m. flexor digitorum brevis; EDL: m. extensor digitorum longus; and Sol: m. soleus were examined. Relevant parts of the original gel images are presented in . Figure 1—figure supplement 1—source data 7. Age-related mRNA expression of Gapdh in newborn and in m. tibialis anterior skeletal muscle type of adult mouse. Figure 1—figure supplement 1—source data 8. Age-related mRNA expression of Septin7 in newborn and in different skeletal muscle types (newborn, m. flexor digitorum brevis, m. externsor digitorum longus, and m. soleus ) of adult mouse. Figure 1—figure supplement 1—source data 9. Age-related mRNA expression of Septin7 in newborn and in m. tibialis anterior skeletal muscle type of adult mouse. Figure 1—figure supplement 1—source data 10. Ontogenesis-dependent Septin7 (SEPT7) protein expression in proliferating and differentiated C2C12 cells, newborn and different muscle types of adult (4 months old) mice; α-actinin was used as control. Relevant parts of the original gel images are presented in . Figure 1—figure supplement 1—source data 11. Screening for the presence of the HSA-MCM transgene from genomic DNA by PCR in Cre+ and Cre- mice. Relevant parts of the original gel images are presented in . Figure 1—figure supplement 1—source data 12. α-Actinin protein expression in Cre+ mice following a tamoxifen diet compared to Cre- and control BL6 mice. α-Actinin was used as a normalizing control. Relevant parts of the original gel images are presented in . Figure 1—figure supplement 1—source data 13. Altered Septin7 protein expression in Cre+ mice following a tamoxifen diet compared to Cre- and control Bl6 mice.

Article Snippet: Cell line ( M. musculus ) , Mouse immortalized C2C12 , ATCC , Cat# CRL-1772; RRID: CVCL_0188 , .

Techniques: Expressing, Control, Cell Culture

Journal: eLife

Article Title: Septin7 is indispensable for proper skeletal muscle architecture and function

doi: 10.7554/eLife.75863

Figure Lengend Snippet: ( A ) Confocal images of Septin7 immunolabeling (green) and actin filaments (red) and their co-localization in control C2C12 cells. Calibration is 20 µm. Transmitted light images of control ( B ) and Septin7 knockout (KO; C ) cells demonstrating that complete KO of Septin7 in C2C12 cells prevents appropriate cell proliferation. Scale bar is 50 µm for both the control and KO cells. ( D ) Partial knockdown (KD) of Septin7 expression at proliferation stage (nondifferentiated [ND]) and 5 days after differentiation induction (D5). 8 µg of protein samples from absolute control (Ctrl), scrambled transfected (Scr), and S7-KD cells in each case were loaded to SDS-PAGE gel, and following electrophoresis and blot transfer into nitrocellulose membrane, Septin7 (50 kDa) and α-actinin (110 kDa) were probed with the appropriate primary antibodies. Numbers 1, 2, and 3 at the bottom of the gels indicate Ctrl, Scr, and S7-KD samples, respectively. First and last lines in the ladders correspond to 35 and 250, and 70 and 250 kDa, for the upper and lower panels, respectively. Immunolabeling of Septin7 filaments in Scr ( E ) and S7-KD cells ( F ) demonstrating altered filament structure, cell size, and shape. Scale bar is 20 µm. Quantification of changes in cell morphology, area ( G ) and circularity ( H ) in S7-KD cultures. Green triangles represent Scr, while red squares represent S7-KD cells. The number of cells investigated was 96 in Scr and 121 in S7-KD cultures; *p<0.05, ***p<0.001 from t-test; experiment was repeated twice (N = 2). ( I ) Decreased proliferation 3 days after the plating assessed as CyQUANT fluorescence (n = 24; N = 3) and ( J ) suppressed differentiation of S7-KD cells determined by the calculation of fusion index during 6 days of investigation (n = 20; N = 2). Horizontal line in ( J ) shows where the difference between Scr and S7-KD cells was statistically significant (*p<0.05 from t-test). See also and . Figure 3—source data 1. Partial knockdown (KD) of Septin7 protein expression at proliferation stage (nondifferentiated [ND]) and following differentiation induction (D1–D2). 8 µg of protein samples from absolute control (Ctrl), scrambled transfected (Scr), and S7-KD cells in each case were loaded to SDS-PAGE gel, and following electrophoresis and blot transfer into nitrocellulose membrane, Septin7 (50 kDa) and α-actinin (110 kDa) were probed with the appropriate primary antibodies. Numbers 1, 2, and 3 at the bottom of the gels indicate Ctrl, Scr, and S7-KD samples, respectively. Relevant parts of the original gel images are presented in . Figure 3—source data 2. Partial knockdown (KD) of Septin7 protein expression following differentiation induction (D3-D6). Figure 3—source data 3. α-actinin protein expression at proliferation stage (ND) and following differentiation induction (D1-D2) in cultured C2C12 cells. Figure 3—source data 4. α-actinin protein expression following differentiation induction (D3-D6) of cultured C2C12 cells.

Article Snippet: Cell line ( M. musculus ) , Mouse immortalized C2C12 , ATCC , Cat# CRL-1772; RRID: CVCL_0188 , .

Techniques: Immunolabeling, Control, Knock-Out, Knockdown, Expressing, Transfection, SDS Page, Electrophoresis, Membrane, CyQUANT Assay, Fluorescence, Cell Culture

Journal: eLife

Article Title: Septin7 is indispensable for proper skeletal muscle architecture and function

doi: 10.7554/eLife.75863

Figure Lengend Snippet: Intracellular localization of Septin7 (green) and actin (red) filaments individually and merged in control (Ctrl, A ), scrambled (Scr, B ), and S7-KD ( C ) C2C12 cells. Calibration is 50 µm. ( D ) Modified expression of Septin7 protein (50 kDa) was followed during the differentiation program of Ctrl, Scr, and S7-KD cells. ND refers to nondifferentiated, D1–D5 to appropriate days (1–5) following the induction of differentiation, while numbers 1, 2, and 3 at the bottom of the gels indicate Ctrl, Scr, and S7-KD samples, respectively. First and last lines in the ladders correspond to 35 and 250 kDa, respectively. ( E ) Quantitation of change in S7-KD cell morphology (area and circularity). Black circles represent data from control, while green triangles from scrambled cells. Individual data points (symbols) and average values (boxes with error bars) are shown for each group. The number of cells investigated was 127 in Ctrl, 96 in Scr, and 121 in S7-KD cultures; the experiment was repeated twice (N = 2). ( F ) Average perimeter was also calculated from different cells, and this data did not show significant difference between Ctrl (black), Scr (green), and S7-KD samples (red). ( G ) Representative confocal images from differentiated (D5) control, scrambled, and Septin7 KD cultures, where desmin (expressed only in myotubes) was fluorescently labeled using specific antibody (green), and nuclei were stained with DAPI (blue). Scale bars represent 50 µm. Differentiation during 6 days of culturing was assessed by calculating the fusion index in Ctrl (black) and Scr (green) cultures revealed to be not significantly different (n = 20; N = 2). Figure 3—figure supplement 1—source data 1. Modified expression of Septin7 protein (50 kDa) in non-differentiated stage and 1-2 days following differentiation induction of Ctrl, Scr, and S7-KD cells. ND refers to nondifferentiated, D1–D5 to appropriate days (1–5) following the induction of differentiation, while numbers 1, 2, and 3 at the bottom of the gels indicate Ctrl, Scr, and S7-KD samples, respectively. Relevant parts of the original gel images are presented in . Figure 3—figure supplement 1—source data 2. Modified expression of Septin7 protein during the differentiation (D3-D6) program of Ctrl, Scr, and S7-KD cells.

Article Snippet: Cell line ( M. musculus ) , Mouse immortalized C2C12 , ATCC , Cat# CRL-1772; RRID: CVCL_0188 , .

Techniques: Control, Modification, Expressing, Quantitation Assay, Labeling, Staining

Journal: eLife

Article Title: Septin7 is indispensable for proper skeletal muscle architecture and function

doi: 10.7554/eLife.75863

Figure Lengend Snippet: Representative confocal images of Septin7 (green) and actin (red) filaments in Scr ( A ) and a S7-KD ( B ) C2C12 cell, where co-localization analysis was also performed. The Pearson’s coefficient was smaller for the S7-KD ( B , 0.648) than for the Scr ( A , 0.833) C2C12 cell. From the original merged images of scrambled ( A ) and S7-KD cells ( B ), Septin7 (in green) and actin filaments (in red) are separately presented for scrambled ( C ) and S7-KD ( D ) cells, respectively. Note the asymmetric distribution of intensities in case of S7-KD cells. Filamentous images ( C, D ) were created with thresholding of the separate channels. Scale bar in panels ( A ) and ( B ) corresponds to 10 μm.

Article Snippet: Cell line ( M. musculus ) , Mouse immortalized C2C12 , ATCC , Cat# CRL-1772; RRID: CVCL_0188 , .

Techniques:

Journal: eLife

Article Title: Septin7 is indispensable for proper skeletal muscle architecture and function

doi: 10.7554/eLife.75863

Figure Lengend Snippet: ( A, B ) Representative electron microscopy (EM) images of myofibrils with mitochondria from cross-sectional samples of m. tibialis anterior (TA) muscles of Cre- and Cre+ mice. ( C ) Perimeter, aspect ratio (AR), and form factor (FF) of the individual mitochondria (average ± SEM; **p<0.01, ***p<0.001 from t-test). ( D ) Statistical distribution of mitochondrial area determined from EM images of Cre- (green) and Cre+ (red) samples. The average area with SE is presented in the inset of this panel. ( E ) The number of mitochondria within 1 µm 2 of an appropriate visual field was calculated from EM images. The total number of mitochondria counted for area, perimeter, AR, and FF was 811 and 866 in Cre- and Cre+, respectively, while the number of visual fields examined was 50 and 43 for calculating the number of mitochondria (***p<0.001 from t-test). ( F, G ) Representative EM images of myofibrils and mitochondria from longitudinal sections of TA muscle samples originating from Cre- and Cre+ mice. ( H ) Perimeter, AR, and FF were determined from the longitudinal sections in both Cre- (n = 1635) and Cre+ (n = 713) samples (average ± SEM; ***p<0.001 from t-test). ( I ) The relative distribution of mitochondrial area, average area of mitochondria in Cre- and Cre+ skeletal muscle fibers is presented in the inset of this panel. The number of visual fields investigated was 42 and 41, respectively (average ± SEM; ***p<0.001 from t-test). Scale bars are equal to 1 µm in all images. ( J ) Mitochondrial DNA content was determined from m. pectoralis (P) and m. quadriceps femoris (Q) of Cre- and Cre+ mice using specific qPCR primers. The number of samples was 8, while in each case experimental triplicate was performed (***p<0.001 from t-test). See also . Changes in mitochondrial morphology following Septin7 depletion were also studied in cultured C2C12 cells. For these results, see .

Article Snippet: Cell line ( M. musculus ) , Mouse immortalized C2C12 , ATCC , Cat# CRL-1772; RRID: CVCL_0188 , .

Techniques: Electron Microscopy, Muscles, Cell Culture

Journal: eLife

Article Title: Septin7 is indispensable for proper skeletal muscle architecture and function

doi: 10.7554/eLife.75863

Figure Lengend Snippet:

Article Snippet: Cell line ( M. musculus ) , Mouse immortalized C2C12 , ATCC , Cat# CRL-1772; RRID: CVCL_0188 , .

Techniques: Recombinant, CRISPR, Plasmid Preparation, Construct, shRNA, Retroviral, CyQUANT Assay, Proliferation Assay, Software, Labeling, Expressing, Blocking Assay, Binding Assay, Immunolabeling, Flow Cytometry, FACS, Single Photon Emission Computed Tomography, Activity Assay, In Vitro, Transmission Assay, Microscopy, Real-time Polymerase Chain Reaction, Cell Culture