Journal: Frontiers in Pharmacology

Article Title: The Mitochondrial Deubiquitinase USP30 Regulates AKT/mTOR Signaling

doi: 10.3389/fphar.2022.816551

Figure Lengend Snippet: PINK1/Parkin-dependent mitophagy induces apoptosis during mitochondrial stress (A) . Western blot analysis of mitophagy proteins and the pro-apoptosis signal in Hela (no Parkin) cells and Hela Parkin cells after the AO (Antimycin A+ oligomycin) treatment. Cells were treated with AO at 5 ug/ml for 0, 3, 6, and 9 h. Beta Actin served as the loading control. These are representative figures from three independent experiments (B) . Cell viability assay of Hela (no Parkin) cells and Hela Parkin cells after AO treatment. Cells were treated with AO at 5 ug/ml for 24 h and then incubated with resazurin for 2 h. Fluorescence was read using 544 nm excitation and 590 nm emission wavelength. It is a representative figure from three independent experiments (C) . Western blot analysis of mitophagy proteins and the pro-apoptosis signal in Hela Parkin cells and Hela Parkin with PINK1 KO cells after the AO treatment. Cells were treated with AO at 5 ug/ml for 0, 3, 6, and 9 h. Beta Actin served as the loading control (D) . Cell viability assay of Hela Parkin cells and Hela Parkin with USP30 overexpression cells after AO treatment with or without ST-539. Cells were treated with AO at 5 ug/ml w/o 10 ug/ml ST-539 for 24 h and then incubated with resazurin for 2 h. Fluorescence was read using544 nm excitation and 590 nm emission wavelength (E) . Western blot analysis of mitophagy proteins and the pro-apoptosis signal in Hela Parkin cells and Hela Parkin with USP30 overexpression cells after the AO treatment w/o ST-539. Cells were treated with AO at 5 ug/ml w/o 10 ug/ml ST-539 for 0, 3, and 6 h. Beta Actin served as the loading control (F) . Cell viability assay of Hela ATG5 knockout cells, Hela ATG5 knockout Parkin cells, and Hela ATG5 knockout Parkin USP30 cells after AO treatment. Cells were treated with AO at 5 ug/ml for 24 h and then incubated with resazurin for 2 h. Fluorescence was read using544 nm excitation and 590 nm emission wavelength.

Article Snippet: Primary antibodies used as described: USP30 (Sino Biological Inc., 14,548-RP01, 1:500); p -AKT (Cell Signaling Technology, 4060S, 1:1000); Parkin (Cell Signaling Technology, 4211S, 1:1000); AKT (Cell Signaling Technology, 4685S, 1:1000); Cleaved PARP (Cell Signaling Technology, 5625S, 1:1000); OPTN (Proteintech, 10837-I-AP, 1:1000); p -mTOR (Cell Signaling Technology, 5536S, 1:1000); mTOR (Cell Signaling Technology, 2983S, 1:1000); TOM20 (Cell Signaling Technology, 42406S, 1:1000); NDP52 (Cell Signaling Technology, 60732S, 1:1000); p-P70S6K (Cell Signaling Technology, 9234P, 1:1000); P70S6K (Cell Signaling Technology, 9202S, 1:1000); LC3A/B (Cell Signaling Technology, 4108S, 1:1000); Beta-Actin (Cell Signaling Technology, 3700S, 1:1000).

Techniques: Western Blot, Control, Viability Assay, Incubation, Fluorescence, Over Expression, Knock-Out

Journal: Frontiers in Pharmacology

Article Title: The Mitochondrial Deubiquitinase USP30 Regulates AKT/mTOR Signaling

doi: 10.3389/fphar.2022.816551

Figure Lengend Snippet: USP30 upregulates AKT/mTOR signal (A) . Western blot analysis of AKT/mTOR pathway proteins in Hela Parkin cells and Hela Parkin with USP30 overexpression cells after the AO treatment. Cells were treated with AO at 5 ug/ml for 0, 3, 6, and 9 h. Beta Actin served as the loading control. 2 (B) . Western blot analysis of AKT signal in Hela Parkin cells and Hela Parkin with USP30 overexpression cells after the AO treatment w/o ST-539. Cells were treated with AO at 5 ug/ml w/o ST-539 for 0, 3, and 6 h. Beta Actin served as the loading control. 2 (C) . Western blot analysis of AKT and cleaved PARP in Hela Parkin and Hela Parkin USP30 cells after the AO treatment w/o chloroquine. Cells were treated with AO at 5 ug/ml for 0, 3, and 6 h with DMSO or 10 uM chloroquine to inhibit autophagy/mitophagy. Beta Actin served as the loading control. 2 (D) . Western blot analysis of AKT and cleaved PARP in Hela ATG5 knockout Parkin and Hela ATG5 knockout Parkin USP30 cells after the AO treatment. Cells were treated with AO at 5 ug/ml for 0, 3, and 6 h. Beta Actin served as the loading control.

Article Snippet: Primary antibodies used as described: USP30 (Sino Biological Inc., 14,548-RP01, 1:500); p -AKT (Cell Signaling Technology, 4060S, 1:1000); Parkin (Cell Signaling Technology, 4211S, 1:1000); AKT (Cell Signaling Technology, 4685S, 1:1000); Cleaved PARP (Cell Signaling Technology, 5625S, 1:1000); OPTN (Proteintech, 10837-I-AP, 1:1000); p -mTOR (Cell Signaling Technology, 5536S, 1:1000); mTOR (Cell Signaling Technology, 2983S, 1:1000); TOM20 (Cell Signaling Technology, 42406S, 1:1000); NDP52 (Cell Signaling Technology, 60732S, 1:1000); p-P70S6K (Cell Signaling Technology, 9234P, 1:1000); P70S6K (Cell Signaling Technology, 9202S, 1:1000); LC3A/B (Cell Signaling Technology, 4108S, 1:1000); Beta-Actin (Cell Signaling Technology, 3700S, 1:1000).

Techniques: Western Blot, Over Expression, Control, Knock-Out

Journal: Frontiers in Pharmacology

Article Title: The Mitochondrial Deubiquitinase USP30 Regulates AKT/mTOR Signaling

doi: 10.3389/fphar.2022.816551

Figure Lengend Snippet: USP30 in cancer treatment. 3 (A) . Cell viability assay of Hela Parkin USP30 cells after AKT/mTOR inhibitors treatment w/o ST. Cells were treated with 10 uM MK2206 or 10uM Rapamycin or 1uM Torin1 for 48 h with DMSO or 10 ug/ml ST-539 and then incubated with resazurin for 2 h. Fluorescence was read using 544 nm excitation and 590 nm emission wavelength. 3 (B) . Cell viability assay on Jurkat T cells after 72 h MK2206 treatment with ST. Cells were treated with MK2206 and ST in the concentration gradient manner for 72 h. After the treatment, cells were incubated with resazurin for 2 h. Fluorescence was read using544 nm excitation and 590 nm emission wavelength. Each dot is the mean value of three biologically independent experiments. Trend lines are non-linear regression fitting curves. 3 (C) . Western blot analysis of AKT, mitophagy, and pro-apoptosis signal in Jurkat T cells treated with DMSO, MK2206, or ST. Cells were treated with DMSO, MK2206, ST, or MK220 + ST for 24 h. Beta Actin served as the loading control.

Article Snippet: Primary antibodies used as described: USP30 (Sino Biological Inc., 14,548-RP01, 1:500); p -AKT (Cell Signaling Technology, 4060S, 1:1000); Parkin (Cell Signaling Technology, 4211S, 1:1000); AKT (Cell Signaling Technology, 4685S, 1:1000); Cleaved PARP (Cell Signaling Technology, 5625S, 1:1000); OPTN (Proteintech, 10837-I-AP, 1:1000); p -mTOR (Cell Signaling Technology, 5536S, 1:1000); mTOR (Cell Signaling Technology, 2983S, 1:1000); TOM20 (Cell Signaling Technology, 42406S, 1:1000); NDP52 (Cell Signaling Technology, 60732S, 1:1000); p-P70S6K (Cell Signaling Technology, 9234P, 1:1000); P70S6K (Cell Signaling Technology, 9202S, 1:1000); LC3A/B (Cell Signaling Technology, 4108S, 1:1000); Beta-Actin (Cell Signaling Technology, 3700S, 1:1000).

Techniques: Viability Assay, Incubation, Fluorescence, Concentration Assay, Western Blot, Control

Journal: International journal of biological sciences

Article Title: Radiosensitizer EXO-miR-197-3p Inhibits Nasopharyngeal Carcinoma Progression and Radioresistance by Regulating the AKT/mTOR Axis and HSPA5-mediated Autophagy.

doi: 10.7150/ijbs.69934

Figure Lengend Snippet: Figure 6. MiR-197-3p downregulating HSPA5 modulates autophagy to control X-ray sensitivity. (A) The expression of autophagy related proteins in transfected cells was detected by Western Blot. (B, C) The changes of autophagosomes after transfection with GFP-LC3 were detected. (D) Western Blot experiments verified that miR-197-3p regulated autophagy by targeting HSPA5. (E) The expression of HSPA5 and autophagy related protein LC3B in transfected cells was detected by cell immunofluorescence. (F) Immunofluorescence showed that tumors infected with LV-miR-197-3p expressed HSPA5 and LC3B in animal tissue. (G) Western Blot experiment showed the expression of LC3BI/LC3BII and HSPA5 in animal tumor protein. (H) Immunohistochemical results showed the expression of HSPA5, Ki-67, BCL2, LC3B and P62.

Article Snippet: The used antibodies are as follows: GAPDH (Transgen, HC301-01), HSPA5 (Proteintect, 11587-1-AP), LC3B (Abcam, ab192890), P62 (BOSTER, M00300-1), Phospho-mTOR (Ser2448) (D9C2) (Cell Signaling Technology, 5536T), mTOR (7C10) (Cell Signaling Technology, 2983T), PhosphoAkt (Ser473) (D9E) (Cell Signaling Technology, 4060S), Akt (pan) (C67E7) (Cell Signaling Technology, 4691S), Exosome Panel (Calnexin, CD9, CD63, CD81, Hsp70, TSG101) (ab275018), Goat Anti-mouse IgG (Transgen, HS201-01), Goat anti-rabbit IgG (Transgen, HS101-01).

Techniques: Control, Expressing, Transfection, Western Blot, Immunofluorescence, Infection, Immunohistochemical staining

Journal: International journal of biological sciences

Article Title: Radiosensitizer EXO-miR-197-3p Inhibits Nasopharyngeal Carcinoma Progression and Radioresistance by Regulating the AKT/mTOR Axis and HSPA5-mediated Autophagy.

doi: 10.7150/ijbs.69934

Figure Lengend Snippet: Figure 8. EXO-miR-197-3p regulates radioresistance and autophagy by targeting HSPA5. (A, B) Colony formation assay was detected the radioresistance of the cells ingested EXO-miR-197-3p. (C, D, E, F) Flow cytometry was detected apoptosis after uptake of exosomes at 4GY. (G) Western Blot was detected the expression of HSPA5 after cell uptake of EXO-miR-197-3p. (H) Animal experiments tested the effect of EXO-miR-197-3p. (I, J) Tumor volume and weight were measured. (K) Immunohistochemical results showed that after EXOs-miR-197-3p was injected; the expressions of HSPA5, Ki-67, BCL2, LC3B and P62 were analyzed. (L) Immunohistofluorescence showed that the expression of HSPA5 and LC3B; Immunohistofluorescence showed that the expression of HSPA5 and LC3B was decreased in the EXO group. (M) Western Blot assay detected the expression of LC3B and HSPA5 in tumor. (N) Western Blot detected the expression of HSPA5 after cell uptake of EXO-miR-197-3p. (O, P) After transfection GFP-LC3, autophagosome formation was detected after EXO-HK-1 ingestion. (Q) The expressions of HSPA5 and autophagy related protein LC3B in cell uptake of EXO-HK-1 was detected by cell immunofluorescence. (R) The correlation between miR-197-3p, HSPA5 and LC3B was detected by Western Blot.)

Article Snippet: The used antibodies are as follows: GAPDH (Transgen, HC301-01), HSPA5 (Proteintect, 11587-1-AP), LC3B (Abcam, ab192890), P62 (BOSTER, M00300-1), Phospho-mTOR (Ser2448) (D9C2) (Cell Signaling Technology, 5536T), mTOR (7C10) (Cell Signaling Technology, 2983T), PhosphoAkt (Ser473) (D9E) (Cell Signaling Technology, 4060S), Akt (pan) (C67E7) (Cell Signaling Technology, 4691S), Exosome Panel (Calnexin, CD9, CD63, CD81, Hsp70, TSG101) (ab275018), Goat Anti-mouse IgG (Transgen, HS201-01), Goat anti-rabbit IgG (Transgen, HS101-01).

Techniques: Colony Assay, Flow Cytometry, Western Blot, Expressing, Immunohistochemical staining, Injection, Immunohistofluorescence, Transfection, Immunofluorescence

Journal: Endocrinology

Article Title: Deficiency of C1QL1 reduced murine ovarian follicle reserve through intraovarian and endocrine control.

doi: 10.1210/endocr/bqac048

Figure Lengend Snippet: Figure 5. Deficiency of C1QL1 promoted apoptosis and inhibited autophagy in the ovary. (A) Representative images of TUNEL staining of ovarian sections. The green signals indicate representative cells with positive TUNEL staining, which was mainly detected in the large atretic follicles. (B) The intensity of TUNEL-positive signals was shown in charts (n = 4 per group). (C) Representative images of immunofluorescence staining of ovarian sections with LC3B antibody. LC3B-positive signals were found in granulosa cells and oocytes and were weakened in C1QL1-deficient ovaries. (D) The intensity of LC3B-positive signals are shown (n = 4 per group). (E) Apoptosis- and autophagy-related proteins in the ovaries were analyzed by Western blotting. Representative blots are shown in the left panel, and the amount of protein normalized to β-tubulin, which is presented as mean ± SEM, is shown in the right panel. n = 6 per group. *P < 0.05, **P < 0.01, ***P < 0.001. NS, no significance.

Article Snippet: The sections and granulosa cells were stained with anti-rabbit LC3B antibody and Alexa Fluor 594-conjugated secondary antibody and mounted with DAPI (Boster).

Techniques: TUNEL Assay, Staining, Immunofluorescence, Western Blot