Journal: International journal of molecular sciences

Article Title: Refining Evaluation of Bone Mass and Adipose Distribution in Dunnigan Syndrome.

doi: 10.3390/ijms241713118

Figure Lengend Snippet: Figure 3. Osteoclast differentiation derived from peripheral blood mononuclear cells from controls and DS patients. (A) An area of osteoclasts developed in a culture in the presence of MCSF + RANKL and MCSF + RANKL + chemerin. (B) The number of osteoclast cells according to the type of medium added in sequence. Each step of the sequence was cultured in 3 distinct wells for each subject analyzed. The final result is the average sum of controls and patients. (C) The appearance of the osteoclast cells developed in a culture supplemented with MCSF + RANKL and (D) MCSF + RANKL + chemerin in a DS subject. Osteoclasts must have 3 or more nuclei. * p < 0.05. Abbreviations: MCSF, macrophage colony-stimulating factor; RANKL, receptor activator of nuclear factor kappa-B ligand; DS, Dunnigan syndrome.

Article Snippet: These monocytes were cultured in 3 × 3 wells in 96-well plates with α-MEM containing 10% FBS added sequentially in triplets with 25 ng/mL of human macrophage colony-stimulating factor (MCSF) (R&D systems, catalog 216-MCC, Minneapolis, MN, USA), 25 ng/mL of receptor activator of nuclear factor kappa-B ligand (RANKL) (R&D systems, catalog 390-TN, Minneapolis, MN, USA) and 50 ng/mL of chemerin (R&D systems, catalog 2324-CM, Minneapolis, MN, USA) to stimulate osteoclasts.

Techniques: Derivative Assay, Sequencing, Cell Culture

Journal: Nature Communications

Article Title: Leucine regulates autophagy via acetylation of the mTORC1 component raptor

doi: 10.1038/s41467-020-16886-2

Figure Lengend Snippet: a Leu metabolic pathway. Red box shows MCCC1 gene. b Autophagy activation by MCCC1 depletion. siRNA knockdown of MCCC1 in HeLa cells was used to determine whether MCCC1 can regulate autophagy (Con; scrambled, nontargeting siRNA, MCCC1; SMARTpool MCCC1 targeted siRNA). Blots are representative of four independent experiments ( N = 4). Two-tailed unpaired t -test. c Immunostaining of HeLa cells treated with MCCC1 SMARTpool siRNA using MCCC1 (red) and LC3 (green) antibodies, nuclei are stained with DAPI (blue). # MCCC1 knockdown cells, *non-knockdown cells. Scale bar, 10 μm. N = 4; 70–80 cells scored per condition per experiment. Two-tailed unpaired t -test. d Reduced HTT (Q74) aggregation in MCCC1 knockdown HeLa cells. HeLa cells were seeded on coverslips in triplicates, transfected with siRNAs targeting control or MCCC1, followed by HA-tagged HTT (Q74) expression. N = 4, 40–60 cells scored per condition per experiment. Two-tailed unpaired t -test. e α-synuclein degradation assay in control or MCCC1 knockdown ATG16L1 WT or CRISPR KO HeLa cells. Cells were transfected with control or MCCC1 siRNAs for 3 days. In the last 24 h, cells were transfected with empty pEGFP (as a “transfection/loading control”) + pEGFP-α-synuclein A53T. Levels of α-synuclein A53T are expressed as a ratio to GFP. N = 3. * p < 0.05 vs. control cells; two-tailed unpaired t -test. Autophagy activation by MCCC1 depletion in SH-SY5Y cells ( f ) and primary neurons ( g ). N = 3. *** p < 0.001 vs. control cells; two-tailed unpaired t -test. Long exposure (LE); Short exposure (SE). h Autophagic flux in mouse primary cortical neurons from mRFP-GFP-LC3 (tfLC3) transgenic mice. Representative confocal z-stack images (right panel) and total number of GFP/mRFP dots (autophagosomes) and mRFP-only dots (autolysosomes). In total, 25–35 cells analyzed per condition per experiment; two-tailed unpaired t -test. Scale bar, 10 μm. Data are presented as mean values ± SEM. Source data are provided as a file.

Article Snippet: The following DNA or siRNA/shRNA constructs were also used in this work: empty pEGFP (Clontech), pEGFP-α-synuclein A53T and pcDNA3.1-myc-6XHis (Invitrogen); pCMV6-XL5-MCCC1 (#SC113201) from Origene; pcDNA3.1-EP300-6XHis (#23252), pRK5-HA-YFP-raptor (#73385), pRK5-HA-raptor (#8513), pcDNA4-BECN1-Flag (#24388) from Addgene.

Techniques: Activation Assay, Two Tailed Test, Immunostaining, Staining, Transfection, Expressing, Degradation Assay, CRISPR, Transgenic Assay

Journal: Nature Communications

Article Title: Leucine regulates autophagy via acetylation of the mTORC1 component raptor

doi: 10.1038/s41467-020-16886-2

Figure Lengend Snippet: a mTORC1 inhibition in MCCC1-depleted cells. HeLa cells were treated with MCCC1 siRNA and immunostained for MCCC1 (green) and phosphorylated S6 (red), nuclei were stained with DAPI (blue). For ease of visualization, the cell outlines are highlighted in white dashed lines. # MCCC1 knockdown cells, *non-knockdown cells. Scale bar, 10 μm. N = 4, 30–40 cells scored per condition per experiment. Two-tailed unpaired t -test. The effect of mTOR inhibitor Torin1 in MCCC1 knockdown HeLa ( b ) or SH-SY5Y ( c ) and Mccc1 knockdown primary neurons ( d ). Control and MCCC1 knockdown cells were treated with 0.5 µM Torin1 (or DMSO) for 4 h. N = 4 for HeLa cells or N = 3 for SH-SY5Y and primary neurons. *** p < 0.001 vs. control cells; two-tailed unpaired t -test. e Autophagy response to changes in Leu levels. HeLa cells were treated with scrambled siRNA or MCCC1 siRNA then incubated in Leu-depleted media for 4 h or incubated in Leu-depleted media for 4 h, followed by the re-addition of 10 µM of Leu to the media for 1 h. N = 3, 50–60 cells scored per condition per experiment. Two-tailed unpaired t -test. f Increased number of WIPI2 dots in Leu-depleted media. N = 3, 40 cells scored per condition per experiment. Scale bar, 5 μm. Two-tailed unpaired t -test. g Rescue of autophagy and mTORC1 activation in MCCC1 knockdown by dichloroacetate (DCA), not Leu or KIC. MCCC1 knockdown HeLa cells were treated with 10 µM Leu, 10 mM KIC, or 10 mM DCA for 1 h. N = 3. Two-tailed unpaired t -test. Data are presented as mean values ± SEM. Source data are provided as a file.

Article Snippet: The following DNA or siRNA/shRNA constructs were also used in this work: empty pEGFP (Clontech), pEGFP-α-synuclein A53T and pcDNA3.1-myc-6XHis (Invitrogen); pCMV6-XL5-MCCC1 (#SC113201) from Origene; pcDNA3.1-EP300-6XHis (#23252), pRK5-HA-YFP-raptor (#73385), pRK5-HA-raptor (#8513), pcDNA4-BECN1-Flag (#24388) from Addgene.

Techniques: Inhibition, Staining, Two Tailed Test, Incubation, Activation Assay

Journal: Nature Communications

Article Title: Leucine regulates autophagy via acetylation of the mTORC1 component raptor

doi: 10.1038/s41467-020-16886-2

Figure Lengend Snippet: a AcCoA levels were assessed using the PicoProbe AcCoA assay kit. HeLa cells were incubated in nutrient-depleted conditions for 4 h followed by lysis and measurement of AcCoA levels. N = 4. Two-tailed unpaired t -test. b Effect of DCA and Leu on mTOR signaling and autophagy modulation caused by MCCC1 knockdown. Control cells, and cells treated with MCCC1 siRNA were treated with 10 mM DCA or 10 µM Leu for 1 h. Cells were lysed and western blots for phosphorylated S6K1 (p-S6K1) and total S6K1, as well as LC3 and MCCC1 are shown. N = 3. Two-tailed unpaired t -test. c Rescue of activated autophagy in MCCC1-depleted cells by DCA. 10 mM DCA for 1 h treatment restored the increased LC3 dots in MCCC1-depleted cells. N = 3, 60–70 cells scored per condition per experiment. Two-tailed unpaired t -test. Scale bar, 10 μm. AcCoA-induced autophagy regulation in mTORC1-dependent manner. Treatment with 0.5 µM Torin1 for 4 h or RRAGA and RRAGB double-knockdown (RRAGA + B) blocked the rescue effect of Leu or DCA on autophagy activation (LC3-II level ( d ) and LC3 dots ( e )) by Leu depletion. HeLa cells were treated with scrambled siRNA or MCCC1 siRNA then incubated in Leu-depleted media for 4 h or incubated in Leu-depleted media for 4 h, followed by the re-addition of 10 µM of Leu or 10 mM DCA to the media for 1 h. N = 3, 40–50 cells scored per condition per experiment. Two-way ANOVA with post-hoc Tukey’s multiple comparison test. Scale bar, 10 μm. Data are presented as mean values ± SEM. Source data are provided as a file.

Article Snippet: The following DNA or siRNA/shRNA constructs were also used in this work: empty pEGFP (Clontech), pEGFP-α-synuclein A53T and pcDNA3.1-myc-6XHis (Invitrogen); pCMV6-XL5-MCCC1 (#SC113201) from Origene; pcDNA3.1-EP300-6XHis (#23252), pRK5-HA-YFP-raptor (#73385), pRK5-HA-raptor (#8513), pcDNA4-BECN1-Flag (#24388) from Addgene.

Techniques: Incubation, Lysis, Two Tailed Test, Western Blot, Activation Assay

Journal: Nature Communications

Article Title: Leucine regulates autophagy via acetylation of the mTORC1 component raptor

doi: 10.1038/s41467-020-16886-2

Figure Lengend Snippet: a HeLa cells were treated with control siRNA or MCCC1 siRNAs, and cytosolic EP300 activity was assessed. N = 4. Two-tailed unpaired t -test. b Reduced raptor acetylation in MCCC1 knockdown cells. HeLa cells were treated with control or MCCC1 siRNA and transfected with HA-raptor. Following incubation in Leu-depleted media for 4 h, cells were lysed and raptor was immunoprecipitated using an anti-HA antibody. N = 4. c HeLa cells were depleted of raptor with siRNA and transfected with cDNA constructs encoding either raptor WT or raptor K1097R mutant (KR) (both HA-tagged). N = 4. d mTORC1 activity (phosphorylated S6) and LC3 dot numbers in HeLa cells expressing raptor WT or raptor KR. *HA-raptor WT or KR-expressing cells, # nontransfected cells. Scale bar, 10 μm. N = 3, 20–30 cells scored per condition per experiment. Two-tailed unpaired t -test. Acetylated PIK3C3 ( e ) and BECN1 levels ( f ) in HeLa cells depleted of raptor then reconstituted with raptor WT or raptor KR. Two-tailed unpaired t -test. HC, heavy chain. N = 4. g Effects of EP300 activator on mTORC1 signaling and autophagy in response to Leu deprivation with or without DCA in HeLa cells depleted of raptor then reconstituted with raptor WT or raptor KR. HeLa cells were incubated in Leu-depleted media for 4 h or incubated in Leu-depleted media with Torin1 for 4 h, followed by re-addition of 10 mM DCA to the media for 1 h. 50 µM CTB for 12 h was used to activate EP300. Blots are representative of three biologically independent experiments ( N = 3). h Leu abundance regulates autophagy in many cell types via its metabolite AcCoA. AcCoA activates EP300, which acetylates raptor, leading to mTORC1 activation, which inhibits autophagy. In Leu deprivation conditions, autophagy activation is mainly mediated by decreased raptor acetylation causing mTORC1 inhibition, rather than by altered acetylation of other autophagy regulators. Data are presented as mean values ± SEM. Source data are in file.

Article Snippet: The following DNA or siRNA/shRNA constructs were also used in this work: empty pEGFP (Clontech), pEGFP-α-synuclein A53T and pcDNA3.1-myc-6XHis (Invitrogen); pCMV6-XL5-MCCC1 (#SC113201) from Origene; pcDNA3.1-EP300-6XHis (#23252), pRK5-HA-YFP-raptor (#73385), pRK5-HA-raptor (#8513), pcDNA4-BECN1-Flag (#24388) from Addgene.

Techniques: Activity Assay, Two Tailed Test, Transfection, Incubation, Immunoprecipitation, Construct, Mutagenesis, Expressing, Activation Assay, Inhibition