Figure 1.
Starvation but not 20-hydroxyecdysone (20E) promotes BmTip60 in addition to autophagy in B. mori fat body. (A) mRNA levels of BmE75a after 20E treatment for 24 h. (B) Protein levels of BmSqstm1, BmTip60, and BmAtg8 after 20E treatment. Two repeats are shown here. (C) LysoTracker red staining and BmAtg8 immunofluorescent staining after 20E treatment. Arrow: BmAtg8 puncta. Scale bar: 10 micron. (D) Protein levels of BmSqstm1, BmTip60, p-AMPKα (T172), and BmAtg8 after starvation for 24 h. Two repeats are shown here. (E) LysoTracker red staining and BmAtg8 immunofluorescent staining after starvation. Arrow: Atg8 puncta in the cytoplasm. Scale bar: 10 micron. (E’) Quantification of BmAtg8 puncta in (E). (F) Immunofluorescent staining of BmTip60 after starvation. White solid box: magnified field. White dashed box: original field. Scale bar: 10 micron. (F’) Quantification of BmTip60 density in (F). One-way analysis of variance was performed by SPSS in the whole figure, * p < 0.05; ** p < 0.01; *** p < 0.001, n = 200 cells.
Figure 1.
Starvation but not 20-hydroxyecdysone (20E) promotes BmTip60 in addition to autophagy in B. mori fat body. (A) mRNA levels of BmE75a after 20E treatment for 24 h. (B) Protein levels of BmSqstm1, BmTip60, and BmAtg8 after 20E treatment. Two repeats are shown here. (C) LysoTracker red staining and BmAtg8 immunofluorescent staining after 20E treatment. Arrow: BmAtg8 puncta. Scale bar: 10 micron. (D) Protein levels of BmSqstm1, BmTip60, p-AMPKα (T172), and BmAtg8 after starvation for 24 h. Two repeats are shown here. (E) LysoTracker red staining and BmAtg8 immunofluorescent staining after starvation. Arrow: Atg8 puncta in the cytoplasm. Scale bar: 10 micron. (E’) Quantification of BmAtg8 puncta in (E). (F) Immunofluorescent staining of BmTip60 after starvation. White solid box: magnified field. White dashed box: original field. Scale bar: 10 micron. (F’) Quantification of BmTip60 density in (F). One-way analysis of variance was performed by SPSS in the whole figure, * p < 0.05; ** p < 0.01; *** p < 0.001, n = 200 cells.
Figure 2.
Phosphorylation at Ser99 is required for BmTip60-mediated autophagy. (A) Amino acid sequences of Tip60 homologs in B. mori and H. sapiens are aligned by DNAMAN software. Dark blue: conserved amino acid sequence, light blue: nonconservative amino acid sequence. Red box: the conserved phosphorylation site. (B) Phosphorylation level of immunoprecipitated BmTip60 after overexpression of BmTip60-V5, BmTip6099SA-V5, and BmTip6099SD-V5 in BmN cells for 48 h, respectively. WT: wild-type; 99SA: Serine99 mutated to Alanine99; 99SD: Serine99 mutated to Aspartic99. (C) Protein levels of BmSqstm1, BmTip60, and BmAtg8 after overexpression of BmTip60-V5, BmTip6099SA-V5, or BmTip6099SD-V5 in BmN cells for 48 h. (D) Observation of EGFP-BmAtg8 and immunostained BmTip60-V5/BmTip6099SA-V5/BmTip6099SD-V5 under nutrient-rich conditions in BmN cells. Scale bar: 10 micron. White solid box: magnified field. White dashed box: original field. (D’) Quantification of EGFP-BmAtg8 puncta in (D). One-way analysis of variance was performed by SPSS in the whole figure; different lowercase letters mean p < 0.05, n = 50 cells.
Figure 2.
Phosphorylation at Ser99 is required for BmTip60-mediated autophagy. (A) Amino acid sequences of Tip60 homologs in B. mori and H. sapiens are aligned by DNAMAN software. Dark blue: conserved amino acid sequence, light blue: nonconservative amino acid sequence. Red box: the conserved phosphorylation site. (B) Phosphorylation level of immunoprecipitated BmTip60 after overexpression of BmTip60-V5, BmTip6099SA-V5, and BmTip6099SD-V5 in BmN cells for 48 h, respectively. WT: wild-type; 99SA: Serine99 mutated to Alanine99; 99SD: Serine99 mutated to Aspartic99. (C) Protein levels of BmSqstm1, BmTip60, and BmAtg8 after overexpression of BmTip60-V5, BmTip6099SA-V5, or BmTip6099SD-V5 in BmN cells for 48 h. (D) Observation of EGFP-BmAtg8 and immunostained BmTip60-V5/BmTip6099SA-V5/BmTip6099SD-V5 under nutrient-rich conditions in BmN cells. Scale bar: 10 micron. White solid box: magnified field. White dashed box: original field. (D’) Quantification of EGFP-BmAtg8 puncta in (D). One-way analysis of variance was performed by SPSS in the whole figure; different lowercase letters mean p < 0.05, n = 50 cells.