TAp63gamma is required for the late stages of myogenesis

Figures & data

Figure 1. C2C7 mouse myoblast cells induced to differentiate express TAp63gamma. (A) RT-qPCR quantification of p53 family members RNA extracts from C2C7 myoblast grown in differentiation condition for 24, 48 and 72 hours. (B) mRNA expression of TAp63 isoform highlights its up regulation during skeletal muscle differentiation. (C) Western blot analysis of proteins extracts from C2C7 cells. A TAp63 isoform specific antibody was used to detect TAp63 isoform. Protein extracts from SAOS transfected cells with TAp63alpha and TAp63gamma plasmids were used as positive controls. Beta-actin was used as loading control. (D) Immunostaining for p63 is in green color while in blue is the DAPI staining. All the images are presented as merge of both the channels. One representative experiment of 3 is shown. (E) RT-qPCR and (F) protein gel blot analysis of myogenic regulator factor. Error bars in RT-qPCR indicate the SD of triplicate experiments.

Figure 2. Knock-down of TAp63gamma affects late stages of myogenic differentiation in C2C7. (A) RT-qPCR and (B) western blot analysis of C2C7 cells confirm p63 transient silencing after 72 h of differentiation. (C) C2C7 cells scramble transfected (Ctrl) or si-TAp63 (si-p63) were seeded and induced to differentiate for 72 h in differentiate medium. Cells were then fixed and immunostained for MHC (red) to visualize myotubes and counterstained with DAPI (blue) to visualize nuclei. One representative experiment of 3 is shown. (D) Quantification of fusion index. The fusion index (percent differentiation) was determined by dividing the number of nuclei in myotubes-positive by the total number of nuclei in a given microscopic field. Error bars represent the SEM of three independent experiments. Asterisks denote significance (*, P < 0.005). (E) Actin filaments evidentiated by phalloidin-stained Ctrl C2C7 cells versus si-p63. (F) Western blot analysis of proliferation and differentiation markers in Ctrl C2C7 cells (Ctrl-scramble) and si-p63 induced to differentiate for 24, 48 and 72 hours.

Table 1. Genes expression modulated upon 48 h si-TAp63

Symbol	Gene name	Fold regulation	Functional gene groups
Genes down-regulated
Actn3	Actinin alpha 3	−2.6528	SMContractility
Adrb2	Adrenergic recept, beta 2	−2.8023	Hypertrophy
Atp2a1	ATPase, Ca++ transporting, cardiac muscle, fast twitch 1	−6.6995	SMContractility
Cav1	Caveolin isoform 1	−2.1306	Myogenesis
Cav3	Caveolin isoform 3	−12.3456	SMContractility
Cryab	Crystallin, alpha B	−3.2976	SMContractility
Cs	Citrate synthase	−2.1938	SMContractility
Dag1	Dystroglycan 1	−2.0579	SMContractility
Dmpk	Dystrophia myotonica-protein kinase	−2.2612	SMContractility
HK2	Hexokinase II	−4,0588	Energy metabolism
Igf2	Insulin-like growth factor 2	−4.1584	Autocrine signaling
Igfbp5	Insulin-like growth factor binding protein 5	−4.3204	Myogenesis Hypertrophy
Il6	Interleukin 6	−2.8418	SMContractility
Mb	Myoglobin	−6.2332	SMContractility
Myh1	Myosin, heavy polypeptide 1	−21.6256	SMContractility
Myh2	Myosin, heavy polypeptide 2	−12.0711	SMContractility
Neb	Nebulin	−8.5486	SMContractility
Ppargc1b	Peroxisome proliferative activated receptor, gamma, coactivator 1 beta	−2.6188	Wasting Atrophy
Prkag3	Protein kinase, AMP-activated, gamma 3 non-catatlytic subunit	−7.271	Metabolic syndrome
Sgca	Sarcoglycan, alpha	−2.0521	SMContractility
Slc2a4	Solute carrier family 2	−5.2538	Metabolic syndrome
Tnnc1	Troponin C, cardiac/slow skeletal	−6.5209	SMContractility
Tnni2	Troponin I, skeletal, fast 2	−4.7124	SMContractility
Tnnt3	Troponin T3, skeletal, fast	−2.6733	SMContractility
Ttn	Titin	−8.0881	SMContractility
Pax7	Pax7	−11	Skeletal myogenesis
Genes up-regulated
Capn3	Calpain 3	2.4538	SMContractility
Des	Desmin	4.8628	SMContractility
Mmp9	Matrix metallopeptidase 9	12.1877	Wasting Atrophy
Nos2	Nitric oxide synthase 2, inducible	2.2585	Wasting Atrophy
Pparg	Peroxisome proliferator activated receptor gamma	2.1007	Metabolic syndrome
Ppargc1a	Peroxisome proliferative activated receptor, gamma, coactivator 1 alpha	2.0689	Metabolic syndrome

To note that the genes down-regulatated upon siTAp63 are the gene POSITIVELY regulated by TAp63 (directly or indirectly); while the genes up-regulated upon so-TAp63 are the one NEGATIVELY regulated by TAp63 (directly or indirectly.

Functional Gene grouping, as identified in RT² Profiler PCR Array:

1- SMContractility: Skeletal Muscle Contractility.

2- Myogenesis: Skeletal Myogenesis.

3- Hypertrophy: Skeletal Muscle Hypertrophy.

4- Autocrine signaling: Skeletal Muscle Autocrine signaling.

5- Metabolic syndrome: Diabetes/Metabolic syndrome.

6- Wasting, Atrophy: Skeletal Muscle Wasting/Atrophy.

Figure 3. Validation of TAp63gamma down-stream genes. mRNA extracted by control (Ctrl-scamble stable clone, Fig. S2A) and stable silenced-p63 (Clone A, Fig. S2A) cells, grown for 48 h in differentiation medium, were analyzed by RT-qPCR. (A) RT-qPCR showing p63 knockdown. (B) RT-qPCR to detect Pax7, “skeletal myogenesis” category. (C-H) RT-qPCR of genes involved in “skeletal muscle contractility” category. (I) RT-qPCR for Igf2, “autocrine signaling” category. (J) RT-qPCR for HK2, “energy metabolism” category. (K-M) RT-qPCR for Prkag3 and Pparg, “metabolic syndrome” category. (L) RT-qPCR for Mmp9, “wasting atrophy” category. The categories indicated are the one described in Table 1. Data are shown as the mean of 3 experiments +/− standard deviation.