Journal: Drug Design, Development and Therapy

Article Title: Attenuation of everolimus-induced cytotoxicity by a protective autophagic pathway involving ERK activation in renal cell carcinoma cells

doi: 10.2147/DDDT.S160557

Figure Lengend Snippet: Everolimus (RAD001) induces autophagy in RCCs. Notes: ( A , B ) 786-O and A498 cell lines were treated by RAD001 with different concentrations (0, 0.01, 0.1, 1, 10, and 20 μmol/L) for 24 hours. ( A ) Immunoblotting showed that RAD001 induced autophagy indicators including upregulation of LC3-II/I and downregulation of p62 in a dose-dependent manner. ( B ) Quantification of the immunoblots. * P <0.05 versus 0 μmol/L group. n=3. ( C , D ) 786-O and A498 cell lines were treated by RAD001 for 0, 1, 2, 4, and 8 hours. ( C ) Immunoblotting showed that RAD001 induced autophagy in 786-O and A498 cells as time prolonged, which peaked at 8 hours. ( D ) Quantification of the immunoblots. * P <0.05 versus 0 hour group. n=3. Abbreviation: RCCs, renal cell carcinoma cells.

Article Snippet: The small molecular inhibitors RAD001 and AZD6244 were obtained from MedChem Express (Monmouth Junction, NJ, USA).

Techniques: Western Blot

Journal: Drug Design, Development and Therapy

Article Title: Attenuation of everolimus-induced cytotoxicity by a protective autophagic pathway involving ERK activation in renal cell carcinoma cells

doi: 10.2147/DDDT.S160557

Figure Lengend Snippet: Inhibition of autophagy enhances RAD001-induced cytotoxicity, RAD001-induced activation of ERK signaling pathway in RCCs. Notes: 786-O and A498 cells were pretreated with CQ (autophagy inhibitor) (10 mmol/L) for 30 minutes and then treated with RAD001 for 8 hours. ( A ) MTT assay showed that RAD001 impaired cell viability of 786-O and A498 cells in a dose-dependent manner. n=3. ( B ) Immunoblotting of LC3-II/I and p62 showed that CQ could restore the downregulation of p62, but would not impair the upregulation of LC3-II/I because CQ blocks autophagy at the lysosomal degradation step. ( C ) Densitometric analysis was performed to quantify the immunoblots. * P <0.05 versus control. # P <0.05 versus RAD001 group. n=3. ( D ) MTT assay displayed that autophagy inhibition by CQ promoted RAD001-induced cytotoxicity in 786-O and A498 cells. * P <0.05 versus control. # P <0.05 versus RAD001 group. n=3. ( E ) The apoptosis indicator cleave-PARP detected by immunoblot indicated that cells apoptosis was increased in 786-O and A498 cells after CQ intervention, compared to the RAD001 group. ( F ) Densitometric analysis was performed to quantify the immunoblots. * P <0.05 versus control group. # P <0.05 versus RAD001 group. n=3. 786-O and A498 cells were treated by RAD001 for 0, 1, 2, 4, and 8 hours. ( G ) Immunoblotting analyzed the JNK, p38, and ERK signaling molecules in 786-O and A498 cells after RAD001 treatment. ( H ) Quantification of the immunoblots. * P <0.05 versus 0 hour group. n=3. Abbreviations: CQ, chloroquine; Con, control; RCCs, renal cell carcinoma cells; ERK, extracellular signal-regulated kinase; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; PARP, poly ADP ribose polymerase; JNK, c-Jun N-terminal kinase.

Article Snippet: The small molecular inhibitors RAD001 and AZD6244 were obtained from MedChem Express (Monmouth Junction, NJ, USA).

Techniques: Inhibition, Activation Assay, MTT Assay, Western Blot, Control

Journal: Drug Design, Development and Therapy

Article Title: Attenuation of everolimus-induced cytotoxicity by a protective autophagic pathway involving ERK activation in renal cell carcinoma cells

doi: 10.2147/DDDT.S160557

Figure Lengend Snippet: Selumetinib (AZD6244) significantly enhances RAD001-induced apoptosis of RCCs. Notes: 786-O and A498 cells were pretreated with AZD6244 (1 μmol/L) for 30 minutes and then treated with RAD001 for 8 hours. ( A ) Immunoblotting showed that AZD6244 significantly inhibited the activation of ERK pathway in 786-O and A498 cells. ( B ) Quantification of the immunoblots. ( C ) MTT assay detected the effect of AZD6244 on cell viability of 786-O and A498 cells, which suggested that AZD6244 effectively blunts RAD001-induced cell viability. ( D ) Immunoblotting demonstrates that AZD6244 could effectively restore upregulation of LC3-II/I and downregulation of p62 induced by RAD001 in 786-O and A498 cells. ( E ) Densitometric analysis was performed to quantify the immunoblots. ( F ) Immunoblot analysis indicated that combination treatment of RAD001 with AZD6244 could increase cleave-PARP level in 786-O and A498 cells, compared to the RAD001 group. ( G ) Densitometric analysis was performed to quantify the cleave-PARP immunoblots. ( H ) The difference of apoptosis determined by flow cytometry showed that AZD6244 significantly increased RAD001-induced cells apoptosis in 786-O and A498 cell lines. ( I ) Quantification of the apoptosis proportion. * P <0.05 versus control group. # P <0.05 versus RAD001 group. n=3. Abbreviations: RCCs, renal cell carcinoma cells; ERK, extracellular signal-regulated kinase; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; PARP, poly ADP ribose polymerase; FITC, fluorescein isothiocyanate; PI, propidium iodide; RAD001, everolimus; AZD6244, selumetinib; Con, control.

Article Snippet: The small molecular inhibitors RAD001 and AZD6244 were obtained from MedChem Express (Monmouth Junction, NJ, USA).

Techniques: Western Blot, Activation Assay, MTT Assay, Flow Cytometry, Control

Journal: Drug Design, Development and Therapy

Article Title: Attenuation of everolimus-induced cytotoxicity by a protective autophagic pathway involving ERK activation in renal cell carcinoma cells

doi: 10.2147/DDDT.S160557

Figure Lengend Snippet: ERK mediated autophagy through upregulating Beclin-1 and p-Bcl-2 expression. Notes: ( A , B ) 786-O and A498 cells were treated by RAD001 for 0, 1, 2, 4, and 8 hours. ( A ) Immunoblotting showed that RAD001 induced upregulation of downstream signaling molecules of the ERK pathway including Beclin-1 and Bcl-2 in 786-O and A498 cells in a time-dependent manner. ( B ) Densitometric analysis was performed to quantify the immunoblots. * P <0.05 versus 0 hour group. n=3. ( C , D ) 786-O and A498 cells were pretreated with AZD6244 (1 μmol/L) for 30 minutes and then treated with RAD001 for 8 hours. ( C ) Immunoblotting showed that AZD6244 significantly decreased protein levels of Beclin-1 and Bcl-2 in 786-O and A498 cells. ( D ) Densitometric analysis was performed to quantify the immunoblots. * P <0.05 versus control group. # P <0.05 versus RAD001 group. n=3. Abbreviations: ERK, extracellular signal-regulated kinase; RAD001, everolimus.

Article Snippet: The small molecular inhibitors RAD001 and AZD6244 were obtained from MedChem Express (Monmouth Junction, NJ, USA).

Techniques: Expressing, Western Blot, Control

Journal: Cell Death & Disease

Article Title: MAGI3 deficiency unleashes β-catenin conformational change to drive metastatic progression and mTOR inhibitor resistance in ccRCC

doi: 10.1038/s41419-026-08563-x

Figure Lengend Snippet: A Knockdown of MAGI3 increased the IC 50 of Everolimus in 786-O and 769-P cells. Dose-response survival curves of MAGI3-knockdown 786-O and 769-P cells exposed to increasing concentrations of Everolimus for 48 h. Mean ± SD; n = 3; two-way ANOVA. B The reduced drug sensitivity caused by MAGI3 knockdown is restored by treatment with XAV-939, an inhibitor of the Wnt/β-catenin pathway. Cell viability of 786-O and 769-P cells was determined using CCK-8 assays. C , D MAGI3 knockdown promotes clonogenic formation in Everolimus-treated 786-O and 769-P cells, while concomitant treatment with XAV-939 and Everolimus markedly inhibits clonogenic formation. Colony formation assays were conducted in 786-O and 769-P cells treated with Everolimus in the absence or presence of XAV-939 for 14 days. Quantification analysis of clone formation assays. E Combination treatment with XAV939 and Everolimus synergistically inhibits cell invasion of ccRCC. Transwell invasion assays were conducted in 786-O and 769-P cells treated with Everolimus in the absence or presence of XAV-939 for 24 h. ( t test; * p < 0.05; ** p < 0.01; *** p < 0.001; ns, no significant difference; values represent mean ± SD, n = 3).

Article Snippet: IC50 values were determined by treating cells with increasing concentrations of everolimus (TargetMol, #T1784; 10 nM to 100 μM) for 48 h. Cell viability was assessed using CCK-8 and analyzed with GraphPad Prism.

Techniques: Knockdown, CCK-8 Assay

Journal: Cell Death & Disease

Article Title: MAGI3 deficiency unleashes β-catenin conformational change to drive metastatic progression and mTOR inhibitor resistance in ccRCC

doi: 10.1038/s41419-026-08563-x

Figure Lengend Snippet: A Representative immunohistochemical staining of MAGI3 and β-catenin in tumor tissues from 53 ccRCC patients treated with everolimus. Sections from responders (stable disease or partial response) and non-responders (progressive disease) are shown. Left: overview (scale bar, 200 μm); right: higher-magnification views of the boxed areas (scale bar, 50 μm). H-score analysis of MAGI3 ( B ) and β-catenin ( C ) expression in tumors from responders and non-responders. Data are presented as mean ± SD. Differences between groups were assessed by the Mann-Whitney U test (* P < 0.05, ** P < 0.01). D Scatter plot showing a significant negative correlation between MAGI3 and β-catenin expression levels (Spearman’s r = –0.63, P < 0.001). E Kaplan-Meier overall survival analysis of patients stratified by high or low MAGI3 expression. Statistical significance was determined by the log-rank test. F – I In vivo efficacy and safety of combined mTOR and Wnt inhibition. F Representative images of tumors resected from each group at the end of the study. G Tumor growth curves of mice bearing shMAGI3 xenografts ( n = 5 per group) treated with vehicle, Everolimus (EVE, 5 mg/kg), XAV-939 (XAV, 10 mg/kg), or the combination (EVE + XAV). The shCtrl group is shown as a reference. H Final tumor weights for each group. I Body weight changes of mice during the treatment period, indicating treatment tolerability. Data in G – I are shown as mean ± SD. Statistical significance was determined by two-way ANOVA ( G , I ) or one-way ANOVA ( H ) followed by post-hoc tests (*** P < 0.001, shCtrl vs. shMAGI3; ### P < 0.001, combination therapy vs. shMAGI3+EVE monotherapy; ns not significant).

Article Snippet: IC50 values were determined by treating cells with increasing concentrations of everolimus (TargetMol, #T1784; 10 nM to 100 μM) for 48 h. Cell viability was assessed using CCK-8 and analyzed with GraphPad Prism.

Techniques: Immunohistochemical staining, Staining, Expressing, MANN-WHITNEY, In Vivo, Inhibition

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