Journal: Molecular medicine reports

Article Title: VHL loss predicts response to Aurora kinase A inhibitor in renal cell carcinoma cells.

doi: 10.3892/mmr.2018.9038

Figure Lengend Snippet: Figure 2. 769‑P cells with VHL re‑expression show resistance to alisertib. (A) VHL and AURKA protein expression in 769‑P cells transfected with control or VHL plasmid was analyzed by immunoblotting. GAPDH was employed as a loading control. (B) The anti‑proliferative activity of alisertib against cells was assessed by Cell Counting Kit ‑8 assay. *P<0.01 vs. vector. VHL, von Hippel‑Lindau tumor suppressor; AURKA, Aurora kinase A; IC50, half‑maximal inhibitory concentration.

Article Snippet: Appropriate antibodies against VHL (1:1,000), AURKA (1:1,000; Cell Signaling Technology, Inc., Danvers, MA, USA), GAPDH (1:5,000; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) were used.

Techniques: Expressing, Transfection, Control, Plasmid Preparation, Western Blot, Activity Assay, Cell Counting, Concentration Assay

Journal: Molecular medicine reports

Article Title: VHL loss predicts response to Aurora kinase A inhibitor in renal cell carcinoma cells.

doi: 10.3892/mmr.2018.9038

Figure Lengend Snippet: Figure 1. VHL expression profiles and alisertib anti‑proliferative activities in RCC cells. (A) VHL and AURKA protein expression was detected by immunob lotting. GAPDH was employed as a loading control. (B) Anti‑proliferative activity was assessed by Cell Counting Kit‑8 assay following exposure to alisertib for 72 h. (C) Alisertib anti‑tumor activity in vivo. VHL, von Hippel‑Lindau tumor suppressor; RCC, renal cell carcinoma; AURKA, Aurora kinase A; IC50, half‑maximal inhibitory concentration; TGI, tumor growth inhibition rate.

Article Snippet: Appropriate antibodies against VHL (1:1,000), AURKA (1:1,000; Cell Signaling Technology, Inc., Danvers, MA, USA), GAPDH (1:5,000; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) were used.

Techniques: Expressing, Control, Activity Assay, CCK-8 Assay, In Vivo, Concentration Assay, Inhibition

Journal: Molecular medicine reports

Article Title: VHL loss predicts response to Aurora kinase A inhibitor in renal cell carcinoma cells.

doi: 10.3892/mmr.2018.9038

Figure Lengend Snippet: Figure 4. VHL regulates AURKA levels via HIF‑dependent and ‑independent pathways. (A) Hypoxia could induce AURKA protein upregulation upon analysis of HIF‑1 and AURKA protein expression in CAK‑I cells by immunoblotting; GAPDH was employed as a loading control. (B) The 26S proteasome inhibitor MG132 (10 µM) rescued the downregulation of AURKA in 769‑P cells re‑expressed with pVHL. *P<0.01 vs. empty vector. (C) pVHL promoted AURKA degradation. 769‑p cells were transfected with either pVHL or vector plasmid. Cells were treated with 100 µg/ml CHX and harvested at the indicated time points, and cell lysates were prepared. Proteins from cell lysates were subjected to western blotting with anti‑AURKA and anti‑GAPDH antibodies. Relative protein levels were plotted from the integrated optical density of the AURKA bands on the western blot (lower panel). VHL, von Hippel‑Lindau tumor suppressor; AURKA, Aurora kinase A; IC50, half‑maximal inhibitory concentration; HIF, hypoxia inducing factor; pVHL, VHL protein; CHX, cycloheximide.

Article Snippet: Appropriate antibodies against VHL (1:1,000), AURKA (1:1,000; Cell Signaling Technology, Inc., Danvers, MA, USA), GAPDH (1:5,000; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) were used.

Techniques: Expressing, Western Blot, Control, Plasmid Preparation, Transfection, Concentration Assay

Journal: Molecular medicine reports

Article Title: VHL loss predicts response to Aurora kinase A inhibitor in renal cell carcinoma cells.

doi: 10.3892/mmr.2018.9038

Figure Lengend Snippet: Figure 3. shRNA knockdown of VHL in CAKI cells confers decreased alisertib sensitivity. (A) VHL and AURKA protein expression in CAKI cells transfected with control or VHL shRNA was analyzed by immunoblotting. GAPDH was employed as a loading control. (B) The anti‑proliferative activity of alisertib against cells was assessed by Cell Counting Kit ‑8 assay. *P<0.01 vs. vector. (C) Inhibition of tumor growth by alisertib in xenografts of CAKI cells transfected with control or VHL shRNA. VHL, von Hippel‑Lindau tumor suppressor; AURKA, Aurora kinase A; IC50, half‑maximal inhibitory concentration; shRNA, short hairpin RNA.

Article Snippet: Appropriate antibodies against VHL (1:1,000), AURKA (1:1,000; Cell Signaling Technology, Inc., Danvers, MA, USA), GAPDH (1:5,000; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) were used.

Techniques: shRNA, Knockdown, Expressing, Transfection, Control, Western Blot, Activity Assay, Cell Counting, Plasmid Preparation, Inhibition, Concentration Assay

Journal: World Journal of Gastroenterology

Article Title: Thymoquinone affects hypoxia-inducible factor-1α expression in pancreatic cancer cells via HSP90 and PI3K/AKT/mTOR pathways

doi: 10.3748/wjg.v30.i21.2793

Figure Lengend Snippet: Expression of von Hippel Lindau protein in von Hippel Lindau overexpression and von Hippel Lindau knockdown cell models under normoxia condition. A: Western blot assay was performed to detect von Hippel Lindau (VHL) protein expression in cell models of control group, overexpressed empty carrier (OE-NC) group and VHL overexpressed (OE-VHL) group under normoxia condition; B: Statistical analysis of VHL protein expression in cell models of control group, OE-NC group and OE-VHL group under normoxia condition; C: Western blot assay was performed to detect VHL protein expression in cell models of control group, knockdown empty vector (sh-NC) group and VHL knockdown (sh-VHL) group under normoxia condition; D: Statistical analysis of VHL protein expression in cell models of control group, sh-NC group and sh-VHL group under normoxia condition. a P < 0.05; b P < 0.01. VHL: von Hippel Lindau; OE-NC: Overexpressed empty carrier; OE-VHL: VHL overexpressed; sh-NC: Knockdown empty vector; sh-VHL: VHL knockdown.

Article Snippet: The following materials were used in this study: TQ (HY-D0803; MCE; New Jersey; United States); 26S proteasome inhibitor (HY-132598; MCE); geldanamycin (GA; HY-15230; MCE); cycloheximide (CHX) (S7418; SELLECK; Houston; United States); dimethyl sulfoxide (DMSO; Sigma; St. Louis; United States); fetal bovine serum (FBS; GIBCO; Invitrogen; Carlsbad; United States); Binding Buffer (XP2; Omega; Norcross; United States); trypsin-EDTA (GNM25200; Gino Biomedical Technology Co, Ltd; Hangzhou; China); Trypsin Solution without EDTA (C0205; Beyotime Biotech Co., Ltd; Shanghai; China); IP cell lysate (AS1003; Aspen Biotechnology Co., Ltd; Wuhan; China); SDS-PAGE gel preparation kit (AS1012; Aspen Biotechnology Co., Ltd; Wuhan; China); RPMI-1640 medium (GIBCO); Trizol (15596026; Ambion; Austin; United States); iScript® II Q RT SuperMix for quantitative real-time polymerase chain reaction (qPCR) (+gDNA wiper) (R233-01; VAZYME; Nangjing; China); HiScript II Q Select RT SuperMix for qPCR (+gDNA wiper) (R233-01; VAZYME); SYBR Green Master Mix (Q111-02; VAZYME); Taq Plus DNA Polymerase (ET105-01; TIANGEN; Beijing; China); DL2000 DNA Marker (MD114-02; TIANGEN); primary antibodies: Anti-HIF1α (20960-1-AP; PROTEINTECH; Chicago; United States), anti-OH-HIF1α (3434T; CST; Danvers; United States), anti-phosphatidyl inositol-4,5-bisphosphate-3-kinase-p85α (PI3K-p85α; 60225-1-Ig; PROTEINTECH), anti-protein kinase B (Akt; 60203-2-Ig; PROTEINTECH), anti-mammalian target of rapamycin (mTOR; 66888-1-Ig; PROTEINTECH), anti-S6 kinase (S6K; 14485-1-AP; PROTEINTECH), anti-eukaryotic translation initiation factor 4E binding protein p70 S6 kinase (4E-BP1; 60246-1-Ig; PROTEINTECH), anti-eukaryotic translation initiation factor 4E (eIF-4E; 66655-1-Ig; PROTEINTECH), and anti-von Hippel Lindau (VHL) (16538-1-AP; PROTEINTECH); Cell counting kit-8 (CCK-8) kit (Beyotime Biotech Co., Ltd; Shanghai; China); Annexin V-FITC Apoptosis Detection Kit (AO2001-02P-G; Tianjin Sanjian Biotechnology Co., Ltd; Tianjin; China); phosphate-buffered saline (PBS, Gino Biomedical Technology Co, Ltd; Hangzhou; China); and tris-buffered saline (TBS; Gino Biomedical Technology Co, Ltd).

Techniques: Expressing, Over Expression, Knockdown, Western Blot, Control, Plasmid Preparation

Journal: World Journal of Gastroenterology

Article Title: Thymoquinone affects hypoxia-inducible factor-1α expression in pancreatic cancer cells via HSP90 and PI3K/AKT/mTOR pathways

doi: 10.3748/wjg.v30.i21.2793

Figure Lengend Snippet: Effects of Thymoquinone on ubiquitination degradation of hypoxia-inducible factor-1α protein mediated by von Hippel Lindau under normoxia condition. A: Western blot analysis was performed to detect the expression of von Hippel Lindau (VHL) protein and hypoxia-inducible factor-1α (HIF-1α) protein in Lenti-VHL group, Lenti-VHL + Thymoquinone (TQ) group, VHL knockdown (sh-VHL) group and sh-VHL+TQ group under normoxia condition; B: Statistical analysis of VHL protein expression in the Lenti-VHL group, Lenti-VHL+TQ group, sh-VHL group and sh-VHL+TQ group under normoxia condition; C: Statistical analysis of HIF-1α expression in Lenti-VHL group, Lenti-VHL+TQ group, sh-VHL group and sh-VHL+TQ group under normoxia condition. a P < 0.05; b P < 0.01. TQ: Thymoquinone; VHL: von Hippel Lindau; sh-VHL: VHL knockdown; HIF-1α: Hypoxia-inducible factor-1α.

Article Snippet: The following materials were used in this study: TQ (HY-D0803; MCE; New Jersey; United States); 26S proteasome inhibitor (HY-132598; MCE); geldanamycin (GA; HY-15230; MCE); cycloheximide (CHX) (S7418; SELLECK; Houston; United States); dimethyl sulfoxide (DMSO; Sigma; St. Louis; United States); fetal bovine serum (FBS; GIBCO; Invitrogen; Carlsbad; United States); Binding Buffer (XP2; Omega; Norcross; United States); trypsin-EDTA (GNM25200; Gino Biomedical Technology Co, Ltd; Hangzhou; China); Trypsin Solution without EDTA (C0205; Beyotime Biotech Co., Ltd; Shanghai; China); IP cell lysate (AS1003; Aspen Biotechnology Co., Ltd; Wuhan; China); SDS-PAGE gel preparation kit (AS1012; Aspen Biotechnology Co., Ltd; Wuhan; China); RPMI-1640 medium (GIBCO); Trizol (15596026; Ambion; Austin; United States); iScript® II Q RT SuperMix for quantitative real-time polymerase chain reaction (qPCR) (+gDNA wiper) (R233-01; VAZYME; Nangjing; China); HiScript II Q Select RT SuperMix for qPCR (+gDNA wiper) (R233-01; VAZYME); SYBR Green Master Mix (Q111-02; VAZYME); Taq Plus DNA Polymerase (ET105-01; TIANGEN; Beijing; China); DL2000 DNA Marker (MD114-02; TIANGEN); primary antibodies: Anti-HIF1α (20960-1-AP; PROTEINTECH; Chicago; United States), anti-OH-HIF1α (3434T; CST; Danvers; United States), anti-phosphatidyl inositol-4,5-bisphosphate-3-kinase-p85α (PI3K-p85α; 60225-1-Ig; PROTEINTECH), anti-protein kinase B (Akt; 60203-2-Ig; PROTEINTECH), anti-mammalian target of rapamycin (mTOR; 66888-1-Ig; PROTEINTECH), anti-S6 kinase (S6K; 14485-1-AP; PROTEINTECH), anti-eukaryotic translation initiation factor 4E binding protein p70 S6 kinase (4E-BP1; 60246-1-Ig; PROTEINTECH), anti-eukaryotic translation initiation factor 4E (eIF-4E; 66655-1-Ig; PROTEINTECH), and anti-von Hippel Lindau (VHL) (16538-1-AP; PROTEINTECH); Cell counting kit-8 (CCK-8) kit (Beyotime Biotech Co., Ltd; Shanghai; China); Annexin V-FITC Apoptosis Detection Kit (AO2001-02P-G; Tianjin Sanjian Biotechnology Co., Ltd; Tianjin; China); phosphate-buffered saline (PBS, Gino Biomedical Technology Co, Ltd; Hangzhou; China); and tris-buffered saline (TBS; Gino Biomedical Technology Co, Ltd).

Techniques: Ubiquitin Proteomics, Western Blot, Expressing, Knockdown

Journal: World Journal of Gastroenterology

Article Title: Thymoquinone affects hypoxia-inducible factor-1α expression in pancreatic cancer cells via HSP90 and PI3K/AKT/mTOR pathways

doi: 10.3748/wjg.v30.i21.2793

Figure Lengend Snippet: Further validation of the effects of Thymoquinone on ubiquitination degradation of hypoxia-inducible factor-1αprotein. A: Western blot assay was performed to detect hypoxia-inducible factor-1α (HIF-1α) expression in von Hippel Lindau (VHL) knockdown PANC-1 cells treated with cycloheximide (CHX) at 0 h, 1 h, 2 h and 3 h under normoxia condition; B: Statistical analysis of HIF-1α protein expression in VHL knockdown PANC-1 cells treated with CHX at 0 h, 1 h, 2 h and 3 h under normoxia condition; C: Western blot assay was performed to detect HIF-1α expression in VHL knockdown PANC-1 cells treated with CHX + Thymoquinone (TQ) at 0 h, 1 h, 2 h and 3 h under normoxia condition; D: Statistical analysis of HIF-1α expression in VHL knockdown PANC-1 cells treated with CHX+TQ at 0 h, 1 h, 2 h and 3 h under normoxia conditions. a P < 0.01. CHX: Cycloheximide; VHL: von Hippel Lindau; sh-VHL: VHL knockdown; HIF-1α: Hypoxia-inducible factor-1α.

Article Snippet: The following materials were used in this study: TQ (HY-D0803; MCE; New Jersey; United States); 26S proteasome inhibitor (HY-132598; MCE); geldanamycin (GA; HY-15230; MCE); cycloheximide (CHX) (S7418; SELLECK; Houston; United States); dimethyl sulfoxide (DMSO; Sigma; St. Louis; United States); fetal bovine serum (FBS; GIBCO; Invitrogen; Carlsbad; United States); Binding Buffer (XP2; Omega; Norcross; United States); trypsin-EDTA (GNM25200; Gino Biomedical Technology Co, Ltd; Hangzhou; China); Trypsin Solution without EDTA (C0205; Beyotime Biotech Co., Ltd; Shanghai; China); IP cell lysate (AS1003; Aspen Biotechnology Co., Ltd; Wuhan; China); SDS-PAGE gel preparation kit (AS1012; Aspen Biotechnology Co., Ltd; Wuhan; China); RPMI-1640 medium (GIBCO); Trizol (15596026; Ambion; Austin; United States); iScript® II Q RT SuperMix for quantitative real-time polymerase chain reaction (qPCR) (+gDNA wiper) (R233-01; VAZYME; Nangjing; China); HiScript II Q Select RT SuperMix for qPCR (+gDNA wiper) (R233-01; VAZYME); SYBR Green Master Mix (Q111-02; VAZYME); Taq Plus DNA Polymerase (ET105-01; TIANGEN; Beijing; China); DL2000 DNA Marker (MD114-02; TIANGEN); primary antibodies: Anti-HIF1α (20960-1-AP; PROTEINTECH; Chicago; United States), anti-OH-HIF1α (3434T; CST; Danvers; United States), anti-phosphatidyl inositol-4,5-bisphosphate-3-kinase-p85α (PI3K-p85α; 60225-1-Ig; PROTEINTECH), anti-protein kinase B (Akt; 60203-2-Ig; PROTEINTECH), anti-mammalian target of rapamycin (mTOR; 66888-1-Ig; PROTEINTECH), anti-S6 kinase (S6K; 14485-1-AP; PROTEINTECH), anti-eukaryotic translation initiation factor 4E binding protein p70 S6 kinase (4E-BP1; 60246-1-Ig; PROTEINTECH), anti-eukaryotic translation initiation factor 4E (eIF-4E; 66655-1-Ig; PROTEINTECH), and anti-von Hippel Lindau (VHL) (16538-1-AP; PROTEINTECH); Cell counting kit-8 (CCK-8) kit (Beyotime Biotech Co., Ltd; Shanghai; China); Annexin V-FITC Apoptosis Detection Kit (AO2001-02P-G; Tianjin Sanjian Biotechnology Co., Ltd; Tianjin; China); phosphate-buffered saline (PBS, Gino Biomedical Technology Co, Ltd; Hangzhou; China); and tris-buffered saline (TBS; Gino Biomedical Technology Co, Ltd).

Techniques: Biomarker Discovery, Ubiquitin Proteomics, Western Blot, Expressing, Knockdown

Journal: World Journal of Gastroenterology

Article Title: Thymoquinone affects hypoxia-inducible factor-1α expression in pancreatic cancer cells via HSP90 and PI3K/AKT/mTOR pathways

doi: 10.3748/wjg.v30.i21.2793

Figure Lengend Snippet: Full text summary. HIF-1α: Hypoxia-inducible factor-1α; HIF-1β: Hypoxia-inducible factor-1β; PI3K: Phosphatidyl inositol-4,5-bisphosphate-3-kinase; Akt: Protein kinase B; mTOR: Mammalian target of rapamycin; 4E-BP1: Eukaryotic translation initiation factor 4E binding protein 1; eIF-4E: Eukaryotic translation initiation factor 4E; S6K: S6 kinase; Ras: Rat sarcoma; Raf: Rapidly accelerated fibrosarcoma; MAPK: Mitogen-activated protein kinases; ERK: Extracellular signal-regulated kinase; MEK: Mitogen extracellular signal-regulated kinas; MNK: MAP kinase interacting kinase; HRE: Hypoxia response elements; HSP90: Heat shock protein 90; pVHL: von Hippel-Lindau protein; Mdm2: Murine double-minute 2.

Article Snippet: The following materials were used in this study: TQ (HY-D0803; MCE; New Jersey; United States); 26S proteasome inhibitor (HY-132598; MCE); geldanamycin (GA; HY-15230; MCE); cycloheximide (CHX) (S7418; SELLECK; Houston; United States); dimethyl sulfoxide (DMSO; Sigma; St. Louis; United States); fetal bovine serum (FBS; GIBCO; Invitrogen; Carlsbad; United States); Binding Buffer (XP2; Omega; Norcross; United States); trypsin-EDTA (GNM25200; Gino Biomedical Technology Co, Ltd; Hangzhou; China); Trypsin Solution without EDTA (C0205; Beyotime Biotech Co., Ltd; Shanghai; China); IP cell lysate (AS1003; Aspen Biotechnology Co., Ltd; Wuhan; China); SDS-PAGE gel preparation kit (AS1012; Aspen Biotechnology Co., Ltd; Wuhan; China); RPMI-1640 medium (GIBCO); Trizol (15596026; Ambion; Austin; United States); iScript® II Q RT SuperMix for quantitative real-time polymerase chain reaction (qPCR) (+gDNA wiper) (R233-01; VAZYME; Nangjing; China); HiScript II Q Select RT SuperMix for qPCR (+gDNA wiper) (R233-01; VAZYME); SYBR Green Master Mix (Q111-02; VAZYME); Taq Plus DNA Polymerase (ET105-01; TIANGEN; Beijing; China); DL2000 DNA Marker (MD114-02; TIANGEN); primary antibodies: Anti-HIF1α (20960-1-AP; PROTEINTECH; Chicago; United States), anti-OH-HIF1α (3434T; CST; Danvers; United States), anti-phosphatidyl inositol-4,5-bisphosphate-3-kinase-p85α (PI3K-p85α; 60225-1-Ig; PROTEINTECH), anti-protein kinase B (Akt; 60203-2-Ig; PROTEINTECH), anti-mammalian target of rapamycin (mTOR; 66888-1-Ig; PROTEINTECH), anti-S6 kinase (S6K; 14485-1-AP; PROTEINTECH), anti-eukaryotic translation initiation factor 4E binding protein p70 S6 kinase (4E-BP1; 60246-1-Ig; PROTEINTECH), anti-eukaryotic translation initiation factor 4E (eIF-4E; 66655-1-Ig; PROTEINTECH), and anti-von Hippel Lindau (VHL) (16538-1-AP; PROTEINTECH); Cell counting kit-8 (CCK-8) kit (Beyotime Biotech Co., Ltd; Shanghai; China); Annexin V-FITC Apoptosis Detection Kit (AO2001-02P-G; Tianjin Sanjian Biotechnology Co., Ltd; Tianjin; China); phosphate-buffered saline (PBS, Gino Biomedical Technology Co, Ltd; Hangzhou; China); and tris-buffered saline (TBS; Gino Biomedical Technology Co, Ltd).

Techniques: Binding Assay

Journal: ACS pharmacology & translational science

Article Title: Aptamer Proteolysis-Targeting Chimeras (PROTACs): A Novel Strategy to Combat Drug Resistance in Estrogen Receptor α-Positive Breast Cancer.

doi: 10.1021/acsptsci.4c00469

Figure Lengend Snippet: Figure 4. Ternary complex competition assay. (a) Single VH032-propargyl, or an aptamer alone, competitively inhibits ubiquitinated degradation of ERα by P7TA. (b) Quantification of the changes in ERα levels under the (a) conditions. (c) Single VH032-propargyl, or aptamer alone, competitively inhibits ubiquitinated degradation of ERα by P9TC. (d) Quantification of the changes in ERα levels under the (c) conditions. (e) Proteasome inhibitor control assay. (f) Quantification of the changes in ERα levels under the (e) conditions. (g) Chiral small molecule of VH032- propargyl was used as a negative control. (h) Quantification of the changes in ERα levels under the (g) conditions (*p < 0.05; ***p < 0.001; ****p < 0.0001).

Article Snippet: The 5′ ends of aptamer P9TA (5′-CCCGGCATGGTTGCGGAGCAGGAGTATAACACTACCATTG-3') and aptamer P9TC (5′- GTCAGGTCACAGTGACCTGATCAAAGTTAATG-3′) were modified by an azide group, and VH032-propargyl (HY126465, MedChemExpress) was modified by an alkyne.

Techniques: Competitive Binding Assay, Control, Negative Control

Journal: Journal of Medicinal Chemistry

Article Title: Leveraging Targeted Protein Degradation for G Protein-Coupled Receptors: The Development of CCR2 Molecular Degraders

doi: 10.1021/acs.jmedchem.5c02920

Figure Lengend Snippet: Compounds 18 and 19 developed as a negative control of CCR2 degrader 7 and 8 maintain CCR2 inhibitory activity and degradation capacity. (A) Chemical structures of compounds 18 and 19 . (B) Inhibition of CCL2-induced cellular response by the indicated compounds (10 μM), determined in xCELLigence assays using U2OS-CCR2 cells. Cellular response was derived from peak-height analysis of the vehicle-corrected, normalized CI xCELLigence traces within the first 6 min after stimulation. For comparison, cellular response was normalized to 10 nM CCL2 in the absence of any compound. Data are shown as mean ± SEM of two independent experiments performed in duplicate. (C, D) Kinetic degradation profiles of CCR2-HiBiT after treatment with multiple concentrations of compound 18 (C) or 19 (D). The kinetic profile was obtained in a real-time HiBiT detection assay, using HEK293-LgBiT cells transiently transfected with 2 μg CCR2-HiBiT. (E) Kinetic degradation profiles of CCR2-HiBiT after treatment with compound 8 in the presence of pomalidomide (100 μM) or CRBN-6-5-5-VHL (1 μM). Cells were pretreated for 2 h before addition of compound 8 and measurement of luminescence. Kinetic profiles were obtained in a real-time HiBiT detection assay, using HEK293-LgBiT cells transiently transfected with 2 μg of CCR2-HiBiT plasmid. (F) Bar graph showing the fractional RLU values after 12 or 24 h of measuring luminescence. Fractional RLU values were obtained from the real-times traces shown in panel (E). Dotted lines indicate either 100 or 50% CCL2 response. Data are shown as mean ± SEM of at least three independent experiments performed in duplicate or triplicate. Statistical differences between fractional RLU values of compound 8 in the absence and presence of pomalidomide or CRBN-6-5-5-VHL at different time points were analyzed using a two-way ANOVA with Dunnett’s posthoc test: * p < 0.05.

Article Snippet: Bortezomib (PS-341), MG-132, bafilomycin-A1 (Baf-A1), and chloroquine diphosphate were purchased from Selleck Chemicals (Bio-Connect, Huissen, The Netherlands); CRBN-6-5-5-VHL from MedChem Express (Bio-Connect, Huissen, The Netherlands); cycloheximide from LKT Laboratories (St. Paul, MN, USA); and chemokine ligand CCL2 from PeproTech (Cranbury, NJ, USA).

Techniques: Negative Control, Activity Assay, Inhibition, Derivative Assay, Comparison, Detection Assay, Transfection, Plasmid Preparation

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: mTORC1 phosphorylates and stabilizes LST2 to negatively regulate EGFR.

doi: 10.1073/pnas.2405959121

Figure Lengend Snippet: Fig. 2. LST2 is stabilized by mTORC1-mediated phosphorylation. (A) Immunoblots upon empty vector and LST2-WT, LST2-F401A, LST2-S670A, LST2-S670E overexpression in HEK293T cells. Media replenished 4 h before cell collection. ACTIN serves as a loading control. (B) Quantification of A. One-way ANOVA, N = 4, ****P < 0.0001. (C) Immunoblots upon LST2-WT, LST2-F401A, LST2-S670A, LST2-S670E overexpression in HeLa cells. Cells were treated with DMSO (Ctrl), 200 nM INK-128, or 10 µM MG132 for 20 h. ACTIN serves as a loading control. (D) Immunoblots upon LST2-WT overexpression in HEK293T cells treated with 100 µg/mL cycloheximide (CHX) with or without 200 nM INK-128 for the indicated times. ACTIN serves as a loading control. (E) Quantification of D. Nonlinear fit. Inhibitor vs. response (three parameters), N = 3. Multiple unpaired t test, N = 3, *P < 0.05, **P < 0.01. Statistical difference is shown comparing CHX + INK-128 to CHX in the illustrated time points. (F) Immunoblots upon LST2-F401A overexpression in HEK293T cells treated with 100 µg/mL cycloheximide (CHX) for the indicated times. ACTIN serves as a loading control. (G) Immunoblots upon LST2-S670A overexpression in HEK293T cells treated with 100 µg/mL cycloheximide (CHX) for the indicated times. ACTIN serves as a loading control. (H) Immunoblots upon LST2-S670E overexpression in HEK293T cells treated with 100 µg/mL cycloheximide (CHX) for the indicated times. ACTIN serves as a loading control. (I) Quantification of D, F, G, and H. Nonlinear fit. Inhibitor vs. response (three parameters), N = 3. Multiple unpaired t test, N = 3, *P < 0.05, ***P < 0.001. Statistical difference is shown for the specific mutant in comparison to WT in the illustrated time points.

Article Snippet: The following LST2 plasmids are available at Addgene: pCMV- MLG- EGFP- LST2 (#220785) (41), pCMVMLG- HMF- LST2 (#220786) (42), and pAceBAC2- HMF- LST2 (#220787) (43).

Techniques: Phospho-proteomics, Western Blot, Plasmid Preparation, Over Expression, Control, Mutagenesis, Comparison

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: mTORC1 phosphorylates and stabilizes LST2 to negatively regulate EGFR.

doi: 10.1073/pnas.2405959121

Figure Lengend Snippet: Fig. 3. Phosphorylation on S670 is required for LST2 ubiquitination. (A) Immunoblots upon empty vector and LST2-WT and LST2-K87R overexpression in HEK293T cells. Media replenished 4 h before cell collection. ACTIN serves as a loading control. (B) Immunoblots upon LST2-WT and LST2-K87R overexpression in HEK293T cells treated with 100 µg/mL cycloheximide (CHX) for the indicated times. ACTIN serves as a loading control. (C) Quantification of B. Nonlinear fit. Inhibitor vs. response (three parameters), N = 3. Multiple unpaired t test, N = 3, *P < 0.05, **P < 0.01. Quantification of WT is as in Fig. 2E. Statistical difference is shown in comparison to WT in the illustrated time points. (D) HMF tagged LST2-WT, LST2-K87R, LST2-F401A, LST2-S670A, and LST2-S670E overexpressed in HEK293T cells. LST2 immunoprecipitated with flag beads. Immunoblot of equally loaded LST2. Input in SI Appendix, Fig S4A. (E) Quantification of D. One-way ANOVA, N = 3, *P < 0.05, **P < 0.01. (F) Immunoblots upon LST2-WT, LST2-K87R, LST2-S670E, LST2-S670E-K87R overexpression in HEK293T cells. Media replenished 4 h before cell collection. ACTIN serves as a loading control. (G) Quantification of F. LST2 levels were first normalized to ACTIN then ratio made as indicated. One-way ANOVA, N = 3, ***P < 0.001, ****P < 0.0001.

Article Snippet: The following LST2 plasmids are available at Addgene: pCMV- MLG- EGFP- LST2 (#220785) (41), pCMVMLG- HMF- LST2 (#220786) (42), and pAceBAC2- HMF- LST2 (#220787) (43).

Techniques: Phospho-proteomics, Ubiquitin Proteomics, Western Blot, Plasmid Preparation, Over Expression, Control, Comparison, Immunoprecipitation

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: mTORC1 phosphorylates and stabilizes LST2 to negatively regulate EGFR.

doi: 10.1073/pnas.2405959121

Figure Lengend Snippet: Fig. 4. LST2 S670 phosphorylation promotes reticular distribution of LST2. (A) HeLa cells overexpressing mCherry tagged EEA1 in combination with GFP tagged LST2-WT, LST2-K87R, LST2-F401A, LST2-S670A, or LST2-S670E. Cells fixed in 4% PFA. In blue, DAPI staining. 2 µm bar scale. (B) Quantification of A. Manders’ coefficient LST2/EEA1, One-way ANOVA, N = 60, ****P < 0.0001. (C) HeLa cells overexpressing mCherry tagged LAMP1 in combination with GFP tagged LST2-WT, LST2-K87R, LST2-F401A, LST2-S670A, or LST2-S670E. Cells fixed in 4% PFA. In blue, DAPI staining. 2 µm bar scale. (D) Quantification of C. Manders’ coefficient LST2/LAMP1, One-way ANOVA, N = 60, ****P < 0.0001.

Article Snippet: The following LST2 plasmids are available at Addgene: pCMV- MLG- EGFP- LST2 (#220785) (41), pCMVMLG- HMF- LST2 (#220786) (42), and pAceBAC2- HMF- LST2 (#220787) (43).

Techniques: Phospho-proteomics, Staining

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: mTORC1 phosphorylates and stabilizes LST2 to negatively regulate EGFR.

doi: 10.1073/pnas.2405959121

Figure Lengend Snippet: Fig. 5. LST2 is a negative regulator of EGFR. (A) Immunoblot of WT and LST2-KO MDA-MB-231 cells. Cells serum-starved for 3 h and 100 ng/mL of EGF provided for the indicated time. CALNEXIN serves as a loading control. (B) Quantification of A. Two-way ANOVA, N = 3, ****P < 0.0001. (C) Immunoblot of WT and LST2-KO MDA-MB-231 cells. Cells were serum-starved for 3 h (Ctrl) and then stimulated for 30 min with 100 ng/mL of EGF. 20 µM AG-1478 or 10 µM PD153035 or 1:500 of DMSO (Vehicle) were provided 1 h before EGF as indicated. ACTIN serves as a loading control. (D) Quantification of C. Two-way ANOVA, N = 3. ns, no significance. (E) Schematic model of LST2 regulation by mTORC1. This figure has been generated with BioRender.com.

Article Snippet: The following LST2 plasmids are available at Addgene: pCMV- MLG- EGFP- LST2 (#220785) (41), pCMVMLG- HMF- LST2 (#220786) (42), and pAceBAC2- HMF- LST2 (#220787) (43).

Techniques: Western Blot, Control, Generated