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:
This image is the copyrighted work of the attributed author or publisher, and ZFIN has permission only to display this image to its users. Additional permissions should be obtained from the applicable author or publisher of the image. Full text @ Autophagy