Journal: Oncotarget

Article Title: AMPK-mediated up-regulation of mTORC2 and MCL-1 compromises the anti-cancer effects of aspirin

doi: 10.18632/oncotarget.7648

Figure Lengend Snippet: ( A ) Western blot analysis of the effect of aspirin on S6K1 phosphorylation. ( B ) HepG2 cells were treated with or without 5 mM aspirin and 10 μM compound C, followed by immunoprecipitation of the rictor-mTOR complex. The immunopreciptates were subjected to in vitro kinase assay using Akt2 as the substrate, followed by western blot analysis of rictor, mTOR, Akt2 and p-Akt2 (S474). ( C ) Western blot analysis of the effects of rictor knockdown on the induction of Akt, ERK1/2 phosphorylation and MCL-1 expression by 5 mM aspirin. ( D ) Western blot analysis of the induction of Akt and ERK1/2 phosphorylation by 5 mM aspirin in MCF-10A and rictor-null MCF-10A cells. A representative of two or more independent experiments was shown.

Article Snippet: The antibodies used were as follows: anti-MCL-1, Akt, p-Akt (S473), ERK1/2, p-ERK1/2(T202/Y204), AMPKα, p-AMPKα (T172), p-ACC (S79), p-mTOR (S2481), p-S6K1 (S371), c-Caspase-7, PARP (Cell Signaling Technology, Beverly, MA); rictor (Merk Millipore Corporation, Darmstadt, Germany); mTOR, Ki67 (Burlingame, CA, USA); actin (Proteintech, Chicago, IL, USA).

Techniques: Western Blot, Phospho-proteomics, Immunoprecipitation, In Vitro, Kinase Assay, Knockdown, Expressing

Journal: Oncology Letters

Article Title: Hypoxia‑induced SREBP1‑mediated lipogenesis and autophagy promote cell survival via fatty acid oxidation in breast cancer cells

doi: 10.3892/ol.2025.14921

Figure Lengend Snippet: Figure 2. SREBP1 enhances lipogenesis in MDA‑MB‑231 cells under hypoxic conditions. (A) Assessment of the amount of lipids using Nile Red staining by immunofluorescence with or without fatostatin in MCF‑7 and MDA‑MB‑231 cells under normoxic and hypoxic conditions for 48 h. Scale bar, 150 µm. The graphs indicate the quantitative analysis of fluorescence intensity. (B) Expression levels of hypoxia indicators, HIF‑1α, autophagy‑related markers, LC3‑I/II and lipogenesis‑related protein, SREBP1, in MCF‑7 and MDA‑MB‑231 cells under normoxic and hypoxic conditions for 48 h. The graphs indicate the semi‑quantitative analysis of HIF‑1α, LC3‑II and SREBP1 protein levels normalized to β‑actin expression. (C) Expression levels of SREBP1 and β‑actin proteins following treatment with different concentrations of fatostatin in MCF‑7 and MDA‑MB‑231 cells under hypoxic conditions for 48 h. *P<0.05; **P<0.01; ***P<0.001. HIF‑1α, hypoxia inducible factor‑1α; LC3, microtubule associated protein 1 light chain 3; SREBP1, sterol regulatory element‑binding protein 1; Nor, normoxia; Hypo, hypoxia; ns, not significant.

Article Snippet: Fatostatin, an SREBP1 inhibitor, and rapamycin, an autophagy inducer, were purchased from Sigma‐Aldrich (Merck KGaA) and Selleck Chemicals, respectively.

Techniques: Staining, Immunofluorescence, Fluorescence, Expressing

Journal: Oncology Letters

Article Title: Hypoxia‑induced SREBP1‑mediated lipogenesis and autophagy promote cell survival via fatty acid oxidation in breast cancer cells

doi: 10.3892/ol.2025.14921

Figure Lengend Snippet: Figure 3. Autophagy is activated via SREBP1 in MDA‑MB‑231 cells under hypoxic conditions. (A) Assessment of autophagy using LC3 staining by immu‑ nofluorescence with or without fatostatin in MCF‑7 and MDA‑MB‑231 cells under normoxic and hypoxic conditions for 48 h. Scale bar, 150 µm. The graphs indicate the quantitative analysis of fluorescence intensity. (B) Expression levels of SREBP1 and LC3‑I/II with or without fatostatin in MCF‑7 and MDA‑MB‑231 cells under hypoxic conditions for 48 h. The graphs indicate the semi‑quantitative analysis of SREBP1 and LC3‑II protein levels normalized to β‑actin expression. *P<0.05; **P<0.01; ***P<0.001. LC3, microtubule associated protein 1 light chain 3; SREBP1, sterol regulatory element‑binding protein 1; ns, not significant.

Article Snippet: Fatostatin, an SREBP1 inhibitor, and rapamycin, an autophagy inducer, were purchased from Sigma‐Aldrich (Merck KGaA) and Selleck Chemicals, respectively.

Techniques: Staining, Fluorescence, Expressing

Journal: Oncology Letters

Article Title: Hypoxia‑induced SREBP1‑mediated lipogenesis and autophagy promote cell survival via fatty acid oxidation in breast cancer cells

doi: 10.3892/ol.2025.14921

Figure Lengend Snippet: Figure 4. Reduced lipogenesis and autophagy in MDA‑MB‑231 cells under hypoxic conditions following SREBP1 knockdown. (A) Expression levels of precursor SREBP1 and LC3‑I/II with or without SREBP1 siRNA in MCF‑7 and MDA‑MB‑231 cells under normoxic and hypoxic conditions for 48 h. The graphs indicate the semi‑quantitative analysis of SREBP1and LC3‑II protein levels normalized to β‑actin expression. Assessment of the co‑localization of lipid accumulation and (B) autophagy using Nile Red and LC3 and (C) lysosomes using Nile Red and LAMP2 staining by immunofluorescence with or without SREBP1 siRNA in MCF‑7 and MDA‑MB‑231 cells under normoxic and hypoxic conditions for 48 h. Scale bar, 75 µm. *P<0.05; **P<0.01. LC3, microtubule associated protein 1 light chain 3; SREBP1, sterol regulatory element‑binding protein 1; LAMP2, lysosomal associated membrane protein 2; ns, not significant.

Article Snippet: Fatostatin, an SREBP1 inhibitor, and rapamycin, an autophagy inducer, were purchased from Sigma‐Aldrich (Merck KGaA) and Selleck Chemicals, respectively.

Techniques: Knockdown, Expressing, Staining, Immunofluorescence, Membrane

Journal: Oncology Letters

Article Title: Hypoxia‑induced SREBP1‑mediated lipogenesis and autophagy promote cell survival via fatty acid oxidation in breast cancer cells

doi: 10.3892/ol.2025.14921

Figure Lengend Snippet: Figure 7. SREBP1, analyzed through SREBF1 mRNA expression, serves as a prognostic marker for survival outcomes among patients with triple nega‑ tive breast cancer. The recurrence‑free survival and distant metastasis‑free survival rates of (A) ER+/PR‑/HER2‑(n=200 and 118, respectively) and (B) ER‑/PR‑/HER2‑(n=392 and 306, respectively) patients with breast cancer were analyzed over 250 months using Kaplan‑Meier plots to assess the impact of SREBF1 mRNA expression. SREBF1, sterol regulatory element‑binding transcription factor 1; ER, estrogen receptor; PR, progesterone receptor; HER2, human epidermal growth factor receptor 2; HR, hazard ratio.

Article Snippet: Fatostatin, an SREBP1 inhibitor, and rapamycin, an autophagy inducer, were purchased from Sigma‐Aldrich (Merck KGaA) and Selleck Chemicals, respectively.

Techniques: Expressing, Marker

Journal: bioRxiv

Article Title: MPS1 localizes to microtubule-attached kinetochores and actively promotes microtubule release

doi: 10.1101/2022.05.23.493048

Figure Lengend Snippet: ( A ) Diagram of the rapamycin-sensitive dimerization module used to recruit INCENP-Aurora B to the outer kinetochore protein Mis12. ( B ) Movie stills of a HeLa cell expressing Myc-MIS12-FKBPx3, mCherry-FRB-INCENP 47-918 and MPS1-GFP, and incubated with SiR-tubulin and Hoechst. Cells were treated with MG132 30 min prior to imaging to enrich for cells with fully attached metaphase plates. Cell at -1 min is at metaphase prior to addition of 500 nM rapamycin. ( C ) Quantification of kinetochore intensities of MPS1-GFP (green), mCherry-FRB-INCENP (red) or misaligned chromosomes (proportion of Hoechst fluorescence signal outside of the metaphase plate) (blue) in cells as shown in (B). Line shows the mean, the shaded areas are S.E.M. ( D ) HeLa cells as in (B), arrested for 2 hours with MG132 (20 μM) and stained with anti-Myc and anti-Astrin. Aurora B inhibitor ZM447439 was added at 10 μM for 10 min prior to fixing, rapamycin was added at 500 nM 2 min prior to fixation. ( E ) Average MPS1 cell intensities, normalized against Myc-Mis12 intensities, at kinetochores in cells treated as in (D) were plotted. Bars represent mean± S.E.M.

Article Snippet: Inhibitors were obtained from Tocris Bioscience (MPS1 inhibitor AZ3146 (3994); Aurora B Kinase Inhibitor ZM447439 (2458); PP1 and PP2A inhibitor Calyculin A (1336)), Sigma-Aldrich (Kinesin-5/Eg5 inhibitor S-trityl-L-cysteine (STLC) (164739-5G); Rapamycin (R0395)), Santa Cruz Biotechnology (Proteasome inhibitor MG132 (sc-201270)), Spirochrome (SiR-Tubulin (SC002)) and Merck Chemicals Ltd (microtubule polymerization inhibitor Nocodazole (484728-10MG)).

Techniques: Expressing, Incubation, Imaging, Fluorescence, Staining

Journal: bioRxiv

Article Title: MPS1 localizes to microtubule-attached kinetochores and actively promotes microtubule release

doi: 10.1101/2022.05.23.493048

Figure Lengend Snippet: ( A ) Live cell imaging stills of HeLa cells expressing MPS1-GFP and incubated with SiR-Tubulin and Hoechst, either with the Aurora-B kinetochore targeting system but without addition of Rapamycin (left), or without the Aurora-B kinetochore targeting system but with Rapamycin added. Cells were treated with 20μM MG132 30 min prior to imaging. Cell at - 1 min is at metaphase before addition of 500nM Rapamycin. In neither case is MPS1-GFP recruited to attached kinetochores, nor is chromosome alignment altered. ( B ) Quantification of kinetochore MPS1-GFP or mCherry-FRB-INCENP intensities and level of chromosome misalignment in cells treated as in (A). Lines represent mean, coloured bands represent S.E.M. ( C ) HeLa MPS1-GFP cells with the Aurora-B kinetochore targeting system were arrested with 0.6 μM Nocodazole or 20μm MG132 for 2 hrs, and 500 nM Rapamycin was added for 2 min prior to fixation. The cells were immuno-stained as indicated. ( D ) Plot of MPS1 kinetochore mean cell intensities from cells in (C). Bars show mean± S.E.M. ( E ) Plot of MAD1 kinetochore mean cell intensities from cells in (C). Bars show mean± S.E.M. ( F ) HeLa MPS1-GFP cells with the Aurora-B kinetochore targeting system were treated as in (C) and then cooled to 4°C for 9 min before fixation and staining as indicated. ( G ) The proportion of mitotic cells with cold-stable bipolar spindles in each condition from 3 experiments is shown. Error bars show S.D.

Article Snippet: Inhibitors were obtained from Tocris Bioscience (MPS1 inhibitor AZ3146 (3994); Aurora B Kinase Inhibitor ZM447439 (2458); PP1 and PP2A inhibitor Calyculin A (1336)), Sigma-Aldrich (Kinesin-5/Eg5 inhibitor S-trityl-L-cysteine (STLC) (164739-5G); Rapamycin (R0395)), Santa Cruz Biotechnology (Proteasome inhibitor MG132 (sc-201270)), Spirochrome (SiR-Tubulin (SC002)) and Merck Chemicals Ltd (microtubule polymerization inhibitor Nocodazole (484728-10MG)).

Techniques: Live Cell Imaging, Expressing, Incubation, Imaging, Staining

Journal: bioRxiv

Article Title: MPS1 localizes to microtubule-attached kinetochores and actively promotes microtubule release

doi: 10.1101/2022.05.23.493048

Figure Lengend Snippet: ( A ) HeLa MPS1-GFP cells expressing the Aurora-B kinetochore targeting system were arrested with 20 μm MG132 for 2 hrs, and 500 nM Rapamycin, in the presence or absence of the indicated kinase inhibitors, was added for the indicated time prior to a 9 mins cold treatment followed by fixation and immuno-staining as indicated. ( B ) The proportion of mitotic cells with cold-stable bipolar spindles in each condition at different times after rapamycin addition is shown.

Article Snippet: Inhibitors were obtained from Tocris Bioscience (MPS1 inhibitor AZ3146 (3994); Aurora B Kinase Inhibitor ZM447439 (2458); PP1 and PP2A inhibitor Calyculin A (1336)), Sigma-Aldrich (Kinesin-5/Eg5 inhibitor S-trityl-L-cysteine (STLC) (164739-5G); Rapamycin (R0395)), Santa Cruz Biotechnology (Proteasome inhibitor MG132 (sc-201270)), Spirochrome (SiR-Tubulin (SC002)) and Merck Chemicals Ltd (microtubule polymerization inhibitor Nocodazole (484728-10MG)).

Techniques: Expressing, Immunostaining

Journal: bioRxiv

Article Title: MPS1 localizes to microtubule-attached kinetochores and actively promotes microtubule release

doi: 10.1101/2022.05.23.493048

Figure Lengend Snippet: ( A ) Movie stills of a HeLa cell expressing Myc-Mis12-FKBPx3, mCherry-FRB-INCENP 47-918 and MPS1-GFP, incubated with SiR-Tubulin (magenta) and Hoechst (blue). Cells were treated with 20 μM MG132 30 min prior to imaging. Cell at -1 min is at metaphase prior to addition of 500 nM rapamycin. MPS1i AZ3146 was added at 2 μM for 10 min prior to imaging. ( B ) Quantification of misaligned chromosomes (blue) in cells as shown in (A). ( C ) Live cell imaging stills of HeLa cells expressing HEC1-mCherry and MPS1-GFP, incubated with SiR-Tubulin and 10 μM STLC prior to imaging. Shown is an example of an initially syntelically attached kinetochore pair, where MPS1-GFP localization precedes error correction and attachment loss at one kinetochore. ( D ) 136 kinetochores of the cells in (C) were assessed for kinetochore attachment status and presence of absence of MPS1-GFP over a 10-minute period. ( E ) Immunofluorescence analysis of control or PP2A-B56 depleted HeLa MPS1-GFP cells treated with the indicated kinase inhibitors before cold-treatment. ( F ) Quantitation of cells treated as in (F), n =3. ( G ) Incoherent feed-forward loop describing MPS1 and PP2A-B56 kinetochore recruitment and effector function. ( H ) Model for MPS1 recruitment and function.

Article Snippet: Inhibitors were obtained from Tocris Bioscience (MPS1 inhibitor AZ3146 (3994); Aurora B Kinase Inhibitor ZM447439 (2458); PP1 and PP2A inhibitor Calyculin A (1336)), Sigma-Aldrich (Kinesin-5/Eg5 inhibitor S-trityl-L-cysteine (STLC) (164739-5G); Rapamycin (R0395)), Santa Cruz Biotechnology (Proteasome inhibitor MG132 (sc-201270)), Spirochrome (SiR-Tubulin (SC002)) and Merck Chemicals Ltd (microtubule polymerization inhibitor Nocodazole (484728-10MG)).

Techniques: Expressing, Incubation, Imaging, Live Cell Imaging, Immunofluorescence, Control, Quantitation Assay

Journal: bioRxiv

Article Title: 3D vascularized microtumors unveil aberrant ccRCC vasculature and differential sensitivity to targeted treatments

doi: 10.1101/2025.03.27.645644

Figure Lengend Snippet: HUVEC (A and B), RCC4 spheroids (C and D) or co-culture of HUVEC and RCC4 spheroids (E-G) were embedded in collagen I hydrogel and treated by temsirolimus (5, 10 or 25 µM), crizotinib (0.05 or 0.5 µM), sunitinib (0.05, 0.5 or 5 µM) or vehicle (0) in NHDF-conditioned medium for 2 days. A: Capillaries immunostained for CD31 (red) and stained bt DAPI for nuclei (blue). Scale bar: 100 µm. B: 3D quantification of total capillary length, branch point number and connectivity index of capillary network. Results were normalized to control condition (vehicle). C: RCC4 spheroids stained for F-actin by phalloidin (green) and for nuclei by DAPI (red). Scale bar: 100 µm. D: Quantification of area and perimeter of spheroids. Results were normalized to control condition (vehicle). E: Vascularized fluorescent RCC4 spheroids (green, mid- and bottom panels) immunostained for CD31 (red, top and bottom panels) and stained by DAPI for nuclei (blue, bottom panels). Scale bar: 100 µm. F: 3D quantification of total capillary length, branch point number and connectivity index of the capillary network in the vascularized microtumors. Results were normalized to control condition (vehicle). G: Quantification of area and perimeter of spheroids. Results were normalized to control condition (vehicle). Graphs represent the mean of 3 independent experiments +/- SEM. Statistical analyses were performed over control condition using Kruskal-Wallis test (B, D, F and G). ns p>0.05, * p<0.05, ** p<0.01, *** p<0.001, $ p<0.00001.

Article Snippet: To assess the effects of drugs on tumor invasion, capillary or pond formation, RCC4 spheroids and HUVECs, either alone or in co-culture were treated with vehicle (dimethyl sulfoxide, DMSO), temsirolimus (ThermoFisher Scientific, #J63654.MB; 5, 10 or 25 µM, crizotinib (Merck Millipore, #PZ0191; 0.05 or 0.5 µM) or sunitinib (Merck Millipore, #1642358; 0.05, 0.5 or 5 µM).

Techniques: Co-Culture Assay, Staining, Control

Journal: Oncology Letters

Article Title: Nucleobindin-2 enhances the epithelial-mesenchymal transition in renal cell carcinoma

doi: 10.3892/ol.2020.11526

Figure Lengend Snippet: AMPK and TORC1 pathways are critical for regulating the NUCB-2-mediated inhibition of migration and invasion. (A) Effect of NUCB-2-KO on proteins involved in the AMPK and TORC1 pathways. (B) Densitometry analysis of the ratio of the phosphorylated variant to the total expression. Expression was first normalized to GAPDH. *P<0.05, **P<0.01. NUCB-2, nucleobindin 2; AMPK, AMP-dependent protein kinase; TORC1, target of rapamycin complex; ACC, acetyl-CoA carboxylase; S6, ribosomal S6 kinases; 4eBP1, 4E-BP1, eIF4E-binding protein 1; p-, phospho; t-, total; KO, knockout.

Article Snippet: Rapamycin, dorsomorphin and MTT were purchased from Sigma-Aldrich; Merck KGaA.

Techniques: Inhibition, Migration, Variant Assay, Expressing, Binding Assay, Knock-Out

Journal: Oncology Letters

Article Title: Nucleobindin-2 enhances the epithelial-mesenchymal transition in renal cell carcinoma

doi: 10.3892/ol.2020.11526

Figure Lengend Snippet: NUCB-2 upregulates epithelial-mesenchymal transition in renal cell carcinoma through the AMPK/TORC1 pathway. (A) SK-RC-52 cells with NUCB-2-KO were treated with 40-µM dorsomorphin or control for 100 min. (B) Effect of the inhibitor on members of the AMPK or TORC1 pathways. Densitometry analysis of the western blots in the left panel. The relative levels of protein levels normalized to those of GAPDH. *P<0.05, **P<0.01 SK-RC-52 vs. NUCB-2 KO cells; # P<0.05, ## P<0.01 NUCB-2 KO vs. NUCB-2 KO cells treated with an AMPK inhibitor. (C) Relative cell migration in the three groups. *P<0.05, # P<0.05. (D) Number of invaded cells transferred in the Transwell assay in the three groups. When cells were treated with an AMPK inhibitor, the migratory and invasive abilities of NUCB-2-KO stable clones was increased. *P<0.05, # P<0.05. NUCB-2, nucleobindin 2; AMPK, AMP-dependent protein kinase; TORC1, target of rapamycin complex; ACC, acetyl-CoA carboxylase; S6, ribosomal S6 kinases; 4eBP1, 4E-BP1, eIF4E-binding protein 1; p-, phospho; t-, total; KO, knockout.

Article Snippet: Rapamycin, dorsomorphin and MTT were purchased from Sigma-Aldrich; Merck KGaA.

Techniques: Control, Western Blot, Migration, Transwell Assay, Clone Assay, Binding Assay, Knock-Out

Journal: Oncology Letters

Article Title: Nucleobindin-2 enhances the epithelial-mesenchymal transition in renal cell carcinoma

doi: 10.3892/ol.2020.11526

Figure Lengend Snippet: Rapamycin inhibits EMT in renal cell carcinoma through the TORC1/ZEB1 pathway. (A) Western blot of EMT markers in SK-RC-52 cells treated with or without rapamycin. (B) Densitometry analysis of western blots normalized to GAPDH or β-tubulin. *P<0.05, **P<0.01. EMT, epithelial-mesenchymal transition; ZEB1, zinc finger E-box binding to homeobox 1.

Article Snippet: Rapamycin, dorsomorphin and MTT were purchased from Sigma-Aldrich; Merck KGaA.

Techniques: Western Blot, Binding Assay