Journal: Journal of Translational Medicine

Article Title: Didymin alleviates metabolic dysfunction-associated fatty liver disease (MAFLD) via the stimulation of Sirt1-mediated lipophagy and mitochondrial biogenesis

doi: 10.1186/s12967-023-04790-4

Figure Lengend Snippet: Antibodies

Article Snippet: ATG5 , Rabbit , Proteintech , 10181-2-ap , 1:1000.

Techniques:

Journal: Journal of Translational Medicine

Article Title: Didymin alleviates metabolic dysfunction-associated fatty liver disease (MAFLD) via the stimulation of Sirt1-mediated lipophagy and mitochondrial biogenesis

doi: 10.1186/s12967-023-04790-4

Figure Lengend Snippet: Didymin enhances lipophagy by activating Sirt1 in PA-treated AML12 cells. A AML12 cells transfected with mCherry-eGFP-PLIN2 plasmid. (Scale bar = 30 μm). B Immunoprecipitation of FoxO3a in AML12 cells showing acetylated FoxO3a level (n = 3). C Western blot analysis of LC3, Beclin1, P62, PLIN2, LAMP1 and ATG5 in AML12 cells (n = 3). Data are expressed as mean ± SD. *P < 0.05, **P < 0.01 PA vs. PA + Didymin. ## P < 0.01, ### P < 0.001, #### P < 0.0001 control vs. PA. PA palmitic acid, FoxO3a Foxkhead Box Class O 3a, PLIN2 perilipin 2, LAMP1 lysosomal associated membrane protein 1

Article Snippet: ATG5 , Rabbit , Proteintech , 10181-2-ap , 1:1000.

Techniques: Transfection, Plasmid Preparation, Immunoprecipitation, Western Blot, Control, Membrane

Journal: Journal of Translational Medicine

Article Title: Didymin alleviates metabolic dysfunction-associated fatty liver disease (MAFLD) via the stimulation of Sirt1-mediated lipophagy and mitochondrial biogenesis

doi: 10.1186/s12967-023-04790-4

Figure Lengend Snippet: Didymin enhances mitochondrial biogenesis and function, promotes lipophagy, and inhibits apoptosis by activating Sirt1 in mice hepatocytes. A Western blot analysis of Sirt1 expression in hepatocytes (n = 3). B Western blot analysis of NRF1, TFAM, NDUFB8, SDHB, and MTCO2 in hepatocytes. C Transmission electron microscopy figures of hepatocytes, arrows show mitochondria (Scale bar = 0.6 μm). D Transmission electron microscopy figures of hepatocytes, arrows show autolysosomes (Scale bar = 0.6 μm). E Western blot analysis of LC3, Beclin1, P62, PLIN2, LAMP1, and ATG5 in hepatocytes (n = 3). F Western blot analysis of Cleaved PARP, Bax, Bcl2, Cleaved Caspase 3, and Caspase 3 proteins in hepatocytes (n = 3). G Immunofluorescence staining of γ-H2AX in hepatocytes (Scale bar = 50 μm). NRF1 nuclear respiratory factor 1, TFAM mitochondrial transcription factor A, PLIN2 perilipin 2, LAMP1 lysosomal associated membrane protein 1, Bax Bcl-2-associated X protein, Bcl2 B-cell lymphoma 2

Article Snippet: ATG5 , Rabbit , Proteintech , 10181-2-ap , 1:1000.

Techniques: Western Blot, Expressing, Transmission Assay, Electron Microscopy, Immunofluorescence, Staining, Membrane

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