Fig. 1.
Fig. 1. uPA-deficient mice show a regeneration defect following muscle injury. / (A) uPA expression is increased in regenerating muscle tissue of wild-type (WT) mice after glycerol injury. RNA was isolated from the gastrocnemius muscle group of WT mice (control) or 3 days after glycerol injury (wound). RT-PCR was performed to detect the expression of uPA, GAPDH, MyoD, and myogenin. One tenth of the reaction volume was removed after 10, 15, 20, 22, 24, 26, and 30 amplification cycles. The number of amplification cycles is indicated at the top of each lane. Purified murine cDNAs corresponding to uPA, MyoD, and myogenin were used as positive controls for the PCR reaction (+). Expression of uPA, MyoD, and myogenin increased several times in regenerating muscle compared with control muscle. (B) Impaired up-regulation of MyoD and myogenin mRNAs in skeletal muscle of uPA-deficient mice after freeze-crush injury. RNA was isolated from the musculus tibialis anterior of WT, uPA-deficient, and tPA-deficient mice (control) or 3 days after freeze-crush injury (wound). Northern blot analysis was performed to detect the expression of uPA, MyoD, myogenin and 18S (top) and of tPA and 18S (bottom). Transcript levels of uPA, tPA, MyoD, and myogenin were induced in regenerating muscle of WT mice. Expression of MyoD and myogenin was lower in regenerating muscle of uPA-deficient mice than in regenerating muscle of WT or tPA-deficient mice. As expected, no uPA or tPA mRNA was detected in uPA- or tPA-deficient mice, respectively, whereas levels of 18S were similar in all lanes. (C) Proteolytic activity of uPA is increased in regenerating muscle tissue of WT mice after glycerol injury. Gel zymographies. Forty micrograms protein from muscle extracts of noninjured (0 days after injury) and injured skeletal muscle (2, 5, 7, and 9 days after injury) was analyzed, after SDS-PAGE, by PA zymography (top). Two nanograms purified human uPA (55 kd) was used as a standard (+). Forty micrograms protein from muscle extracts of noninjured (lane 1) and 5-day injured skeletal muscle (lane 2) of uPA-deficient mice and from 2-day injured muscle of WT mice (lane 3) was analyzed by PA zymography (bottom). The approximately 45-kd molecular mass species, indicated by an arrow, corresponds to murine uPA and was calculated according to standard molecular mass markers electrophoresed in an adjacent lane and stained with Coomassie blue. Photographs were taken after overnight incubation at 4°C and 4 hours at 37°C.

uPA-deficient mice show a regeneration defect following muscle injury.

(A) uPA expression is increased in regenerating muscle tissue of wild-type (WT) mice after glycerol injury. RNA was isolated from the gastrocnemius muscle group of WT mice (control) or 3 days after glycerol injury (wound). RT-PCR was performed to detect the expression of uPA, GAPDH, MyoD, and myogenin. One tenth of the reaction volume was removed after 10, 15, 20, 22, 24, 26, and 30 amplification cycles. The number of amplification cycles is indicated at the top of each lane. Purified murine cDNAs corresponding to uPA, MyoD, and myogenin were used as positive controls for the PCR reaction (+). Expression of uPA, MyoD, and myogenin increased several times in regenerating muscle compared with control muscle. (B) Impaired up-regulation of MyoD and myogenin mRNAs in skeletal muscle of uPA-deficient mice after freeze-crush injury. RNA was isolated from the musculus tibialis anterior of WT, uPA-deficient, and tPA-deficient mice (control) or 3 days after freeze-crush injury (wound). Northern blot analysis was performed to detect the expression of uPA, MyoD, myogenin and 18S (top) and of tPA and 18S (bottom). Transcript levels of uPA, tPA, MyoD, and myogenin were induced in regenerating muscle of WT mice. Expression of MyoD and myogenin was lower in regenerating muscle of uPA-deficient mice than in regenerating muscle of WT or tPA-deficient mice. As expected, no uPA or tPA mRNA was detected in uPA- or tPA-deficient mice, respectively, whereas levels of 18S were similar in all lanes. (C) Proteolytic activity of uPA is increased in regenerating muscle tissue of WT mice after glycerol injury. Gel zymographies. Forty micrograms protein from muscle extracts of noninjured (0 days after injury) and injured skeletal muscle (2, 5, 7, and 9 days after injury) was analyzed, after SDS-PAGE, by PA zymography (top). Two nanograms purified human uPA (55 kd) was used as a standard (+). Forty micrograms protein from muscle extracts of noninjured (lane 1) and 5-day injured skeletal muscle (lane 2) of uPA-deficient mice and from 2-day injured muscle of WT mice (lane 3) was analyzed by PA zymography (bottom). The approximately 45-kd molecular mass species, indicated by an arrow, corresponds to murine uPA and was calculated according to standard molecular mass markers electrophoresed in an adjacent lane and stained with Coomassie blue. Photographs were taken after overnight incubation at 4°C and 4 hours at 37°C.

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