Tnfsf12 inhibition affects MMP-9 protein levels independently of NF-κB but reducing MMP-9 does not reduce muscle degeneration. (A–G) Anterior left, dorsal top, side-mounted 3.5 dpf larvae. (A–D) Merged phalloidin and MMP-9 stained (pseudo-colored fuchsia) channels in WT and spns1−/− larvae, MMP-9 staining alone is in black inset boxes. Zebrafish were treated with either 2% EtOH from 30 hpf–3.5 dpf or 100 μM TWEAK inhibitor Aurintricarboxylic acid (ATA) from 30 hpf–3.5 dpf. (A,B) Absence of MMP-9 protein in WT zebrafish treated with EtOH and with ATA. (C) Presence of MMP-9 protein in spns1−/− zebrafish treated with EtOH. White arrowheads indicate MMP-9 positive staining. (D) Absence of MMP-9 protein in spns1−/− zebrafish treated with ATA. (E–G) Merged phalloidin (white) and EGFP (green) channels in EtOH treated and untreated Tg(NF-κB-EGFP); WT and Tg(NF-κB-EGFP); spns1−/− zebrafish. (E1–G1) EGFP single channel. White arrows indicate NF-κB-EGFP positive muscle fibers in (F1) EtOH treated WT zebrafish, but NF-κB-EGFP is not upregulated in spns1−/− mutants. (H) Changes in muscle structure, as measured by mean gray value, were insignificant in 3 dpf spns1−/− zebrafish treated with ATA or EtOH vehicle control. (I) Treatment with ATA and the subsequent reduction in MMP-9 does not improve muscle structure in spns1−/− mutants as assayed by either birefringence (H) or qualitative assessment of muscle structure after phalloidin staining (I). Categories are normal (healthy tissue), mild (fiber disorganization), or severe (fiber disorganization and fiber balls/MTJ failure). **** p < 0.0001. Scale bars 50 µm.
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