Journal: Advanced Science

Article Title: RONIN/HCF1‐TFEB Axis Protects Against D‐Galactose‐Induced Cochlear Hair Cell Senescence Through Autophagy Activation

doi: 10.1002/advs.202407880

Figure Lengend Snippet: HEI‐OC1 cells were subjected to senescence induction by treatment with D‐gal. A) HEI‐OC1 cells following treatment with D‐gal. B) The results of the CCK‐8 assay demonstrate the survival of cells following treatment with different doses of D‐gal for a duration of 72 h, n = 6. Error bars are ± S. D., ns: no significant difference, *** p < 0.001 and **** p < 0.0001. C,D) Western blot analysis to identify senescence markers post 72 h treatment with different D‐gal concentrations in HEI‐OC1 cells. E–H) Quantitative examination of the western blot data, n = 6 for E, n = 4 for F, n = 3 for G, n = 4 for H. Error bars are ± S.D., * p < 0.05, ** p < 0.01 and *** p < 0.001. I) Immunofluorescence staining depicting the distribution of γ‐H2A.X foci in the nuclei (stained with DAPI) of HEI‐OC1 cells treated with D‐gal for 72 h, Scale bar: 5 µm. J) Quantitative analysis of γ‐H2A.X foci in the nuclei, n = 6. Error bars are ± S.D., ** p < 0.01, *** p < 0.001, and **** p < 0.0001.

Article Snippet: After HEI‐OC1 cells were treated with 15 mg mL −1 D‐gal, SA‐β‐gal staining was performed according to the protocol specified in the Cellular Senescence Detection Kit (SPiDER‐βGal, DOJINDO, SG03).

Techniques: CCK-8 Assay, Western Blot, Immunofluorescence, Staining

Journal: Advanced Science

Article Title: RONIN/HCF1‐TFEB Axis Protects Against D‐Galactose‐Induced Cochlear Hair Cell Senescence Through Autophagy Activation

doi: 10.1002/advs.202407880

Figure Lengend Snippet: Senescence in cochlear explants treated with D‐gal and aging mice. A) Cochleae cultured in vitro and treated with D‐gal. B) Immunofluorescence staining for Myosin7a, demonstrating cochlear HC loss after 72 h of D‐gal treatment (20 and 40 mg mL −1 ), Scale bar: 20 µm. C,D) Measurement of cell numbers expressing Myosin7a in the middle and basal turns of cochlea, n = 4. Error bars are ± S.D., ns: no significant difference, *** p < 0.001 and **** p < 0.0001. E–G) Western blot results showing the expression of senescence‐related markers (γ‐H2A.X, SMP30, and P21) in cochleae following 72 h of treatment with D‐gal. H‐J) Quantitative analysis of western blot data, n = 4 for H, n=6 for I, n=4 for J. Error bars are ± S.D., * p < 0.05, ** p < 0.01, *** p < 0.001. K) ABR threshold in 1 M (1 month) and 12 M (12 months) C57BL/6J mice. n = 3. Error bars are ± S.D., *** p < 0.001. L,M) The results of western blots show the expression of senescence‐related markers in cochleae of aged mice. N,O) Quantification of SMP30 and LaminB1 protein levels using western blot, n = 3. Error bars are ± S.D., ** p < 0.01.

Article Snippet: After HEI‐OC1 cells were treated with 15 mg mL −1 D‐gal, SA‐β‐gal staining was performed according to the protocol specified in the Cellular Senescence Detection Kit (SPiDER‐βGal, DOJINDO, SG03).

Techniques: Cell Culture, In Vitro, Immunofluorescence, Staining, Expressing, Western Blot

Journal: Advanced Science

Article Title: RONIN/HCF1‐TFEB Axis Protects Against D‐Galactose‐Induced Cochlear Hair Cell Senescence Through Autophagy Activation

doi: 10.1002/advs.202407880

Figure Lengend Snippet: Activation of autophagy by Ronin overexpression rescues cellular senescence. A–C) Western blot examination shows alterations in the protein levels of P16, γ‐H2AX, and P21 in HEI‐OC1 cells treated with 15 mg mL −1 for 72 h D‐gal after transfection with Ronin ‐GFP plasmids. D–F) Densitometric quantification of the western blot bands for protein as indicated in (A–C), n = 3. Error bars are ± S.D., * p < 0.05 and *** p < 0.001. G–I) Western blots illustrating alterations in the levels of autophagy markers LC3B‐II, CTSB, and CTSD in HEI‐OC1 cells treated with 15 mg mL −1 D‐gal post‐transfection with Ronin ‐GFP. J–L) Quantification of LC3B‐II, CTSB, and CTSD expression, n = 4 for J, n = 5 for K, n = 3 for L. Error bars are ± S.D., * p < 0.05, ** p < 0.01. M) Images of mCherry‐GFP‐LC3B punctae in HEI‐OC1 cells. Cells were transfected with pcsLenti‐CMV‐mCherry‐GFP‐LC3B (Lentivirus) and Ronin ‐HA plasmids, followed by exposure to 15 mg mL −1 D‐gal for 72 h with or without 100 nM bafliomycinA1 (BafA1) for 6 h. Autophagosomes and autolysosomes are represented by yellow and red dots, respectively. N) Quantification of mCherry‐GFP‐LC3B yellow and red punctae in M, n = 24. Error bars are ± S.D., * p < 0.05 and **** p < 0.0001.

Article Snippet: After HEI‐OC1 cells were treated with 15 mg mL −1 D‐gal, SA‐β‐gal staining was performed according to the protocol specified in the Cellular Senescence Detection Kit (SPiDER‐βGal, DOJINDO, SG03).

Techniques: Activation Assay, Over Expression, Western Blot, Transfection, Expressing

Journal: Molecular and cellular endocrinology

Article Title: Role of cAMP/PKA pathway and T-type calcium channels in the mechanism of action of serotonin in human adrenocortical cells

doi: 10.1016/j.mce.2016.10.008

Figure Lengend Snippet: Coupling of 5-HT receptors to T-type Ca2+ currents in human cultured adrenocortical cells. (A) Superimposed Ca2+ currents elicited by increasing depolarizing pulses (from −60 mV to −30 mV) from a holding potential of −80 mV (upper panel). Current-voltage relationships of the peak Ca2+ currents (lower panel). (B) Superimposed Ca2+ currents elicited by depolarizing pulses from −80 mV to −50 mV and −40 mV recorded in the absence (control) or presence of 5-HT (10−5 M). (C) Effects of graded concentrations of 5-HT on cortisol secretion by adrenocortical cells cultured in the absence or presence of the T-type Ca2+ channel blockers mibefradil (10−6 M; ■) and NiCl2 (8 × 10−5 M; ●). (D) Effects of the L-type channel blocker nifedipine (10−5 M), N-type channel blocker ω-conotoxin (10−6 M) and P-type channel blocker sFTX-3.3 (10−6 M) on basal and 5-HT-induced cortisol production. BL, Basal level. *, p < 0.05; ***, p < 0.001 versus basal production in the absence of test substance. ###, p < 0.001 versus production in the presence of 5-HT.

Article Snippet: Serotonin, dibutyryladenosine 3′,5′-cyclic monophosphate (dbcAMP), 1-methyl-3-isobutylxanthine (IBMX), chelerythrine, H-89, GR113808, zacopride, EGTA, nifedipine, mibefradil, NiCl 2 were purchased from Sigma-Aldrich (Saint-Quentin Fallavier, France). ω-conotoxin and sFTX-3.3 were obtained from Alomone Labs (Jerusalem, Israel).

Techniques: Cell Culture