Journal: Aging Cell

Article Title: DLK / JNK3 Upregulation Aggravates Hair Cell Senescence in Mice Cochleae via Excessive Autophagy

doi: 10.1111/acel.70099

Figure Lengend Snippet: DLK/JNK3 pathway was upregulated in the senescent hair cell line model. (a, c) After HEI‐OC1 cell lines were treated with d ‐galactose, the proportion of galactosidase‐positive cells increased. (b, d) After HEI‐OC1 cells were treated with d ‐galactose, the mitochondrial membrane potential and ratio of green to red fluorescence intensity increased. (e–h) Western blotting showing that the expression of DLK, JNK3, and p‐JNK increased significantly after d ‐galactose treatment. (i–l) Immunofluorescence staining showing that the expression of DLK and p‐JNK increased after d ‐galactose treatment. Data are expressed as mean ± SD, n = 3. * p < 0.05, ** p < 0.01, **** p < 0.0001; mo, month. Scale bar, 20 μm.

Article Snippet: A Cell Senescence β‐galactosidase Staining Kit (Yeasen, 40754ES60) was used for staining.

Techniques: Membrane, Fluorescence, Western Blot, Expressing, Immunofluorescence, Staining

Journal: Aging Cell

Article Title: DLK / JNK3 Upregulation Aggravates Hair Cell Senescence in Mice Cochleae via Excessive Autophagy

doi: 10.1111/acel.70099

Figure Lengend Snippet: Hair cell senescence can be affected by DLK/JNK3 signal regulation. (a–c) Anisomycin further reduced the survival rate of senescent cells, while DLK inhibitor GNE3511 and JNK3 inhibitor J30‐8 increased the survival rate of senescent cells. (d) After senescent HEI‐OC1 cells were treated with GNE3511 or J30‐8, western blotting showed that the expression of DLK, p‐JNK, JNK3, p21, p53, and γ‐H2A.X decreased and that of LaminB1 increased. (e, f) After treating senescent HEI‐OC1 cells with anisomycin (E), the staining intensity of galactosidase increased, and the mitochondrial membrane potential decreased. Treatment with DLK inhibitor GNE3511 or JNK3 inhibitor J30‐8 (f) produced contrasting results. * indicates comparison with the control group, and # indicates comparison with the d ‐galactose‐treated group. Data are presented as mean ± SD, n = 3. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Scale, 50 μm.

Article Snippet: A Cell Senescence β‐galactosidase Staining Kit (Yeasen, 40754ES60) was used for staining.

Techniques: Western Blot, Expressing, Staining, Membrane, Produced, Comparison, Control

Journal: Aging Cell

Article Title: DLK / JNK3 Upregulation Aggravates Hair Cell Senescence in Mice Cochleae via Excessive Autophagy

doi: 10.1111/acel.70099

Figure Lengend Snippet: DLK/JNK alleviated hair cell senescence by inhibiting autophagy activation and activating the ubiquitin degradation process, and inhibition of DLK or JNK3 in vivo can alleviate ARHL in mice. (a) In d ‐galactose‐induced senescent HEI‐OC1 cells, the positively stained area of galactosidase decreased, and the mitochondrial membrane potential increased after 3‐MA treatment. Contrasting results were observed following rapamycin treatment. (b) Western blot showing that the expression of p53 and p21 decreased after adding 3‐MA to DLK‐overexpressing HEI‐OC1 cells, while that of p21 increased after adding rapamycin. (c) Quantitative and statistical analysis of the expression of each protein in the western blot in (b). (d) Western blot showing that the expression of ubiquitin decreased after DLK overexpression in HEI‐OC1 cells, increased after DLK inhibitor GNE3511 or JNK3 inhibitor J30‐8 treatment, and further decreased after anisomycin treatment. (e) In DLK‐overexpressing HEI‐OC1 cells, the positively stained area of galactosidase staining decreased, and the mitochondrial membrane potential increased after 3‐MA treatment. Contrasting results were observed following rapamycin treatment. (f) Schematic diagram of how DLK inhibitor GNE3511 was administered to C57BL/6J mice. (g) ABR threshold of the GNE3511‐treated group remained the same as that of the 6‐month‐old wild‐type group. (h) ABR threshold of the JNK3 inhibitor J30‐8 group ( n = 7) was significantly lower than that of the 9‐month‐old wild‐type group ( n = 5). (i) HE staining showed that the hair cells, spiral ganglion, and stria vascularis in the middle turn of the cochlea in the GNE3511 group were better preserved than those in the 9‐month‐old wild‐type group. (j, k) Number of OHC and IHC in per 100 × 100 pixels area was counted. (l–o) Immunohistochemical staining showing that the expression of ULK1 (l, n) and ATG5 (m, o) in the hair cells, spiral ganglion, and stria vascularis in the GNE3511‐treated group was significantly lower than that in the 9‐month‐old wild‐type group. * indicates comparison with the control group, and # indicates comparison with the DLK‐overexpressing group. Data are presented as mean ± SD, n = 3. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Scale, 50 μm.

Article Snippet: A Cell Senescence β‐galactosidase Staining Kit (Yeasen, 40754ES60) was used for staining.

Techniques: Activation Assay, Ubiquitin Proteomics, Inhibition, In Vivo, Staining, Membrane, Western Blot, Expressing, Over Expression, Immunohistochemical staining, Comparison, Control