Journal: bioRxiv

Article Title: mTORC1-Plin3 pathway is essential to activate lipophagy and protects against hepatosteatosis

doi: 10.1101/812990

Figure Lengend Snippet: A) Depiction of the methodological approach: Oleic acid (OA) treatment induces lipid droplet (LD) formation. Lipophagy is analysed by immunofluorescence and western blotting in homogenates and isolated LDs. B) Autophagy pathway proteins and perilipins expression in total homogenates and LD isolations at 6 hours of OA treatment with or without lysosomal inhibitors (Lys inh) to test autophagy flux. LC3 flux in total homogenates shows active autophagy, supporting selective degradation of LDs. All autophagy proteins tested accumulate in the LD isolations after lysosomal inhibitors treatment. Plin3 is selectively degraded by autophagy, shown both in homogenates and isolated LDs. C) Total homogenates and LDs were subjected to Phos-tag gel electrophoresis and immunoblotted for Plin3. Phosphorylated Plin3 (p-Plin3: black arrowhead) is mainly found in LDs. Phosphorylation is increased after blocking autophagy with lysosomal inhibitors. D-G) LC3 and Lamp1 (magenta) recruitment to LDs (green) is enhanced after blocking autophagy in the OA-treated primary hepatocytes (D and F, quantified in E and G). H, I) Plin3 (magenta) recruitment to LDs (green) is also enhanced after blocking autophagy (H, quantified in I). J, K) Plin3 recruitment is also enhanced after blocking autophagy using Atg7 knockdown in OA-treated NIH-3T3 cells. Scale bar: 20μm. Bars are mean ± SEM. *p < 0.05, **p < 0.01, and ***p < 0.001 (differences caused by lysosomal inhibitors treatment), # p<0.05, ## p<0.01, and ### p<0.001 (differences caused by treatment).

Article Snippet: Antibodies for Atg16l (PM040) from MBL; Atg7 (2631), Beclin 1 (3495), FIP200 (12436), LC3B (2775), mTOR (2983), phopho-mTOR (5536), Rag A (4357), Rag C (3360), Raptor (2280), Rictor (2114), S6 (2217), phopho-S6 (4858), TSC1 (6935) and anti-rabbit (7074) were from Cell Signaling Technology; RagB (NBP1-85801) from Novus Biologicals; Plin3 (3883,for WB) from ProSci; LAMP1 (1D4B, for WB) from Developmental Studies Hybridoma Bank; PLIN3 (GP30 for CoIP) from ProGen Biotechnik; β-actin (A5441) and anti-mouse (AP130P) and anti-rat (AP136P) from SIGMA; GAPDH (ab8245), Lamp1 (ab24170 for IF) and normal IgG (ab188776) from Abcam; Fluorescence secondary antibodies (Alexa Fluor 488 and/or Alexa Fluor 647 conjugated) and anti-guinea pig (A18775) from Fisher Scientific and Beclin 1-HRP(sc-48341 HRP) and Plin 3-HRP (sc-390968 HRP) form Santa Cruz Biotechnology.

Techniques: Immunofluorescence, Western Blot, Isolation, Expressing, Nucleic Acid Electrophoresis, Blocking Assay

Journal: bioRxiv

Article Title: mTORC1-Plin3 pathway is essential to activate lipophagy and protects against hepatosteatosis

doi: 10.1101/812990

Figure Lengend Snippet: A-D) Levels of Plin3 and autophagy pathway proteins in total homogenates from control and Plin3 silenced cells with and without OA and with and without treatment with lysosomal inhibitors. There are no differences in the expression of autophagy proteins between control and silenced cells (quantified in C (protein expression) and D (protein flux). E-H) LC3 and Lamp1 (magenta) recruitment to LDs (green) is reduced after silencing Plin3 in NIH-3T3 fibroblasts treated with OA (showed in C and E, quantified in D and F) indicating impairment of lipophagy in siPlin3 cells. I) Binding between Plin3 and the autophagy initiator proteins Fip200 and ATG16l assessed by the co-immunoprecipitation with endogenous Plin3 is increased after OA treatment. J) OA treatment increases the binding between Fip200 and Beclin, this binding is reduced after silencing Plin3. Scale bar: 20μm. Bars are mean ± SEM. *p < 0.05, **p < 0.01, and ***p < 0.001 (differences caused by lysosomal inhibitors treatment), # p<0.05, ## p<0.01, and ### p<0.001 (differences caused by treatment).

Article Snippet: Antibodies for Atg16l (PM040) from MBL; Atg7 (2631), Beclin 1 (3495), FIP200 (12436), LC3B (2775), mTOR (2983), phopho-mTOR (5536), Rag A (4357), Rag C (3360), Raptor (2280), Rictor (2114), S6 (2217), phopho-S6 (4858), TSC1 (6935) and anti-rabbit (7074) were from Cell Signaling Technology; RagB (NBP1-85801) from Novus Biologicals; Plin3 (3883,for WB) from ProSci; LAMP1 (1D4B, for WB) from Developmental Studies Hybridoma Bank; PLIN3 (GP30 for CoIP) from ProGen Biotechnik; β-actin (A5441) and anti-mouse (AP130P) and anti-rat (AP136P) from SIGMA; GAPDH (ab8245), Lamp1 (ab24170 for IF) and normal IgG (ab188776) from Abcam; Fluorescence secondary antibodies (Alexa Fluor 488 and/or Alexa Fluor 647 conjugated) and anti-guinea pig (A18775) from Fisher Scientific and Beclin 1-HRP(sc-48341 HRP) and Plin 3-HRP (sc-390968 HRP) form Santa Cruz Biotechnology.

Techniques: Expressing, Binding Assay, Immunoprecipitation

Journal: bioRxiv

Article Title: mTORC1-Plin3 pathway is essential to activate lipophagy and protects against hepatosteatosis

doi: 10.1101/812990

Figure Lengend Snippet: A) Depiction of the methodological approach: 1h rapamycin treatment before the induction of LD formation by OA treatment. Lipophagy is tested as previously described. B) Autophagy and mTORC1 pathway protein levels in total homogenates and LD isolations at 6 hours of OA treatment with and without rapamycin pretreatment. Note that levels of autophagy proteins are decreased after rapamycin treatment; active mTOR and Rheb are also reduced when mTORC1 activity is inhibited. C-H) LC3 and Lamp1 (magenta) recruitment to LDs (green) is impaired after blocking mTORC1 activity (C and E, quantified in D and F). However, Plin3 (magenta) recruitment to LDs (green) is enhanced after rapamycin pretreatment in OA-treated primary hepatocytes (G, quantified in H). I) Immunoprecipitation of endogenous Plin3 from LD isolations indicates its reduced binding to Fip200 and ATG16l after rapamycin treatment. J) Plin3 phosphorylation in LDs is diminished after rapamycin treatment. Scale bar: 20μm. Bars are mean ± SEM. *p < 0.05, **p < 0.01, and ***p < 0.001 (differences caused by lysosomal inhibitors treatment), # p<0.05, ## p<0.01, and ### p<0.001 (differences caused by treatment)

Article Snippet: Antibodies for Atg16l (PM040) from MBL; Atg7 (2631), Beclin 1 (3495), FIP200 (12436), LC3B (2775), mTOR (2983), phopho-mTOR (5536), Rag A (4357), Rag C (3360), Raptor (2280), Rictor (2114), S6 (2217), phopho-S6 (4858), TSC1 (6935) and anti-rabbit (7074) were from Cell Signaling Technology; RagB (NBP1-85801) from Novus Biologicals; Plin3 (3883,for WB) from ProSci; LAMP1 (1D4B, for WB) from Developmental Studies Hybridoma Bank; PLIN3 (GP30 for CoIP) from ProGen Biotechnik; β-actin (A5441) and anti-mouse (AP130P) and anti-rat (AP136P) from SIGMA; GAPDH (ab8245), Lamp1 (ab24170 for IF) and normal IgG (ab188776) from Abcam; Fluorescence secondary antibodies (Alexa Fluor 488 and/or Alexa Fluor 647 conjugated) and anti-guinea pig (A18775) from Fisher Scientific and Beclin 1-HRP(sc-48341 HRP) and Plin 3-HRP (sc-390968 HRP) form Santa Cruz Biotechnology.

Techniques: Activity Assay, Blocking Assay, Immunoprecipitation, Binding Assay

Journal: bioRxiv

Article Title: mTORC1-Plin3 pathway is essential to activate lipophagy and protects against hepatosteatosis

doi: 10.1101/812990

Figure Lengend Snippet: A) Schematic diagram of the metabolic function of lipophagy. Fatty acids (FAs) produced by lysosomes are used by mitochondria to produce ATP. Etomoxir inhibits the CPT1a, which transports FAs to the mitochondria. B) Mitochondrial respiration in primary hepatocytes under basal condition and after 6 hours of OA treatment with and without etomoxir (100nM), rapamycin or lysosomal inhibitors pretreatment. OA treatment increases mitochondrial respiration in hepatocytes in basal state, which is prevented in the presence of mitochondrial FA transport (etomoxir), autophagy (Lys inh) or mTORC1 (rapamycin) inhibitors. C and D) Maximal respiration and ATP production are increased in hepatocytes in response to OA treatment, which is suppressed by etomoxir, lysosomal inhibitors or rapamycin. E) Respiration of control or Plin3 silenced NIH-3T3 cells under basal condition and after 6 hours of OA treatment. Plin3 silencing prevents the increase in mitochondrial respiration. F and G) Maximal respiration and ATP production are increased in control NIH-3T3 cells after OA treatment, whilst Plin3 silencing ameliorates this effect. Bars are mean ± SEM. *p < 0.05, **p < 0.01, and ***p < 0.001 (differences caused by lysosomal inhibitors treatment), # p<0.05, ## p<0.01, and ### p<0.001 (differences caused by treatment)

Article Snippet: Antibodies for Atg16l (PM040) from MBL; Atg7 (2631), Beclin 1 (3495), FIP200 (12436), LC3B (2775), mTOR (2983), phopho-mTOR (5536), Rag A (4357), Rag C (3360), Raptor (2280), Rictor (2114), S6 (2217), phopho-S6 (4858), TSC1 (6935) and anti-rabbit (7074) were from Cell Signaling Technology; RagB (NBP1-85801) from Novus Biologicals; Plin3 (3883,for WB) from ProSci; LAMP1 (1D4B, for WB) from Developmental Studies Hybridoma Bank; PLIN3 (GP30 for CoIP) from ProGen Biotechnik; β-actin (A5441) and anti-mouse (AP130P) and anti-rat (AP136P) from SIGMA; GAPDH (ab8245), Lamp1 (ab24170 for IF) and normal IgG (ab188776) from Abcam; Fluorescence secondary antibodies (Alexa Fluor 488 and/or Alexa Fluor 647 conjugated) and anti-guinea pig (A18775) from Fisher Scientific and Beclin 1-HRP(sc-48341 HRP) and Plin 3-HRP (sc-390968 HRP) form Santa Cruz Biotechnology.

Techniques: Produced

Journal: bioRxiv

Article Title: mTORC1-Plin3 pathway is essential to activate lipophagy and protects against hepatosteatosis

doi: 10.1101/812990

Figure Lengend Snippet: A) Depiction of the methodological approach: Human liver slices are pre-treated with or without rapamycin for 1 hour before the OA treatment, then slices are treated with or without lysosomal inhibitors. Slices without any treatment but the lysosomal inhibitors are used as a control. B-C) Levels of autophagy proteins and perilipins in total homogenates from all the conditions were analysed. Autophagy flux is elevated with the OA treatment. Rapamycin pre-treatment increases autophagy flux but leads to an accumulation of Plin3. D) Volume of LDs (measure from LD staining in green) is increased after blocking autophagy in the OA-treated slices. Rapamycin treatment results in even stronger accumulation of lipids. E-I) Recruitment of LC3 (E), Lamp1 (F) or mTOR (G) (magenta) to LDs (green) is decreased after blocking mTORC1 activity whilst Plin3 (H) (magenta) accumulates on the LD (green) surface in response to rapamycin in OA-treated human liver slices (see E to H, quantified in I). Scale bar: 20μm. Bars are mean ± SEM. *p < 0.05, **p < 0.01, and ***p < 0.001 (differences caused by lysosomal inhibitors treatment), # p<0.05, ## p<0.01, and ### p<0.001 (differences caused by treatment)

Article Snippet: Antibodies for Atg16l (PM040) from MBL; Atg7 (2631), Beclin 1 (3495), FIP200 (12436), LC3B (2775), mTOR (2983), phopho-mTOR (5536), Rag A (4357), Rag C (3360), Raptor (2280), Rictor (2114), S6 (2217), phopho-S6 (4858), TSC1 (6935) and anti-rabbit (7074) were from Cell Signaling Technology; RagB (NBP1-85801) from Novus Biologicals; Plin3 (3883,for WB) from ProSci; LAMP1 (1D4B, for WB) from Developmental Studies Hybridoma Bank; PLIN3 (GP30 for CoIP) from ProGen Biotechnik; β-actin (A5441) and anti-mouse (AP130P) and anti-rat (AP136P) from SIGMA; GAPDH (ab8245), Lamp1 (ab24170 for IF) and normal IgG (ab188776) from Abcam; Fluorescence secondary antibodies (Alexa Fluor 488 and/or Alexa Fluor 647 conjugated) and anti-guinea pig (A18775) from Fisher Scientific and Beclin 1-HRP(sc-48341 HRP) and Plin 3-HRP (sc-390968 HRP) form Santa Cruz Biotechnology.

Techniques: Staining, Blocking Assay, Activity Assay

Journal: bioRxiv

Article Title: mTORC1-Plin3 pathway is essential to activate lipophagy and protects against hepatosteatosis

doi: 10.1101/812990

Figure Lengend Snippet: Oleic acid treatment activates mTORC1 activity to directly or indirectly phosphorylate Plin3 at LDs. Phosphorylated Plin3 binds the autophagosome formation machinery (FIP200 and ATG16l) to induce lipophagy. The autophagic machinery mobilises LDs to lysosomes, which generate fatty acids used for energy production by the mitochondria.

Article Snippet: Antibodies for Atg16l (PM040) from MBL; Atg7 (2631), Beclin 1 (3495), FIP200 (12436), LC3B (2775), mTOR (2983), phopho-mTOR (5536), Rag A (4357), Rag C (3360), Raptor (2280), Rictor (2114), S6 (2217), phopho-S6 (4858), TSC1 (6935) and anti-rabbit (7074) were from Cell Signaling Technology; RagB (NBP1-85801) from Novus Biologicals; Plin3 (3883,for WB) from ProSci; LAMP1 (1D4B, for WB) from Developmental Studies Hybridoma Bank; PLIN3 (GP30 for CoIP) from ProGen Biotechnik; β-actin (A5441) and anti-mouse (AP130P) and anti-rat (AP136P) from SIGMA; GAPDH (ab8245), Lamp1 (ab24170 for IF) and normal IgG (ab188776) from Abcam; Fluorescence secondary antibodies (Alexa Fluor 488 and/or Alexa Fluor 647 conjugated) and anti-guinea pig (A18775) from Fisher Scientific and Beclin 1-HRP(sc-48341 HRP) and Plin 3-HRP (sc-390968 HRP) form Santa Cruz Biotechnology.

Techniques: Activity Assay