Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

Article Title: Everolimus Triggers Cytokine Release by Macrophages

doi: 10.1161/atvbaha.112.245381

Figure Lengend Snippet: Figure 1. mTOR inhibition affects de novo protein synthesis. A and B, Mouse macrophages were incubated in serum-containing RPMI medium (control), Earle Balanced Salt Solution (EBSS, starvation) or RPMI supplemented with 10 mmol/L everoli mus. Phospho-p70 S6 kinase (P-p70S6K, Thr389) (A), and phospho-eukaryotic initiation factor 2 (P-eIF2, Ser51) (B) were analyzed by Western blotting. **P0.01, ***P0.001 versus con trol (0 minutes) (1-way ANOVA, followed by Dunnett test, n3). C, Mouse macrophages were incubated in serum-containing RPMI (control), EBSS (starvation), or RPMI supplemented with 10 mmol/L everolimus or 10 mg/mL cycloheximide (CHX) for 2 hours in the presence of L-35S-methionine/cysteine. 35S-labeled proteins were measured with scintillation counting. *P0.05, ***P0.001 versus control (1-way ANOVA, followed by Dunnett test, n7).

Article Snippet: Macrophages were then starved in Earle Balanced Salt Solution (EBSS; Invitrogen, Carlsbad, CA) or treated with 10 mol/L everolimus (Novartis, Basel, Switzerland) in RPMI 1640 medium (Invitrogen).

Techniques: Inhibition, Incubation, Control, Western Blot, Labeling

Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

Article Title: Everolimus Triggers Cytokine Release by Macrophages

doi: 10.1161/atvbaha.112.245381

Figure Lengend Snippet: Figure 2. Everolimus-induced mTOR inhibition stimulates phosphorylation of p38 MAPK and its substrates ATF2 and cPLA2. Mouse macrophages were starved in Earle Balanced Salt Solution (EBSS) or treated with 10 mmol/L everoli mus in RPMI medium for up to 8 hours. Phosphorylation of p38 MAPK (Thr180/ Tyr182) (A), activating transcription factor 2 (ATF2, Thr71) (B), cellular phospho lipase A2 (cPLA2) (C), and NF-B p65 (Ser536) (D) was analyzed by Western blotting. Treatment of cells with 1 mg/ mL LPS was used as positive control for NF-B p65 activation. *P0.05, ***P0.001 versus control (0 minutes) (1-way ANOVA, followed by Dunnett test, n53).

Article Snippet: Macrophages were then starved in Earle Balanced Salt Solution (EBSS; Invitrogen, Carlsbad, CA) or treated with 10 mol/L everolimus (Novartis, Basel, Switzerland) in RPMI 1640 medium (Invitrogen).

Techniques: Inhibition, Phospho-proteomics, Western Blot, Positive Control, Activation Assay, Control

Journal: Oncogene

Article Title: Stem cell-like transcriptional reprogramming mediates metastatic resistance to mTOR inhibition

doi: 10.1038/onc.2016.427

Figure Lengend Snippet: mTORC1 activity concurrent with enhanced metastatic potential. ( a ) Left panels, representative immunohistochemical scores (0, negative, to 3, highest expression) of pS6 staining in the tissue microarray (TMA) of primary breast tumors. Right panel, results for the association between pS6 staining and distant metastasis. ( b ) Increased expression of mTORC1 pathway components with enhanced metastatic potential of MDA-MB-231 cells. The loading control (α-tubulin, TUBA) is shown. Bottom panel, graph showing quantifications of protein levels relative to parental and TUBA (per sample). ( c ) Increased pS6 expression in lung metastases developed by LM2 cells. The arrows mark magnified fields. Right panel, box-and-whisker plots for the quantification (pixels/area, p/a) of pS6 intensity; three mice and three similar lung metastases were analyzed in each setting. The P -value of the two-tailed Mann–Whitney test is shown. ( d ) Left panel, graph showing the in vivo photon flux quantification in mice injected with LM2 and treated with DMSO or everolimus. Representative images from bioluminescence in lungs from DMSO- or everolimus-treated mice are shown. The scale bar depicts the range of photon flux values as a pseudo-color display, with red and blue representing high and low values, respectively. Right top panels, quantification of lung colonization (total metastasis area normalized per total lung area, based on HE). Right bottom panels, representative immunohistochemical results for pS6 and quantification of normalized intensities.

Article Snippet: Balb/C mice were injected subcutaneously with 5 × 10 4 4T1 cells, measured for tumor size at day 10, and randomly organized in two equivalent groups that were treated with DMSO solution (the same concentration as for the compound test) or everolimus (5 mg/kg; SC-218452, Santa Cruz Biotechnology) daily by intraperitoneal injection.

Techniques: Activity Assay, Immunohistochemical staining, Expressing, Staining, Microarray, Control, Whisker Assay, Two Tailed Test, MANN-WHITNEY, In Vivo, Injection

Journal: Oncogene

Article Title: Stem cell-like transcriptional reprogramming mediates metastatic resistance to mTOR inhibition

doi: 10.1038/onc.2016.427

Figure Lengend Snippet: Metastatic resistance to mTOR inhibition. ( a ) Left panels, graphs showing the average and standard deviation of micro- and macro-metastases observed in the lungs of the DMSO- and sirolimus- or everolimus-treated orthoxenografts and 4T1 tumors, respectively. The results correspond to the last day of treatment, and micro- versus macro-metastases were defined using a 2 mm width threshold, and by examining at least three tissue levels separated by > 20 μ M . Right panels, growth rates of the DMSO- and sirolimus- or everolimus-treated tumors. ( b ) Representative immunohistochemical results for pS6 at the invasive tumor fronts (magnifications; top right panels) and the lung metastases (right panels) of DMSO- or sirolimus/everolimus-treated mice. The middle panels show quantifications, which correspond to three tumors, three equal front areas, and three metastases in each case. ( c ) Representative immunohistochemical tumor results for FSCN1/Fscn1 in DMSO- or sirolimus/everolimus-treated mice; quantifications are shown in right panels. ( d ) Recovered pS6 signal with concurrent FSCN1 overexpression through adaptation to everolimus in MCF7 and HCC1937 cells. Days of treatment are shown. ( e ) Left panel, graph showing the quantification of colonies from untreated and everolimus-adapted cells (12 culture fields were analyzed). The one-tailed t -test P -values are shown. Representative images of cell cultures are shown in right panels. ( f ) Flow cytometry results showing the cell counts for CD49f/EpCAM and of CD44/CD24 positivity in untreated or everolimus-adapted MCF7 and HCC1937 cells, respectively.

Article Snippet: Balb/C mice were injected subcutaneously with 5 × 10 4 4T1 cells, measured for tumor size at day 10, and randomly organized in two equivalent groups that were treated with DMSO solution (the same concentration as for the compound test) or everolimus (5 mg/kg; SC-218452, Santa Cruz Biotechnology) daily by intraperitoneal injection.

Techniques: Inhibition, Standard Deviation, Immunohistochemical staining, Over Expression, One-tailed Test, Flow Cytometry

Journal: Oncogene

Article Title: Stem cell-like transcriptional reprogramming mediates metastatic resistance to mTOR inhibition

doi: 10.1038/onc.2016.427

Figure Lengend Snippet: Co-expression analysis and stem cell-like signatures. ( a ) TCGA network of significant co-expression levels (PCC P -values <0.05) between TSC1 or TSC2 and signatures derived from stem cell-like cell studies . The nodes represent TSC1/2 and the signatures, and the edges positive (red) or negative (green) correlations. Edge width is proportional to the corresponding PCC value. ( b ) Clustering correlation of sESC, sMRS and sMYC. The ortho-xenografts are differentially clustered relative to the treatment, and a similar trend is observed for 4T1 tumors. ( c ) The master regulators of the sMRS (that is, Core-9) are found to be relatively overexpressed upon mTOR inhibition. ( d ) Significant overexpression of sESC, sMRS and sMYC in regrown ortho-xenografts after sirolimus treatment. The GSEA ESs and the nominal P -values are shown. ( e ) Most of the regulators of sMRS are relatively overexpressed in everolimus-adapted HCC1937 cells. ( f ) HMGA1, which is encoded in Core-9, is upregulated upon adaptation to mTOR inhibition, particularly in HCC1937 cells.

Article Snippet: Balb/C mice were injected subcutaneously with 5 × 10 4 4T1 cells, measured for tumor size at day 10, and randomly organized in two equivalent groups that were treated with DMSO solution (the same concentration as for the compound test) or everolimus (5 mg/kg; SC-218452, Santa Cruz Biotechnology) daily by intraperitoneal injection.

Techniques: Expressing, Derivative Assay, Inhibition, Over Expression

Journal: Oncogene

Article Title: Stem cell-like transcriptional reprogramming mediates metastatic resistance to mTOR inhibition

doi: 10.1038/onc.2016.427

Figure Lengend Snippet: EVI1 couples stemness, metastatic potential and resistance to mTOR inhibition. ( a ) TCGA network of significant co-expression (PCC P -values <0.05) between EVI1 and signatures derived from stem cell-like cells and/or metastatic settings . ( b ) Distributions of PCCs between EVI1 and the commonly overexpressed 79 genes across the studied models or the complete microarray gene list as background control. The P -value of the Mann–Whitney test for the comparison of the distributions is shown. ( c ) Reduced pS6 levels with EVI1 depletion in cell models. The quantification of pS6/S6 signal ratios is show at the bottom (relative to siControl). ( d ) Ectopic overexpression of GFP-EVI1 in MCF7 (left panels) and HCC1937 (right panels) cells provides higher viability upon exposure to everolimus, relative to GFP-only overexpression. Also shown are the western blot results for defined markers across the drug-exposed cell cultures. The quantification of pS6/S6 signal ratios is show at the bottom (relative to TUBA per sample). ( e ) Increased EVI1 binding at predicted target promoters/gene loci with adaptation to everolimus. The fold changes are relative to the immunoglobulin control and the promoter gene targets are shown in the X axis. ( f ) Relative overexpression of RAPTOR and/or RHEB with adaptation to everolimus in MCF7 and HCC1937 cells. The quantification is show at the bottom (relative to untreated and TUBA per sample). ( g ) Relative reduction of RAPTOR and RHEB expression following EVI1 depletion, in particular in the everolimus-adapted setting.

Article Snippet: Balb/C mice were injected subcutaneously with 5 × 10 4 4T1 cells, measured for tumor size at day 10, and randomly organized in two equivalent groups that were treated with DMSO solution (the same concentration as for the compound test) or everolimus (5 mg/kg; SC-218452, Santa Cruz Biotechnology) daily by intraperitoneal injection.

Techniques: Inhibition, Expressing, Derivative Assay, Microarray, Control, MANN-WHITNEY, Comparison, Over Expression, Western Blot, Binding Assay

Journal: Oncogene

Article Title: Stem cell-like transcriptional reprogramming mediates metastatic resistance to mTOR inhibition

doi: 10.1038/onc.2016.427

Figure Lengend Snippet: EVI1 cooperates with SOX9 and regulates its expression. ( a ) TCGA network of significant co-expression (PCC P -values <0.05) between EVI1 or SOX9 and signatures derived from stem cell-like cells and/or metastatic settings . ( b ) Increased SOX9 expression in ortho-xenograft tumor fronts of mice treated with sirolimus; the results correspond to at least three ortho-xenografts of each group. ( c ) Increased SOX9 and ALDH1 expression in everolimus-adapted cells. ( d ) Graph showing the results from the analysis of the complete drug panel for the correlation between IC 50 profiles and the expression of the V$EVI1_02 gene set; drugs are ranked according to PCC log P -values. Negative and positive PCCs are indicated with different colors, and the mTOR inhibitors in the panel are denoted. ( e ) Left panel, unsupervised clustering and correlation analysis of the difference in EVI1 ChIP results at the SOX9 locus between everolimus-adapted and untreated cells. Right panels, results of ChIP assays targeting a predicted EVI1-binding site in the SOX9 promoter ; the input, control immunoglobulin immunoprecipitation (IP), and EVI1-IP results are shown. The control results for the binding site in FSCN1 are also shown. ( f ) Depletion of EVI1 leads to a reduction of SOX9 expression in three cell conditions (the results correspond to ; the ratios are relative to siControl and TUBA per sample). ( g ) Depletion of EVI1 leads to a reduction of colony-forming capacity. The results of the one-tailed t -test are shown. ( h ) Depletion of Evi1 impairs the tumorigenic potential of 4T1 cells. The log-rank P -value is shown for the comparison between the shControl and short hairpin RNA (shRNA)- EVI1 #1; note that transduction with shRNA- EVI1 #2 completely impaired tumor formation so a P- value could not be computed (n.a.).

Article Snippet: Balb/C mice were injected subcutaneously with 5 × 10 4 4T1 cells, measured for tumor size at day 10, and randomly organized in two equivalent groups that were treated with DMSO solution (the same concentration as for the compound test) or everolimus (5 mg/kg; SC-218452, Santa Cruz Biotechnology) daily by intraperitoneal injection.

Techniques: Expressing, Derivative Assay, Binding Assay, Control, Immunoprecipitation, One-tailed Test, Comparison, shRNA, Transduction

Journal: Oncogene

Article Title: Stem cell-like transcriptional reprogramming mediates metastatic resistance to mTOR inhibition

doi: 10.1038/onc.2016.427

Figure Lengend Snippet: In vivo assessment of the role of EVI1 and SOX9. ( a , b ) Depletion of EVI1 / Evi11 expression (using two different short hairpin RNAs (shRNAs)) in LM2 and 4T1 cells reduced lung colonization, and Sox9 overexpression partially recovered this potential, left panels. ( a ) Right panels show representative images of lungs and their respective HE staining. ( c ) Depletion of SOX9 and overexpression of Sox9 reduced and increased, respectively, lung colonization of LM2 cells. Treatment with everolimus of shControl LM2 cells also reduced lung colonization. ( d ) Depletion of Sox9 or treatment with everolimus of 4T1 cells reduced lung colonization. ( e , f ) Depletion of FSCN1 / Fscn1 expression in LM2 and 4T1 cells reduced lung colonization, and concurrent treatment with everolimus further impaired this potential.

Article Snippet: Balb/C mice were injected subcutaneously with 5 × 10 4 4T1 cells, measured for tumor size at day 10, and randomly organized in two equivalent groups that were treated with DMSO solution (the same concentration as for the compound test) or everolimus (5 mg/kg; SC-218452, Santa Cruz Biotechnology) daily by intraperitoneal injection.

Techniques: In Vivo, Expressing, Over Expression, Staining

Journal: Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology

Article Title: Molecular Pathways Leading to Induction of Cell Death and Anti-Proliferative Properties by Tacrolimus and mTOR Inhibitors in Liver Cancer Cells.

doi: 10.33594/000000230

Figure Lengend Snippet: Fig. 5. LC3II/LC3I ratio and Beclin protein expressions in Tacrolimus-, Sirolimus- and Everolimus-treated HepG2 (A and B, respectively) and Huh7 (C and D, respectively) cells. Treatments were administered at different concentrations (0, 10 nM, 10 µM, and 100 µM). The protein expression of LC3II, LC3I and Beclin (6 h) was determined by Western‐blot analysis described in Material and Methods. Results are expressed as mean ± SEM, and blots are representative of three to five independent experiments. *p ≤ 0.05, **p ≤ 0.01, and ***p ≤ 0.001 between control and immunosuppressant‐treated cells. The groups with different letters (a, b, c, d, or e) were significantly different (p ≤ 0.05).

Article Snippet: Tacrolimus (Ref AT23293, Carbosynth Limited, Berkshire, United Kingdom), Sirolimus (Ref 37094, Sigma-Aldrich, Sant Louis, Missouri, USA), and Everolimus (Ref FE23209, Carbosynth Limited) were dissolved in DMSO (95.8, 91.5 and 80.4 μg/μl, respectively) in order to obtain working solution useful for all the experimental work.

Techniques: Expressing, Western Blot, Control

Journal: Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology

Article Title: Molecular Pathways Leading to Induction of Cell Death and Anti-Proliferative Properties by Tacrolimus and mTOR Inhibitors in Liver Cancer Cells.

doi: 10.33594/000000230

Figure Lengend Snippet: Fig. 6. Thr172P-Cdk4/Cdk4 (A) and Ser15P-p53/p53 (B) ratios in Tacrolimus-, Sirolimus- and Everolimus-treated HepG2 cells; as well as p21 protein expression in Tacrolimus-, Sirolimus- and Everolimus-treated HepG2 (C) and Huh7 (D). Treatments were administered at different concentrations (0, 10 nM, 10 µM, and 100 µM). The protein expression of Thr172P-Cdk4 and Cdk4 (6 h), Ser15P-p53 and p53 (12 h), and p21 (12 h) was determined by Western‐blot analysis. Results are expressed as mean ± SEM, and blots are representative of three to five independent experiments. *p ≤ 0.05, **p ≤ 0.01 and ***p ≤ 0.001 between control and immunosuppressant‐ treated cells. The groups with different letters (a, b, c, d or e) were significantly different (p ≤ 0.05).

Article Snippet: Tacrolimus (Ref AT23293, Carbosynth Limited, Berkshire, United Kingdom), Sirolimus (Ref 37094, Sigma-Aldrich, Sant Louis, Missouri, USA), and Everolimus (Ref FE23209, Carbosynth Limited) were dissolved in DMSO (95.8, 91.5 and 80.4 μg/μl, respectively) in order to obtain working solution useful for all the experimental work.

Techniques: Expressing, Western Blot, Control

Journal: Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology

Article Title: Molecular Pathways Leading to Induction of Cell Death and Anti-Proliferative Properties by Tacrolimus and mTOR Inhibitors in Liver Cancer Cells.

doi: 10.33594/000000230

Figure Lengend Snippet: Fig. 7. FKBP12 and FKBP51 protein expressions in Tacrolimus-, Sirolimus- and Everolimus-treated HepG2 (A and B, respectively) and Huh7 (C and D, respectively) cells. Treatments were administered at different concentrations (0, 10 nM, 10 µM, and 100 µM). The protein expression of FKBP12 and FKBP51 was evaluated by Western‐blot analysis as described in Material and Methods. Results are expressed as mean ± SEM, and blots are representative of four to six independent experiments. *p ≤ 0.05 between control and immunosuppressant‐treated cells. The groups with different letters (a, b, c or d) were significantly different (p ≤ 0.05).

Article Snippet: Tacrolimus (Ref AT23293, Carbosynth Limited, Berkshire, United Kingdom), Sirolimus (Ref 37094, Sigma-Aldrich, Sant Louis, Missouri, USA), and Everolimus (Ref FE23209, Carbosynth Limited) were dissolved in DMSO (95.8, 91.5 and 80.4 μg/μl, respectively) in order to obtain working solution useful for all the experimental work.

Techniques: Expressing, Western Blot, Control

Journal: Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology

Article Title: Molecular Pathways Leading to Induction of Cell Death and Anti-Proliferative Properties by Tacrolimus and mTOR Inhibitors in Liver Cancer Cells.

doi: 10.33594/000000230

Figure Lengend Snippet: Fig. 9. Impact of FKBP51 downregulation on BrdU incorporation (A) and caspase-3 activity (B) in Tacrolimus-, Sirolimus- and Everolimus-treated HepG2 cells. The downregulation of FKBP51 was carried using siRNA technologies. Cell proliferation and apoptosis were determined using commercial BrdU incorporation and caspase‐3 activity assays respectively as described in Material and Methods. Results are expressed as mean ± SEM of six independent experiments. *p ≤ 0.05 and **p ≤ 0.01 between control and immunosuppressant‐ treated cells. The groups with different letters (a, b, c, d, e or f) were significantly different (p ≤ 0.05).

Article Snippet: Tacrolimus (Ref AT23293, Carbosynth Limited, Berkshire, United Kingdom), Sirolimus (Ref 37094, Sigma-Aldrich, Sant Louis, Missouri, USA), and Everolimus (Ref FE23209, Carbosynth Limited) were dissolved in DMSO (95.8, 91.5 and 80.4 μg/μl, respectively) in order to obtain working solution useful for all the experimental work.

Techniques: BrdU Incorporation Assay, Activity Assay, Control

Journal: Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology

Article Title: Molecular Pathways Leading to Induction of Cell Death and Anti-Proliferative Properties by Tacrolimus and mTOR Inhibitors in Liver Cancer Cells.

doi: 10.33594/000000230

Figure Lengend Snippet: Fig. 10. Up- (red) and down- regulation (green) of miRNA expression in Tacrolimus-, Sirolimus- and Everolimus- treated HepG2 cells. Treatments were administered at 10 µM final concentration. The expression of miRNA was analysed using a commercial TaqMan® OpenArray® Human miRNA Panel as described in Material and Methods. miRNA data are obtained from three independent experiments.

Article Snippet: Tacrolimus (Ref AT23293, Carbosynth Limited, Berkshire, United Kingdom), Sirolimus (Ref 37094, Sigma-Aldrich, Sant Louis, Missouri, USA), and Everolimus (Ref FE23209, Carbosynth Limited) were dissolved in DMSO (95.8, 91.5 and 80.4 μg/μl, respectively) in order to obtain working solution useful for all the experimental work.

Techniques: Expressing, Concentration Assay

Journal: Oncogene

Article Title: mTOR inhibition suppresses Myc-driven polyposis by inducing immunogenic cell death

doi: 10.1038/s41388-023-02706-6

Figure Lengend Snippet: APC Min/+ mice were fed control (Ctrl) diet from 4–12 weeks to established polyps followed by Ctrl diet with or without Everolimus (Ever) till week 48. A Schematic of polyp establishment, treatment, and harvest. The small intestine (SI), colon and polyps were analyzed on week 12 (12 w), day 3 or 14 of the treatment (3d, 14d later), and week 48 (48 w, 36 w treatment). B Kaplan–Meier curve of APC Min /+ mice at 48 w. Log-Rank test. C Representative images of whole mount small intestine and colon from mice. Bar = 1 cm. Polyps are indicated with arrows. D Quantitation of total macroscopic adenomas from ( C ). E Quantitation of macroscopic adenomas in different regions from ( C ). n = 10 or 6 mice/group. F Representative Ki67 IHC staining at 3d. Bar = 100 µm. G Quantitation of positive cells per 400× field from ( F ). H Representative IF staining of the indicated proteins in polyps at d3. Bars = 100 µm. I Quantitation of positive cells per 400× field from ( H ). Nuclear Myc was scored. D , E n = 6–10 mice/group. G , I , n = 3 mice/group, and 3–4 randomly chosen polyps/mouse. *** P < 0.001, **** P < 0.0001 (Student’s T -Test, two-tailed). + P < 0.05, ++++ P < 0.0001 (One-way ANOVA and Tukey Post-Hoc test).

Article Snippet: For drug treatment, cells were plated in 12-well plates at ∼30% density 24 h before Everolimus (LC Laboratories) treatment at 10 μM.

Techniques: Control, Quantitation Assay, Immunohistochemistry, Staining, Two Tailed Test

Journal: Oncogene

Article Title: mTOR inhibition suppresses Myc-driven polyposis by inducing immunogenic cell death

doi: 10.1038/s41388-023-02706-6

Figure Lengend Snippet: APC Min/+ mice with established polyps were treated with Everolimus for 3 days [3d]. Small intestinal polyps were analyzed. A Representative phospho-eIF2α (S51, p-eIF2α) and Cleaved-caspase-8 (c-Cas8) IF staining in the polyps. Higher magnification images are shown in insets. Bar = 100 µm. B Quantitation of p-eIF2α+ and c-Cas8+ per 400× field from A. Bar = 100 µm. C Representative TUNEL and phospho-β-catenin (S552) (p-βcat) IF staining. Bar = 100 µm. D Quantitation of TUNEL+ and p-βcat+ cells per 400× field from ( C ). E Representative TUNEL/p-βcat double IF staining. Bar = 100 µm. F Quantitation of cells per 400× field from ( E ). G qRT-PCR analysis of the indicated genes. cDNA was made from pooled polyps, 3–5 polyps/mouse, and 3 mice/group. B , D , F , G , n = 3 mice/group. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.001 (Student’s T -test, two-tailed). + P < 0.05, +++ P < 0.0001 (one-way ANOVA and Tukey Post-Hoc test).

Article Snippet: For drug treatment, cells were plated in 12-well plates at ∼30% density 24 h before Everolimus (LC Laboratories) treatment at 10 μM.

Techniques: Staining, Quantitation Assay, TUNEL Assay, Quantitative RT-PCR, Two Tailed Test

Journal: Oncogene

Article Title: mTOR inhibition suppresses Myc-driven polyposis by inducing immunogenic cell death

doi: 10.1038/s41388-023-02706-6

Figure Lengend Snippet: APC Min/+ mice with established polyps were treated with Everolimus for 3 days or 14 days (Fig. ). The intestinal polyps were analyzed. A Representative CD3ε and CD8 IF staining in the polyps. Bar = 100 µm. B Quantitation of cells per 400× field from ( A ). C Representative Ly-6B.2 and CD68 IF staining. Higher magnification images are shown in insets ( A , C ). D Quantitation of cells per 400× field from ( D ). E qRT-PCR analysis of the indicated immune genes. cDNA was made from pooled polyps, 3–5 polyps/mouse, and 3 mice/group. B , D , E , n = 3 mice/group. * P < 0.05, *** P < 0.001 (Student’s T -Test, two-tailed). +++ P < 0.001 (one-way ANOVA and Tukey Post-Hoc test).

Article Snippet: For drug treatment, cells were plated in 12-well plates at ∼30% density 24 h before Everolimus (LC Laboratories) treatment at 10 μM.

Techniques: Staining, Quantitation Assay, Quantitative RT-PCR, Two Tailed Test

Journal: Oncogene

Article Title: mTOR inhibition suppresses Myc-driven polyposis by inducing immunogenic cell death

doi: 10.1038/s41388-023-02706-6

Figure Lengend Snippet: Human NCM356 cells were transfected with control or the indicated siRNA duplexes and treated with Everolimus (10 μm) for the indicated times. A Attached cells at 48 h were visualized by crystal violet staining. B Apoptosis was measured at 48 h by Hoechst staining of fragmented nuclei. C qRT-PCR analysis of the indicated genes at 24 h. D Western blots of the indicated proteins at 2 h. E Western blots of the indicated proteins at 24 h and F Apoptosis at 48 h measured by Hoechst staining of fragmented nuclei with Myc siRNA. G qRT-PCR analysis of the indicated genes at 24 h with Myc siRNA. H Western blots of the indicated proteins at 24 h and I apoptosis at 48 h measured by Hoechst staining of fragmented nuclei with BID siRNA. Arrows indicated cleaved bands. B , C , F , G , I , n = 3 independent experiments. * P < 0.05, ** P < 0.01, *** P < 0.001 (Student’s T -Test, two-tailed).

Article Snippet: For drug treatment, cells were plated in 12-well plates at ∼30% density 24 h before Everolimus (LC Laboratories) treatment at 10 μM.

Techniques: Transfection, Control, Staining, Quantitative RT-PCR, Western Blot, Two Tailed Test

Journal: Oncogene

Article Title: mTOR inhibition suppresses Myc-driven polyposis by inducing immunogenic cell death

doi: 10.1038/s41388-023-02706-6

Figure Lengend Snippet: APC Min/+ or APC Min/+ /4ES209A (KI) mice with established polyps were treated with Everolimus for 3 days. The intestinal polyps were analyzed. A Representative Myc, TUNEL, p-βcat, p-eIF2α IF staining. Bar = 100 µm. B Quantitation of markers per 400× field from A. Nuclear Myc was scored. The counts in polyps from APC Min/+ mice were included as controls for those in KI mice. C qRT-PCR analysis of the indicated genes. The relative levels were normalized control polyps in APC Min/+ mice. D Representative Ly-6B.2 and CD68 IF staining. E Quantitation of cells from ( D ). B , C , E , n = 3 mice. ++ P < 0.01, +++ P < 0.001, ++++ P < 0.0001 (one-way ANOVA and Tukey Post-Hoc test).

Article Snippet: For drug treatment, cells were plated in 12-well plates at ∼30% density 24 h before Everolimus (LC Laboratories) treatment at 10 μM.

Techniques: TUNEL Assay, Staining, Quantitation Assay, Quantitative RT-PCR, Control

Journal: Oncogene

Article Title: mTOR inhibition suppresses Myc-driven polyposis by inducing immunogenic cell death

doi: 10.1038/s41388-023-02706-6

Figure Lengend Snippet: APC Min/+ /BID −/− mice with established polyps on week 12 were treated with Everolimus for 3 days ( A – G ), or till week 48 (48 w). A Representative TUNEL IF in the polyps. Bar = 100 µm. B Quantitation of TUNEL+ cells in the polyps. C Representative c-Cas8 and p-eiF2α IF in the polyps. Bar = 100 µm. D Quantitation of indicated cells in the polyps. E Quantitation of Ly-6B.2+ cells and CD68+ cells in the polyps. F Survival. APC Min/+ mice were included as controls for BID KO mice. Log-rank test. *** P < 0.001, APC Min/+ /BID -/- Ctrl vs . Ever, **** P < 0.0001 APC Min/+ vs. APC Min/+ /BID −/− . G Quantitation of CD3+ and CD8+ cells in the polyps of indicated genotypes at 36 w (24 w treatment). B , D , E , G , n = 3 mice/group. *** P < 0.001, **** P < 0.0001 (Student’s T -Test, two-tailed). H Working model. Left, elevated Myc and mTOR maintain metabolic and immune adaptation in mutant APC -driven polyposis. mTOR inhibition breaks adaptation through Myc-dependent and -independent targets to induce immunogenic cell death required for long-term tumor control. Right, cell death is induced upon inhibition of p-4EBP1 and S6K1, leading to Myc reduction, AKT hyperactivation (p-AKT), ER stress (p-eIF2α/CHOP), and DR5-Cas8-tBid signaling.

Article Snippet: For drug treatment, cells were plated in 12-well plates at ∼30% density 24 h before Everolimus (LC Laboratories) treatment at 10 μM.

Techniques: TUNEL Assay, Quantitation Assay, Two Tailed Test, Mutagenesis, Inhibition, Control