ZFIN ID: ZDB-FIG-161104-2
Saera-Vila et al., 2016 - Autophagy Regulates Cytoplasmic Remodeling During Cell Reprogramming in a Zebrafish Model of Muscle Regeneration. Autophagy   12(10):1864-1875 Full text @ Autophagy
ADDITIONAL FIGURES
EXPRESSION / LABELING:
Genes:
Antibodies:
Fish:
Conditions:
Knockdown Reagents:
Anatomical Terms:
Stage: Adult
PHENOTYPE:
Fish:
Conditions:
Knockdown Reagents:
Observed In:
Stage: Adult

Fig. 3

Autophagy is required for EOM regeneration. (A) Western blot of fish treated with increasing CQ concentrations showed a dose-dependent increase of Sqstm1 in the injured muscle and Lc3-II in both injured and uninjured muscles (see Figure S1), indicating real autophagy flux. Protein loading was assayed with an anti-Tubg1 antibody. U, uninjured; I, injured. (B o G) The lack of LysoTracker Red staining in injured LR muscles of transgenic actc1b/α-actin:EGFP zebrafish (to visualize the muscles) using our craniectomy technique confirmed that CQ effectively blocked autophagy. (H to J) Craniectomy of an untreated (H) and CQ-treated fish (I) at 3 dpi; note the difference in length of the injured muscle between both groups (pictures are representative examples of 5 fish per group). (J) Quantification of LR muscle regeneration of fish shown in H and I; values are averages ± SD (Student t test, *, P < 0.05, n = 5). (K) Quantification of LR muscle regeneration at 5 dpi treated with different CQ concentrations; values are averages ± SD (One-way ANOVA, P < 0.0001, n=5; **, P < 0.01; ***, P < 0.001; Newman-Keuls multiple comparisons test). (L) Regeneration of LR muscles injected with control or atg5 translation-blocking (TB) MO was measured at 5 dpi; values are averages ± SD (Student t test, *, P < 0.05, n = 5). (M) Regeneration of LR muscles injected with control or becn1 MOs (translation-blocking, TB; splicing-blocking, SB; combination of both, TB+SB) was measured at 5 dpi; values are averages ± SD (Student t test; *, P < 0.05; **, P < 0.01, n = 5). (J to M) Since myectomy left approximately 50% of the muscle remaining, the measurements are shown for the entire regenerating muscle with a gray box indicating the approximate location of the myectomy site. (N) Fluorescence intensity of LR muscles of GFP-Lc3 fish injected with control, atg5 TB or becn1 TB+SB MOs was measured at 18 hpi; values are averages ± SD (Student t test; *, P < 0.05, n = 8). (O) Diagram of a craniectomized zebrafish head; muscles visualized by this technique are shown, and LR muscles are highlighted in green. Green and red boxes show approximately the picture used for regeneration or LysoTracker Red (or GFP-Lc3) assessment, respectively. * Skull base where the pituitary is located.

Gene Expression Details
Gene Antibody Fish Conditions Stage Anatomy Assay
EGFP zf155Tg + MO1-atg5 resection: lateral rectus Adult lateral rectus regenerating tissue IHC
zf155Tg + MO4-becn1 + MO5-becn1 resection: lateral rectus Adult lateral rectus regenerating tissue IHC
GFP zf13Tg resection: lateral rectus, chemical treatment by environment: chloroquine Adult lateral rectus regenerating tissue IHC
Adult muscle IHC
zf13Tg resection: lateral rectus Adult lateral rectus regenerating tissue IHC
Adult muscle IHC
Antibody Labeling Details
Antibody Assay Fish Conditions Stage Anatomy
Ab1-map1lc3 WB WT resection: lateral rectus, chemical treatment by environment: chloroquine Adult lateral rectus regenerating tissue
WB WT resection: lateral rectus Adult lateral rectus regenerating tissue
Ab6-sqstm1 WB WT resection: lateral rectus, chemical treatment by environment: chloroquine Adult lateral rectus regenerating tissue
WB WT resection: lateral rectus Adult lateral rectus regenerating tissue
Phenotype Details
Fish Conditions Stage Phenotype
WT resection: lateral rectus, chemical treatment by environment: chloroquine Adult autophagy disrupted, abnormal
Adult autophagy increased occurrence, abnormal
Adult lateral rectus regenerating tissue ab1-map1lc3 labeling increased amount, abnormal
Adult lateral rectus regenerating tissue Ab6-sqstm1 labeling increased amount, abnormal
WT resection: lateral rectus Adult lateral rectus regenerating tissue ab1-map1lc3 labeling increased amount, abnormal
Adult lateral rectus regenerating tissue Ab6-sqstm1 labeling increased amount, abnormal
WT + MO1-atg5 resection: lateral rectus Adult lateral rectus regenerating tissue decreased length, abnormal
Adult lateral rectus skeletal muscle tissue regeneration decreased process quality, abnormal
WT + MO4-becn1 resection: lateral rectus Adult lateral rectus regenerating tissue decreased length, abnormal
Adult lateral rectus skeletal muscle tissue regeneration decreased process quality, abnormal
WT + MO4-becn1 + MO5-becn1 resection: lateral rectus Adult lateral rectus regenerating tissue decreased length, abnormal
Adult lateral rectus skeletal muscle tissue regeneration decreased process quality, abnormal
WT + MO5-becn1 resection: lateral rectus Adult lateral rectus regenerating tissue decreased length, abnormal
Adult lateral rectus skeletal muscle tissue regeneration decreased process quality, abnormal
zf13Tg resection: lateral rectus, chemical treatment by environment: chloroquine Adult autophagy disrupted, abnormal
Adult autophagy increased occurrence, abnormal
Adult lateral rectus regenerating tissue decreased length, abnormal
zf155Tg + MO1-atg5 resection: lateral rectus Adult autophagy disrupted, abnormal
Adult lateral rectus regenerating tissue EGFP expression decreased amount, abnormal
zf155Tg + MO4-becn1 + MO5-becn1 resection: lateral rectus Adult autophagy disrupted, abnormal
Adult lateral rectus regenerating tissue EGFP expression decreased amount, abnormal
Acknowledgments:
ZFIN wishes to thank the journal Autophagy for permission to reproduce figures from this article. Please note that this material may be protected by copyright. Full text @ Autophagy