Journal: Molecular Biology of the Cell

Article Title: Weak membrane interactions allow Rheb to activate mTORC1 signaling without major lysosome enrichment

doi: 10.1091/mbc.E19-03-0146

Figure Lengend Snippet: Rheb is not enriched on lysosomes. (A, B) Rheb and LAMP1 (late endosomes/lysosomes) immunofluorescence in HeLa cells that were transfected with control and Rheb siRNA, respectively. (C) Comparison of Rheb and LAMP1 colocalization in the insets from A before and after rotating the LAMP1 image by 90° to estimate the extent to which colocalization occurs by chance. (D) Quantification of the Pearson’s R value when comparing Rheb vs. LAMP1 in the original and rotated configurations as described in C ( n = 3 biological replicates, 15 images quantified per replicate, t test). (E, F) Representative immunofluorescence images of anti-HA and anti-LAMP1 staining in genome edited 2xHA-Rheb HeLa cells and control HeLa cells, respectively. Scale bars, 10 μm.

Article Snippet: Control siRNA (5′-CGUUAAUCGCGUAUAAUACGCGUAT-3′) was purchased from Integrated DNA Technologies (IDT, Coralville, IA) and a previously described Rheb siRNA ( Menon et al. , 2014 ) was purchased from Cell Signaling Technology (#14267; CST, Danvers, MA).

Techniques: Immunofluorescence, Transfection, Control, Comparison, Staining

Journal: Molecular Biology of the Cell

Article Title: Weak membrane interactions allow Rheb to activate mTORC1 signaling without major lysosome enrichment

doi: 10.1091/mbc.E19-03-0146

Figure Lengend Snippet: Regulated recruitment of mTOR to lysosomes is not accompanied by significant colocali­zation with Rheb. (A, B) Immunofluorescence analysis of mTOR and LAMP1 localization in starved and amino acid refed cells, respectively. (C, D) Immunofluorescence analysis of mTOR and Rheb localization in starved and amino acid refed cells, respectively. (E) Quantification of the colocali­zation observed in experiments related to A–D ( n = 3 biological replicates, 15 images quantified per repli­cate, one-way analysis of variance (ANOVA) with a Sidak’s multiple comparisons test). (F) Immuno­blot analysis of phospho-S6K and S6K levels in starved and amino acid refed cells. Scale bars, 10 μm.

Article Snippet: Control siRNA (5′-CGUUAAUCGCGUAUAAUACGCGUAT-3′) was purchased from Integrated DNA Technologies (IDT, Coralville, IA) and a previously described Rheb siRNA ( Menon et al. , 2014 ) was purchased from Cell Signaling Technology (#14267; CST, Danvers, MA).

Techniques: Immunofluorescence

Journal: Molecular Biology of the Cell

Article Title: Weak membrane interactions allow Rheb to activate mTORC1 signaling without major lysosome enrichment

doi: 10.1091/mbc.E19-03-0146

Figure Lengend Snippet: GFP-Rheb localizes to the ER and cytosol. (A, B) Spinning disk confocal live-cell imaging of GFP-Rheb in HeLa cells and COS-7 cells, respectively. (C) GFP-Rheb and mRFP-Sec61B (ER marker) localization in COS-7 cells. (D) GFP-Rheb and LAMP1-mCherry (late endosomes and lysosomes) localization in COS-7 cells. Scale bars, 10 μm.

Article Snippet: Control siRNA (5′-CGUUAAUCGCGUAUAAUACGCGUAT-3′) was purchased from Integrated DNA Technologies (IDT, Coralville, IA) and a previously described Rheb siRNA ( Menon et al. , 2014 ) was purchased from Cell Signaling Technology (#14267; CST, Danvers, MA).

Techniques: Live Cell Imaging, Marker

Journal: Molecular Biology of the Cell

Article Title: Weak membrane interactions allow Rheb to activate mTORC1 signaling without major lysosome enrichment

doi: 10.1091/mbc.E19-03-0146

Figure Lengend Snippet: Development of Rheb Depleted cells as a tool for testing relationships between Rheb localization and function. (A) Immunoblot analysis of Rheb levels and phosphorylation status of mTORC1 substrates (S6K, ULK1, and 4EBP1) in Control, Rheb siRNA-treated, Rheb Edited (Rheb hypomorph+RhebL1 KO), and Rheb Depleted (Rheb hypomorph+RhebL1 KO+Rheb siRNA) HeLa cells. (B) Quantification of phospho-S6K levels under the indicated conditions where phospho-S6K levels were divided by S6K and normalized to Control ( **P < 0.01; ** **P < 0.0001; ANOVA with Dunnett’s multiple comparisons test, n = 4). (C) Immunoblot analysis of phospho-S6K levels in Rheb Depleted cells transfected with the indicated plasmids. (D) Quantification of phospho-S6K levels from C. The phospho-S6K levels were divided by S6K and GFP values to control for loading and transfection efficiency. Values were normalized to GFP-Rheb. Statistics were calculated in comparison to the GFP transfection (** **P < 0.0001; ANOVA with Dunnett’s multiple comparisons test; n = 3). (E–G) Live-cell images of GFP-Rheb, GFP-Rheb ΔCaaX , and GFP-RhebL1 in a COS-7 cells, respectively. (H) Schematic of GFP-Rheb-ER chimera that contains N-terminal GFP, Rheb ΔCaaX , and the transmembrane domain of cytochrome b5 (TMD). (I) Live-cell imaging of GFP-Rheb-ER localization. The leftmost image displays a low magnification view of GFP-Rheb-ER in a COS-7 cell. The three subsequent panels show higher magnifications of GFP-Rheb-ER and mRFP-Sec61B from the inset region. (J) Immunoblot analysis of phospho-S6K signaling in Rheb Depleted cells transfected with the indicated plasmids. (K) Quantification of phospho-S6K levels in I. The phospho-S6K levels were divided by S6K and GFP values to control for loading and transfection. Values were normalized to GFP-Rheb. Statistics were calculated in comparison to GFP (** **P < 0.0001; ANOVA with Dunnett’s multiple comparisons test; n = 3). (L) Image showing the spatial relationship between GFP-Rheb-ER on ER tubules and LAMP1-positive lysosomes. Scale bars, 10 μm.

Article Snippet: Control siRNA (5′-CGUUAAUCGCGUAUAAUACGCGUAT-3′) was purchased from Integrated DNA Technologies (IDT, Coralville, IA) and a previously described Rheb siRNA ( Menon et al. , 2014 ) was purchased from Cell Signaling Technology (#14267; CST, Danvers, MA).

Techniques: Western Blot, Phospho-proteomics, Control, Transfection, Comparison, Live Cell Imaging

Journal: Molecular Biology of the Cell

Article Title: Weak membrane interactions allow Rheb to activate mTORC1 signaling without major lysosome enrichment

doi: 10.1091/mbc.E19-03-0146

Figure Lengend Snippet: Weak membrane interactions are optimal for Rheb-dependent mTORC1 activation. (A) Alignment of the hypervariable regions of Rheb, RhebL1, and H-Ras proteins with CaaX motifs highlighted in bold and palmitoylated cysteines in green. These cysteines were mutated to alanines (red) in the GFP-Rheb-HRas C→A mutant. (B–D) Live-cell images of GFP-Rheb, GFP-Rheb-HRas, and GFP-Rheb-HRas C→A in COS-7 cells, respectively. Scale bars, 10 µm. (E) Schematic of GFP-Rheb-H-Ras chimera that contains N-terminal GFP, Rheb ΔHVR , and the HVR of H-Ras. (F) Immunoblot analysis of phospho-S6K levels in Rheb Depleted cells transfected with the indicated plasmids. (G) Quantification of phospho-S6K levels from F. Phospho-S6K levels were divided by S6K and GFP values to control for loading and transfection (*** P < 0.001; **** P < 0.0001; ANOVA with Tukey’s multiple comparisons test; n = 5). (H) Schematic of myr-Rheb chimera that contains Rheb ΔCaaX and a myristoylation consensus sequence. (I) Immunoblot analysis of phospho-S6K levels in Rheb Depleted cells transfected with the indicated plasmids. (J) Quantification of phospho-S6K levels from I. Phospho-S6K levels were divided by S6K and Rheb values to control for loading and transfection (**** P < 0.0001; unpaired t test). (K) Immunoblot analysis of proteins identified in membrane and cytosolic fractions of HeLa cells. (L) Immunoblot analysis of phospho-S6K levels in HeLa cells treated with DMSO vehicle and 5 μM deltarasin for the times indicated.

Article Snippet: Control siRNA (5′-CGUUAAUCGCGUAUAAUACGCGUAT-3′) was purchased from Integrated DNA Technologies (IDT, Coralville, IA) and a previously described Rheb siRNA ( Menon et al. , 2014 ) was purchased from Cell Signaling Technology (#14267; CST, Danvers, MA).

Techniques: Membrane, Activation Assay, Mutagenesis, Western Blot, Transfection, Control, Sequencing

Journal: Science signaling

Article Title: Covalent inhibitors of EGFR family protein kinases induce degradation of human Tribbles 2 (TRIB2) pseudokinase in cancer cells.

doi: 10.1126/scisignal.aat7951

Figure Lengend Snippet: Fig. 3. Afatinib promotes rapid degradation of FLAG-TRIB2 in an inducible HeLa model. (A) Uninduced (−TET) or TET-induced (+TET) HeLa cells containing a stably integrated FLAG-TRIB2 transgene were serum-starved for 16 hours before the addition of serum and lysed at the indicated times. Whole-cell extracts were blotted with FLAG antibody to detect FLAG-TRIB2 and pSer473 AKT. Total AKT and total GAPDH served as loading controls. (B) A selection of clinically approved kinase inhibitors, including dual EGFR/HER2- and EGFR-specific compounds, were added to TET- induced cells at a final concentration of 10 M. Stable cells were induced to express FLAG-tagged TRIB2 with TET for 16 hours before inhibitor treatment for 4 hours. Whole-cell extracts were immunoblotted with FLAG, phosphoryl ated ERK (pERK 1/2), total ERK, or GAPDH antibodies. (C) Stable HeLa cells were incubated with TET for 16 hours and then incubated with DMSO (top panels) or 10 M afatinib (bottom panels) before lysis at the indicated time. One- hour and 16-hour samples were pretreated with protein phosphatase (PP) before SDS-PAGE. Whole-cell extracts were immunoblotted with FLAG or GAPDH antibodies. Blots are representative of three independent experiments.

Article Snippet: 6His–horseradish peroxidase (HRP) and -FLAG antibodies were purchased from SigmaAldrich GAPDH antibody was purchased from Proteintech, and a polyclonal rabbit -TRIB2 antibody was raised toward a unique Nterminal human TRIB2 sequence and affinity-purified before evaluation with recombinant TRIB2 and a variety of human cellular extracts.

Techniques: Stable Transfection, Selection, Concentration Assay, Incubation, Lysis, SDS Page

Journal: bioRxiv

Article Title: Mast-Cell Expressed Membrane Protein-1 (MCEMP1) is expressed in classical monocytes and alveolar macrophages in Idiopathic Pulmonary Fibrosis and regulates cell chemotaxis, adhesion, and migration in a TGFβ dependent manner

doi: 10.1101/2023.10.07.561349

Figure Lengend Snippet: Analysis of the sequence of a 1.5kbp promoter region upstream of the human MCEMP1 gene ATG start codon resulted in the identification of three putative SBE/CAGA sites at locations -1,158/-1,152 (TCT AGAC AGA), -937/-931 ( AGAC AGA) and -325/-314 ( AGAC AC AGAC ACACACAG AGAC ) and a GC box at position -79/73 (GGGCGGG) (A). TGFβ failed to upregulate MCEMP1 expression in the presence of the SMAD3 inhibitor SiS3 (B) or SP1 inhibitor mithramycin A (MitA) (C). Similarly, THP-1 treatment with trichostatin A (TSA), also blocked MCEMP1 upregulation following TGFβ stimulation (D). Chromatin immunoprecipitation followed by qPCR shows that neither SMAD3 nor SP1 can bind to the -1,158/-1,152 SBE/CAGA site (E). SMAD3 binds to the -937/-931 SBE/CAGA site, but not SP1 (F). Concomitant binding of SMAD3 and SP1 is observed at locations -325/-314 and -79/-73 following THP-1 stimulation with TGFβ. This effect is inhibited in the presence of SiS3 or MitA (G, H).

Article Snippet: For proximity ligation assay, we used the anti-SP1 antibody mentioned above and a monoclonal mouse anti-SMAD3 antibody from Proteintech (Rosemont, IL).

Techniques: Sequencing, Expressing, Chromatin Immunoprecipitation, Binding Assay

Journal: bioRxiv

Article Title: Mast-Cell Expressed Membrane Protein-1 (MCEMP1) is expressed in classical monocytes and alveolar macrophages in Idiopathic Pulmonary Fibrosis and regulates cell chemotaxis, adhesion, and migration in a TGFβ dependent manner

doi: 10.1101/2023.10.07.561349

Figure Lengend Snippet: TGFβ treatment resulted in an important increase of fluorescent spots following TGFβ treatment compared to control, particularly in nuclei where both transcription factors translocate to regulate gene expression (A, B, E). Both SiS3 and MitA drastically reduced the formation of SMAD3-SP1 complexes in THP-1 nuclei, demonstrating the specificity of the PLA signal (C, D, E). DAPI (Blue), SMAD3-SP1 complexes (Red). ****p<0.0001, ***p<0.001, *p<0.05.

Article Snippet: For proximity ligation assay, we used the anti-SP1 antibody mentioned above and a monoclonal mouse anti-SMAD3 antibody from Proteintech (Rosemont, IL).

Techniques: Control, Gene Expression