Journal: Autophagy

Article Title: TRIM44 links the UPS to SQSTM1/p62-dependent aggrephagy and removing misfolded proteins

doi: 10.1080/15548627.2021.1956105

Figure Lengend Snippet: TRIM44 promtoes aggregates deaggregation and clearance via autophagy. (a) Cells were treated with MG132 and immunostained with antibodies to ubiquitin (red) and LC3B-II (green). Arrows indicate ubiquitin-positive aggregates that colocalize with LC3B-positive autophagosomes. Scale bars: 10 μm. (b) Confocal images of TRIM44[OE-CON] and TRIM44[OE] U266 cells after treatment with MG132 (0.5 µM) for 16 h followed by a 24-h chase period in normal culture media with DMSO (vehicle), or 3-MA. Arrows indicate cells with remaining aggregates. The relative level of remaining aggregates is determinded by quantifying the percentage of cells with remaining aggregates after a 24-h chase period in normal culture media with DMSO (vehicle), or 3-MA (10 mM) and normalized to the percentage of cells with aggregates formed by the 16 h MG132 (0.5 µM) treatment in corresponding cells. *, P < 0.05; **, P < 0.01; ***, P < 0.001. Scale bars: 10 μm. (c) Confocal images of TRIM44[KD-CON] and TRIM44[KD] U266 cells after treatment with MG132 (0.5 µM) for 16 h followed by a 24-h chase period in normal culture media with DMSO (vehicle), or PP242 (10 nM). Arrows indicate cells with remaining aggregates. Scale bars: 10 µm. The relative level of remaining aggregates is determinded by quantifying the percentage of cells with remaining aggregates after a 24-h chase period in normal culture media with DMSO (vehicle), or PP242 (10 nM) and normalized to the percentage of cells with aggregates formed by the 16 h MG132 (0.5 µM) treatment in corresponding cells. *, P < 0.05; **, P < 0.01; ***, P < 0.001. Scale bars: 10 μm. (d, e) Confocal images of WT or ATG5 KO TRIM44[OE-CON] and TRIM44[OE] U266 cells transfected with NC or ATG5 siRNA after treatment with MG132 (0.5 µM) for 16 h. Aggregates (marked by arrows) were identified by staining with the antibody against ubiquitin (d). Scale bars: 10 µm. The status of aggregates after MG132 treatment was quantified in the histogram. *, P < 0.05; **, P < 0.01; ***, P < 0.001. Scale bars: 10 μm. The protein level of ATG5 and TRIM44 were assayed by western blots (e). (f, g) Confocal images of WT or BECN1 KO TRIM44[OE-CON] and TRIM44[OE] U266 cells were treated with MG132 (0.5 µM) for 16 h. Aggregates (marked by arrows) were identified by staining with the antibody against ubiquitin (f). Scale bars: 10 µm. The status of aggregates after MG132 treatment was quantified in the histogram. *, P < 0.05; **, P < 0.01; ***, P < 0.001. Scale bars: 10 μm. The protein level of BECN1 and TRIM44 were assayed by western blot (g). (h, i) Confocal images of RPMI-TRIM44[Tet-on] cells treated with or without DOX (1 µg/mL) transfected with NC or ATG5 siRNA after treatment with MG132 (0.5 µM) for 16 h. The status of aggregates after MG132 treatment was quantified in the histogram. Scale bars: 10 µm. The status of aggregates after MG132 treatment was quantified in the histogram. *, P < 0.05; **, P < 0.01; ***, P < 0.001. Scale bars: 10 μm. The protein level of ATG5 and TRIM44 were assayed by western blots (h). (j) Confocal images of RPMI-TRIM44[Tet-on] cells treated with or without DOX (1 µg/mL) together with MG132 (5 µM) for 16 h followed by a 24-h chase period in normal culture media with DMSO (vehicle), or 3-MA. Arrows indicate cells with remaining aggregates. The relative level of remaining aggregates is determinded by quantifying the percentage of cells with remaining aggregates after a 24-h chase period in normal culture media with DMSO (vehicle), or 3-MA and normalized to the percentage of cells with aggregates formed by the 16 h MG132 (5 µM) treatment in corresponding cells. *, P < 0.05; **, P < 0.01; ***, P < 0.001. Scale bars: 10 μm.

Article Snippet: Anti-TRIM44 polyclonal antibody (Proteintech Group, 11,511-1-AP); anti-Ub antibody (Biolegend, 646,301); anti-mCherry antibody (ThermoFisher, PA5-34,974); anti-VIM antibody (ThermoFisher, MA5-11,883); anti-NFE2L2/NRF2 antibody (ThermoFisher, PA5-27,882); anti-20S proteasome antibody (MilliporeSigma, ST1049); anti-TUBG/γ-Tubulin antibody (MilliporeSigma, T5326); anti-ATG5 antibody (Novus Biologicals, NBP2-24,389); Anti-GFP antibody (Santa Cruz Biotechnology, sc-9996); anti-ACTB/β-Actin antibody (Santa Cruz Biotechnology, sc-47,778), anti-HA antibody (Santa Cruz Biotechnology, sc-805); anti-BECN1 antibody (Cell Signaling Technology, 4122); anti-HDAC6 antibody (Cell Signaling Technology, 7558); anti-LC3B antibody (Cell Signaling Technology, 3868); anti-SQSTM1/p62 antibody (Cell Signaling Technology, 88,588); anti-cleaved PARP antibody (Cell Signaling Technology, 5625); Alexa Fluor 594 donkey anti rabbit IgG(H + L) (Invitrogen, A21207); Alexa Fluor 546 goat anti mouse IgG(H + L) (Invitrogen, A11003).

Techniques: Ubiquitin Proteomics, Transfection, Staining, Western Blot

Journal: Autophagy

Article Title: Autophagy is differentially induced in prostate cancer LNCaP, DU145 and PC-3 cells via distinct splicing profiles of ATG5

doi: 10.4161/auto.22397

Figure Lengend Snippet: Figure 6. The inhibitory effect of VPA on cell proliferation was not changed by ATG5 rescue in DU145 cells or ATG5 knockdown in LNCaP and PC-3 cells ( A ) Valproic acid (VPA) dose-dependently inhibited the proliferation of LNCaP, DU145 and PC-3 cells. Logarithmic phase cells were incubated with indicated concentrations of VPA for 48 h. ( B ) DU145 cells were transfected with a plasmid containing the wild-type ATG5 gene and then treated with indicated concentrations of VPA for 48 h. ( C and D ) LNCaP and PC-3 cells were transfected with ATG5 siRNA or negative control (NC) siRNA for 48 h, and then treated with indicated concentrations of VPA for another 48 h. Cell proliferation was evaluated using MTS assay ( A–D ). One representative data of three independent experiments with similar results are shown as mean ± SD. The efficient knockdown of ATG5 protein in LNCaP and PC-3 cells was confirmed by western blotting ( C and D , lower panels; VPA = 3.3 mM). The relative levels of ATG5 were normalized to TUBB and the value of NC siRNA-treated control of each group was set as 1.00, respectively.

Article Snippet: ATG5 siRNA I (6345) and negative control siRNA (6568) were also obtained from Cell Signaling Technology.

Techniques: Knockdown, Incubation, Transfection, Plasmid Preparation, Negative Control, MTS Assay, Western Blot, Control

Journal: International Journal of Biological Sciences

Article Title: FOXO1-mediated nuclear sequestration of STAT3 and AKT1 triggers FOXO3-dependent autophagic death in hypoxic granulosa cells

doi: 10.7150/ijbs.101309

Figure Lengend Snippet: FOXO3 acts through ATG3 to promote autophagy and autophagy-dependent apoptosis upon hypoxia exposure. (A) GCs were grown with 1% O 2 for 0, 2, 4, 6, or 12 h, and western blot was performed to determine the levels of autophagy-related proteins, including ATG3, ATG5, Beclin1, and ATG7. (B) GCs treated with FOXO3-siRNA were cultured under normoxia (21% O 2 ) or hypoxia (1% O 2 ) for 12 h, followed by immunoblotting analysis of the expression levels of ATG3, ATG5, Beclin1, and ATG7. (C and D) ChIP assay utilizing the FOXO3 antibody was performed to assess the interaction of FOXO3 with the ATG3 promoter in GCs subjected to either normoxic (21% O 2 ) or hypoxic (1% O 2 ) conditions for 12 h. DNA recovered from the complexes provided the template for subsequent qRT-PCR assays. The amplified qRT-PCR fragments were evaluated using a 2% agarose gel (C) and measured for density with ImageJ software (D) . (E) For 12 h, GCs were exposed to conditions of standard (21% O 2 ) or reduced (1% O 2 ) oxygen, after which the expression levels of ATG3 mRNA were evaluated using qRT-PCR. (F) GCs transfected with FOXO3 -siRNA were incubated under either normoxic (21% O 2 ) or hypoxic (1% O 2 ) conditions for 12 h, after which ATG3 mRNA expression was measured via qRT-PCR analysis. (G) After transfection with ATG3 -siRNA for 24 h, GCs were collected to determine ATG3 levels by western blot. (H-N) GCs were transfected with either ATG3 -siRNA or a non-targeting siRNA control for 12 h, followed by culturing for another 12 or 24 h under conditions of either normoxia (21% O 2 ) or hypoxia (1% O 2 ). Following another 12-h incubation period, the cells were collected to evaluate the protein expression of SQSTM1, MAP1LC3B (H) , and the activated form of caspase-3 (K) by western blot. The data were quantified using densitometry with ImageJ 1.42q software (I, J, and L) , the apoptosis rate was evaluated using TUNEL staining (M) , and the percentage of TUNEL-positive cells was quantified (N) .

Article Snippet: Antibodies against FOXO1 (2880), FOXO3 (2497), p-FOXO3 (Ser253; 9466), cleaved caspase-3 (9664), 14-3-3 (8312), STAT3 (9139), p-STAT3 (Tyr705; 9145), AKT1 (9272), p-JNK1/2 (Thr183/Tyr185) (4668), JNK1/2 (9252), ATG3 (3415), ATG5 (9980), Beclin1 (3738), ATG7 (8558), SQSTM1 (88588), α-tubulin (2125), MAP1LC3B (3868), and FLAG-tag (14793) were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Western Blot, Cell Culture, Expressing, Quantitative RT-PCR, Amplification, Agarose Gel Electrophoresis, Software, Transfection, Incubation, Control, TUNEL Assay, Staining