Journal: Acta Neuropathologica Communications

Article Title: DNA hypomethylator phenotype reprograms glutamatergic network in receptor tyrosine kinase gene-mutated glioblastoma

doi: 10.1186/s40478-024-01750-x

Figure Lengend Snippet: mTORC2 downregulates de novo DNA methyltransferase DNMT3A. A RNA-sequencing-based transcript expression of DNA methylating and demethylating enzymes in U87-EGFRvIII cells with siRNA against Scramble sequence or Rictor. Bar graph showed the expression level of de novo DNA methyltransferases including DNMT3A and DNMT3B in U87-EGFRvIII cells with siScramble or siRictor. KD, knockdown; ND, not detected; NS, not significant; RPKM, reads per kilobase per million mapped reads. B Transcript expression of DNMT3A gene in various types of cancers, based on TCGA datasets. GBM is highlighted in a red box. C Relative protein expression of DNMT3A in U87-EGFRvIII cells treated with drugs targeting mTORC2 substrates including Akt (Akti-1/2: 2.5 µM), SGK1 (GSK650394: 2.0 µM) and PKC-α (Bis-I: 10 µM) for 48 h

Article Snippet: Lentiviral shRNA vectors targeting human Rictor and scramble sequences were obtained from Addgene (shRictor#1) and Santa Cruz (shRictor#2).

Techniques: RNA Sequencing, Expressing, Sequencing, Knockdown

Journal: Acta Neuropathologica Communications

Article Title: DNA hypomethylator phenotype reprograms glutamatergic network in receptor tyrosine kinase gene-mutated glioblastoma

doi: 10.1186/s40478-024-01750-x

Figure Lengend Snippet: mTORC2 redistributes EZH2 in the DNMT3A promoter to suppress its expression. A Immunoblot detection of DNMT3A and H3 p.K27me3 in U87-EGFRvIII cells treated with GSK126 (EZH2 inhibitor: 2.5 µM) and GSKJ4 (JmjC inhibitor: 10 µM) for 48 h. B , C ChIP-qPCR analysis on H3 p.K27me3 ( B ) and EZH2 ( C ) enrichment in DNMT3A promoter regions of U87-EGFRvIII cells transfected with siRNAs against Scramble sequence or Rictor. D Immunoblot analyses of acetylated EZH2 (Ac-EZH2) in U87-EGFRvIII cells with shScramble or shRictor. Ac-K, acetylated-lysine; IB, immunoblotting; IP, immunoprecipitation. E Immunoblot analyses of acetylated EZH2 (Ac-EZH2) in U87 cells with overexpression of GFP or Rictor. Ac-K, acetylated-lysine; IB, immunoblotting; IP, immunoprecipitation. F Analyses on acetylation and redistribution of EZH2 on the DNMT3A promoter in U87-EGFRvIII cells with addition of TSA (1.0 µM) and acetate (10 mM) for 48 h. Ac, acetate. G mRNA expression of DNMT3A in U87-EGFRvIII cells treated by PP242 (mTORC1/C2 inhibitor: 5 uM) along with supplementation of TSA (1.0 µM) and acetate (10 mM) for 48 h. H mTORC2 drives protein acetylation to redistribute EZH2 into the DNMT3A promoter region, and increases H3 p.K27me3 to suppress the expression of DNMT3A in GBM. Ac, acetyl-group

Article Snippet: Lentiviral shRNA vectors targeting human Rictor and scramble sequences were obtained from Addgene (shRictor#1) and Santa Cruz (shRictor#2).

Techniques: Expressing, Western Blot, ChIP-qPCR, Transfection, Sequencing, Immunoprecipitation, Over Expression

Journal: Acta Neuropathologica Communications

Article Title: DNA hypomethylator phenotype reprograms glutamatergic network in receptor tyrosine kinase gene-mutated glioblastoma

doi: 10.1186/s40478-024-01750-x

Figure Lengend Snippet: mTORC2-induced DNA hypomethylation is dependent on the downregulation of DNMT3A. A Enzymatic activities of fractionated nuclear DNMT in U87-EGFRvIII cells with shScramble or shRictor. OD, optical density; U, unit of DNMT enzyme control. B RNA-sequencing analysis of potential DNMT3A target genes regarding cell proliferation and differentiation in U87-EGFRvIII cells with siScramble or siRictor. Note that mTORC2 activation (Scramble) upregulates proliferation-related genes, but downregulates differentiation-related genes. KD, knockdown. C A shift in global DNA methylation level, represented by LINE-1 methylation in Scramble- or Rictor-depleted U87-EGFRvIII cells, with concurrent knockdown of DNMT3A. D Immunohistochemistry for mTORC2 activation marker (p-Akt S473), DNMT3A and 5-mC in human GBM tissue (n = 21). The scatter plots showed the negative or positive correlation between DNMT3A and mTORC2 marker (p-Akt S473: upper panel) or 5-mC (lower panel) respectively, based on quantitative immunohistochemistry. Scale bars, 40 µm (for pAKT and DNMT3A) and 80 µm (for 5-mC)

Article Snippet: Lentiviral shRNA vectors targeting human Rictor and scramble sequences were obtained from Addgene (shRictor#1) and Santa Cruz (shRictor#2).

Techniques: Control, RNA Sequencing, Activation Assay, Knockdown, DNA Methylation Assay, Methylation, Immunohistochemistry, Marker

Journal: Acta Neuropathologica Communications

Article Title: DNA hypomethylator phenotype reprograms glutamatergic network in receptor tyrosine kinase gene-mutated glioblastoma

doi: 10.1186/s40478-024-01750-x

Figure Lengend Snippet: mTORC2-driven global DNA hypomethylation reprograms glutamatergic network in GBM. A Heatmap of the DNA methylation profile (Infinium HumanMethylation 850 K BeadChip) in U87-EGFRvIII cells with shScramble or shRictor. DMP, differential methylation probes; KD, knockdown; SD, standard deviation. B Differential DNA-methylated regions (DMRs) in U87-EGFRvIII cells with shScramble or shRictor, including CpG-islands. ExonBnd, exon boundaries; IGR, intergenic region; TSS, transcription start sites; UTR, untranslated region. C GO term analyses on David_RHyper10perGenes on mTORC2 inhibition.”Chemical synaptic transmission (GO:0007268)” suggest that mTORC2-dependent hypomethylator could regulate the expression of genes related to EAA metabolism. D mRNA expression of glutamate transporters (SLC1A1, SLC1A3, SLC1A6) in Rictor knockdown U87-EGFRvIII GBM cells. E Measurement of EAA (glutamate and aspartate) indicated that Rictor knockdown reduced intracellular glutamate (Glu) and aspartate (Asp) in U87-EGFRvIII GBM cells. Conc, concentration

Article Snippet: Lentiviral shRNA vectors targeting human Rictor and scramble sequences were obtained from Addgene (shRictor#1) and Santa Cruz (shRictor#2).

Techniques: DNA Methylation Assay, Methylation, Knockdown, Standard Deviation, Inhibition, Transmission Assay, Expressing, Concentration Assay

Journal: Acta Neuropathologica Communications

Article Title: DNA hypomethylator phenotype reprograms glutamatergic network in receptor tyrosine kinase gene-mutated glioblastoma

doi: 10.1186/s40478-024-01750-x

Figure Lengend Snippet: Reprogramming of glutamate metabolism drives invasive phenotypes in GBM. A Lower methylation signals by MeDIP-qPCR on the GRIA1 promoter and higher expression of GRIA1 transcripts were observed in U87 cells with lentivirus-mediated overexpression of human Rictor (Rictor OE) in comparison with control (GFP). Met, methylation (5-mC). B GBM tumors from rats infected with PDGFB-HA-IRES-EGFP retroviral vectors (n = 4) were probed by immunohistochemistry against GRIA1. Note intratumoral heterogeneity of GRIA1 immunoreactivity in accordance with the status of mTORC2 activation (pAkt) and 5-mC expression. Peri-necrotic area indicates pAkt_low/5-mC_high region, and non-necrotic to pAkt_high/5-mC_low region. Refer to Fig. B. Nec, necrosis. Scale bar, 20 µm. C Scratch assays using the co-culture of U87-EGFRvIII (GBM) cells with siRNA-mediated knockdown of GRIA1 and SH-SY5Y (neuroblastoma) cells. The area of gap was calculated 24 h after scratch. Cells were colored in red with the binary mode (red) of ImageJ software. Scale bar, 100 µm. D Knockdown of GRIA1 significantly ( p < 0.01) affected GBM cell migration in the co-culture of U87-EGFRvIII and SH-SY5Y cells. E Measurement of glutamate (Glu) indicated that GRIA1 knockdown increased extracellular Glu (reduced intracellular Glu) in U87-EGFRvIII GBM cells. Conc, concentration. F Wound healing/migration assay on the co-culture of SH-SY5Y neuroblastoma cells with U87-EGFRvIII GBM cells treated by PhTx-74 (GRIA1/GRIA2 inhibitor: 20 μM) or a combination of PhTx-74 (20 μM) with GSK2256098 (FAK inhibitor: 100 nM). Cells were colored in red with the binary mode (red) of ImageJ software. Scale bar, 100 µm. G PhTx-74 treatment increased extracellular Glu (reduced intracellular Glu), and FAK inhibitor decreased phosphorylation of FAK (Tyr397) in U87-EGFRvIII GBM cells. Conc, concentration. H TCGA datasets on overall survival and progression free survival of GBM cases stratified by the expression level of GRIA1 transcripts

Article Snippet: Lentiviral shRNA vectors targeting human Rictor and scramble sequences were obtained from Addgene (shRictor#1) and Santa Cruz (shRictor#2).

Techniques: Methylation, Methylated DNA Immunoprecipitation, Expressing, Over Expression, Comparison, Control, Infection, Retroviral, Immunohistochemistry, Activation Assay, Co-Culture Assay, Knockdown, Software, Migration, Concentration Assay, Phospho-proteomics

Journal: Acta Neuropathologica Communications

Article Title: DNA hypomethylator phenotype reprograms glutamatergic network in receptor tyrosine kinase gene-mutated glioblastoma

doi: 10.1186/s40478-024-01750-x

Figure Lengend Snippet: mTORC2 activation correlates with global DNA hypomethylation phenotypes in RTK-mutated GBM. A Immunohistochemical (IHC) staining of human GBM tissue (n = 20) with antibodies against mutant EGFR (EGFRvIII) and a DNA methylation marker (5-mC). EGFR amplification was assessed by FISH with probes for EGFR (7p11.2, Red) and CEP7 (7p11.1-q11.1, Green). Scale bar, 40 µm. B Cerebral and brainstem tissue with GBM tumors was harvested from rats infected with PDGFB-HA-IRES-EGFP retroviral vectors (n = 4). Immunohistochemistry was performed on paraffin-embedded tissue sections against a DNA methylation marker (5-mC) and an mTORC2 marker (p-Akt S473). Nec, necrosis. Scale bars, 50 µm (upper panels) and 20 µm (lower panels). C Immunofluorescent staining of 5-mC in U87-EGFRvIII cells transfected with shRNAs against control sequence (scramble) or Rictor (shRictor#1). Scale bar, 10 µm. D Dot blot analysis of 5-mC in U87-EGFRvIII cells transfected with shScramble versus shRictor#1 (upper panel), or overexpressed (OE) with GFP versus Rictor (lower panel). Total DNA for each sample was determined by methylene blue staining. E Detection of global DNA methylation (ELISA-based assay), represented by methylation of LINE-1 retrotransposable elements in U87-EGFRvIII cells transfected with shScramble or shRictor. OD, optical density; STD, standard

Article Snippet: Lentiviral shRNA vectors targeting human Rictor and scramble sequences were obtained from Addgene (shRictor#1) and Santa Cruz (shRictor#2).

Techniques: Activation Assay, Immunohistochemical staining, Immunohistochemistry, Mutagenesis, DNA Methylation Assay, Marker, Amplification, Infection, Retroviral, Staining, Transfection, Control, Sequencing, Dot Blot, Enzyme-linked Immunosorbent Assay, Methylation

Journal: Microorganisms

Article Title: Endothelial Mechanistic Target of Rapamycin Activation with Different Strains of R. rickettsii : Possible Role in Rickettsial Pathogenesis

doi: 10.3390/microorganisms12020296

Figure Lengend Snippet: Effect of Raptor and Rictor siRNA on rickettsial replication. ECs were transfected with Raptor or Rictor siRNA (25 or 50 nM), along with control siRNA (50 nM), using Lipofectamine RNAiMAX 24 h prior to infection with R. rickettsii (SS) for 48 h. DNA was isolated, and rickettsial copy number was measured ( A ). Total protein lysates were prepared and subjected to Westen blot using antibodies against Raptor and Rictor to determine the extent of Raptor knockdown ( B ) and Rictor knockdown ( C ). The α-tubulin antibody was used as a loading control, and a representative blot ( n ≥ 3) is shown. In some experiments, ( C ) statistically significant changes are shown as ** = p ≤ 0.001 and *** = p ≤ 0.0001.

Article Snippet: In some experiments, mTORC1-specific Raptor siRNA (25 or 50 nM, catalog #sc-44069), mTORC2-specific Rictor siRNA (25 or 50 nM, catalog #sc-61478), or control siRNA (50 nM, catalog #sc-44230) (Santa Cruz Biotechnology, Dallas, TX, USA) were transfected into endothelial cells using Lipofectamine RNAiMAX (ThermoFisher, catalog #13778075) according to the manufacturer’s instructions for 48 h prior to infection with R. rickettsii , and total cell lysates were prepared as described above.

Techniques: Transfection, Control, Infection, Isolation, Knockdown

Journal: Microorganisms

Article Title: Endothelial Mechanistic Target of Rapamycin Activation with Different Strains of R. rickettsii : Possible Role in Rickettsial Pathogenesis

doi: 10.3390/microorganisms12020296

Figure Lengend Snippet: Effect of mTOR on cytokine expression in R. rickettsii (SS)-infected ECs. ECs were transfected with either control, Raptor, or Rictor siRNA 48 h prior to infection with R. rickettsii (SS) for 24 h. Total RNA was then isolated and cytokine expression (IL-6 ( A ), IL-8 ( B ), IL-1α ( C )) was measured via qRT-PCR using a specific primer pair for each cytokine. Mock-infected cells are displayed as controls. Statistically significant changes are shown as * = p ≤ 0.05, ** = p ≤ 0.01, and *** = p ≤ 0.001; NS = not significant.

Article Snippet: In some experiments, mTORC1-specific Raptor siRNA (25 or 50 nM, catalog #sc-44069), mTORC2-specific Rictor siRNA (25 or 50 nM, catalog #sc-61478), or control siRNA (50 nM, catalog #sc-44230) (Santa Cruz Biotechnology, Dallas, TX, USA) were transfected into endothelial cells using Lipofectamine RNAiMAX (ThermoFisher, catalog #13778075) according to the manufacturer’s instructions for 48 h prior to infection with R. rickettsii , and total cell lysates were prepared as described above.

Techniques: Expressing, Infection, Transfection, Control, Isolation, Quantitative RT-PCR

Journal: Autophagy

Article Title: A novel MTORC2-AKT-ROS axis triggers mitofission and mitophagy-associated execution of colorectal cancer cells upon drug-induced activation of mutant KRAS

doi: 10.1080/15548627.2024.2307224

Figure Lengend Snippet: MTORC2 mediates phosphorylation of AKT S473 that triggers intracellular ROS. (A) Schematic diagram of the crosstalk between MTORC1, MTORC2 and AKT phosphorylation. Image created with BioRender.com. (B) HCT116 cells were pre-treated for 2 h with rapamycin or torin 1 (both 250 nM) followed by incubation with C1 (50 μg/mL for 3 h and 6 h) and lysates were immunoblotted for AKT p-S473, total AKT, LC3B-II, SQSTM1 and OPA1 (L and S forms). GAPDH was used as the loading control. (C) HCT116 cells were transfected with si RPTOR or si RICTOR or non-targeting si RNA control (10 nM for 48 h) and exposed to 50 μg/mL of C1 for 3 h and 6 h. Lysates were immunoblotted for AKT p-S473, total AKT, LC3B-II, SQSTM1, OPA1 (L and S forms), RPTOR and RICTOR. GAPDH was used as the loading control. (D and E) Immunoblotting analysis of mitochondria and cytosolic fractions from HCT116 cells pre-treated with rapamycin (250 nM) or torin 1 (100 nM) (D) or transfected with si RPTOR , si RICTOR or non-targeting negative si RNA control (50 nM for 48 h) (E) and then treated with C1 (50 μg/mL) for 6 h. GAPDH, HSPA1A and HSP90AA1 were used as loading controls. SOD2 and SOD1 were used as mitochondria and cytosolic specific controls respectively. (F) HCT116 cells were preincubated with torin 1 (100 nM) for 2 h, then pre-stained with 1 µM Mtphagy Dye and treated with C1 (25 and 50 μg/mL) for 18 h. At least 10,000 cells were analyzed by flow cytometry as described in materials and methods. MFI = mean fluorescence intensity. (G) Mtphagy Dye fluorescence changes are plotted by prism graph using MFI of cells upon the indicated treatments compared to untreated cells (ratio). Data are representative of at least 4 independent experiments. Two-way ANOVA was employed for statistical analysis (* = p < 0.05). (H) HCT116 cells were pre-treated for 2 h with torin 1 (100 nM) followed by treatment with 50 μg/mL of C1 for 18 h. Lysates were immunoblotted for SQSTM1 and LC3B-II. GAPDH was used as the loading control. (I) HCT116 cells were transfected with si RICTOR non-targeting si RNA control (50 nM) for 48 h or pre-incubated with torin 1 (100 nM) for 2 h before exposure to 50 μg/mL of C1 for 6 h. Lysates were immunoblotted for DNM1L p-S616, DNM1L p-S637, DNM1L, AKT p-S473, total AKT, and rictor. GAPDH was used as the loading control. (J) Representative images of HCT116 cells transfected with si RICTOR , si AKT1 and si AKT2 or non-targeting si RNA control (50 nM) for 48 h and then treated with C1 (25 μg/mL for 12 h) before staining with MitoTracker™ green FM and viewed via confocal imaging under 100 X magnification. Scale bar: 10 µm. (K-M) for each sample, 20 cell images were analyzed and mitochondrial footprint, mean summed branch lengths and mean network size (branches) were assessed by MiNA, an ImageJ based analysis used for studying morphological changes in mitochondria. Data were plotted on GraphPad prism V 8.0. C1-treated group were compared against control group using paired t-test (** = p < 0.01, *** = p < 0.001 and **** = p < 0.0001).

Article Snippet: Primary Antibodies: LC3B (Cell Signaling Technology, 3868), GAPDH (Cell Signaling Technology, 2118), anti-phospho-AKT (S473; Cell Signaling Technology, 4060), anti-phospho-AKT (T450; Cell Signaling Technology, 9267), AKT (Cell Signaling Technology, 9272), anti-phospho-MAPKAP1/SIN1 (T86; Cell Signaling Technology 14716), MAPKAP1/SIN1 (Cell Signaling Technology 12860), anti-phospho-DNM1L/DRP1 (S616; Cell Signaling Technology, 4494), anti-phospho-DNM1L/DRP1 (S637; Cell Signaling Technology, 6319), DNM1L/DRP1 (Cell Signaling Technology, 5391), anti-phospho-MAPK/ERK (Y202/Y204; Cell Signaling Technology, 9101), MAPK/ERK (Cell Signaling Technology, 9102), SOD1 (Cell Signaling Technology, 2770), VDAC1 (Cell Signaling Technology, 4661), VDAC2 (Cell Signaling Technology, 9412), HSPA1A/HSP70–1 (Cell Signaling Technology, 4872), BAK1 (Cell Signaling Technology, 3814), TOMM20 (Cell Signaling Technology 13929), OMA1 (Cell Signaling Technology 95,473), human IGF1 (Cell Signaling Technology, 8917), TUBA1A/α-tubulin (Cell Signaling Technology, 2125), KRAS (Bio-Rad Laboratories, MCA-3223Z), anti-phospho-AKT (T308; BD Biosciences 558316), SOD2 (BD Biosciences 611580), OPA1 (Thermo Fisher Scientific, MA5–16149), SQSTM1/p62 (Santa Cruz Biotechnology, sc-28359), RICTOR (Santa Cruz Biotechnology, sc-270181), RPTOR/raptor (Santa Cruz Biotechnology, sc-81537), TIMM23 (Santa Cruz Biotechnology, sc-514463), HSP90AA1A/HSP90AA1B (Santa Cruz Biotechnology, sc-13110) and DNM1L/DRP1 (Santa Cruz Biotechnology, sc-271583).

Techniques: Phospho-proteomics, Incubation, Control, Transfection, Western Blot, Staining, Flow Cytometry, Fluorescence, Imaging

Journal: Autophagy

Article Title: A novel MTORC2-AKT-ROS axis triggers mitofission and mitophagy-associated execution of colorectal cancer cells upon drug-induced activation of mutant KRAS

doi: 10.1080/15548627.2024.2307224

Figure Lengend Snippet: Preventing MTORC2 function rescues cells from drug-induced execution. (A to C) HCT116 cells were treated with 50 μg/mL of C1 for 3 and 6 h and whole cell lysates were probed for (A) AKT p-T308, (B) AKT p-T450 and (C) MAPKAP1 p-T86. Also, in (C), HCT116 cells were exposed to a solution of human insulin (10 ng/mL) + IGF1 (60 ng/mL) for 1 h as a positive control for MAPKAP1 p-T86. Total cell lysates were obtained from (D) HCT116 cells (parental and KRAS WT/- cells) or HT29 cells treated with 50 µg/mL of C1 for 3 h and 6 h, (E) HCT116 cells transiently (48 h) transfected with si KRAS or non-targeting control si RNA (50 nM) and treated with 50 μg/mL of C1 for 3 h, (F) HCT116 cells pre-treated for 2 h with AKT inhibitor VIII (10 µM) followed by incubation with 50 μg/mL of C1 for 3 h or (G) HCT116 cells (parental and AKT DKO) treated with 50 µg/mL of C1 for 3 h. Whole cell lysates were probed for MAPKAP1 p-T86 (antibody picks up MAPKAP1 p-T86.1 of 78 kDa and occasionally the lower MAPKAP1 p-T86.2 of 74 kDa), total MAPKAP1 and KRAS. GAPDH was used as the loading control. (H) HCT116 cells were pre-treated with torin 1 (100 nM) for 2 h before treatment with 25 µg/mL or 50 μg/mL of C1 for 18 h. Cell viability was measured by the Trypan blue exclusion assay. Two-way ANOVA was employed for statistical analysis (** = p < 0.01, *** = p < 0.001). Data are representative of at least 3 independent experiments and shown as mean ± SD of biological triplicates. (I) HCT116 cells were pre-treated with rapamycin (250 nM) or torin 1 (100 nM) for 2 h before treatment with 50 μg/mL of C1 for 18 h. HCT116 cells were also transfected with si RPTOR , si RICTOR or non-targeting si RNA control (10 nM for 48 h) and then treated with 50 μg/mL of C1 for 18 h. Cells (2000) were reseeded to assess colony forming ability. (J) HCT116 cells were pre-treated with rapamycin (250 nM) or torin 1 (100 nM) for 2 h before treatment with 25 µg/mL or 50 μg/mL of C1 for 18 h and long-term 3D spheroid formation was carried out, as described in materials and methods. Results shown are representative of 3 independent experiments. Scale bar: 200 µm. (K) HCT116 cells were pre-treated with torin 1 (100 nM) for 2 h before treatment with 25 µg/mL or 50 μg/mL of C1 for 18 h and 75,000 cells were reseeded into ThinCert® cell culture inserts for 48 h and then stained with crystal violet and viewed under microscope (scale bar: 50 µm) and (L) quantified by dissolving with 33% (v:v) acetic acid and read at absorbance of 590 nm as described in materials and methods. Migration rates are plotted in percentages with respect to control cells. Data are representative of at least 3 independent experiments and shown as mean ± SD of biological triplicates. Two-way ANOVA was employed for statistical analysis (** = p < 0.01, **** = p < 0.0001).

Article Snippet: Primary Antibodies: LC3B (Cell Signaling Technology, 3868), GAPDH (Cell Signaling Technology, 2118), anti-phospho-AKT (S473; Cell Signaling Technology, 4060), anti-phospho-AKT (T450; Cell Signaling Technology, 9267), AKT (Cell Signaling Technology, 9272), anti-phospho-MAPKAP1/SIN1 (T86; Cell Signaling Technology 14716), MAPKAP1/SIN1 (Cell Signaling Technology 12860), anti-phospho-DNM1L/DRP1 (S616; Cell Signaling Technology, 4494), anti-phospho-DNM1L/DRP1 (S637; Cell Signaling Technology, 6319), DNM1L/DRP1 (Cell Signaling Technology, 5391), anti-phospho-MAPK/ERK (Y202/Y204; Cell Signaling Technology, 9101), MAPK/ERK (Cell Signaling Technology, 9102), SOD1 (Cell Signaling Technology, 2770), VDAC1 (Cell Signaling Technology, 4661), VDAC2 (Cell Signaling Technology, 9412), HSPA1A/HSP70–1 (Cell Signaling Technology, 4872), BAK1 (Cell Signaling Technology, 3814), TOMM20 (Cell Signaling Technology 13929), OMA1 (Cell Signaling Technology 95,473), human IGF1 (Cell Signaling Technology, 8917), TUBA1A/α-tubulin (Cell Signaling Technology, 2125), KRAS (Bio-Rad Laboratories, MCA-3223Z), anti-phospho-AKT (T308; BD Biosciences 558316), SOD2 (BD Biosciences 611580), OPA1 (Thermo Fisher Scientific, MA5–16149), SQSTM1/p62 (Santa Cruz Biotechnology, sc-28359), RICTOR (Santa Cruz Biotechnology, sc-270181), RPTOR/raptor (Santa Cruz Biotechnology, sc-81537), TIMM23 (Santa Cruz Biotechnology, sc-514463), HSP90AA1A/HSP90AA1B (Santa Cruz Biotechnology, sc-13110) and DNM1L/DRP1 (Santa Cruz Biotechnology, sc-271583).

Techniques: Positive Control, Transfection, Control, Incubation, Trypan Blue Exclusion Assay, Cell Culture, Staining, Microscopy, Migration

Journal: Autophagy

Article Title: A novel MTORC2-AKT-ROS axis triggers mitofission and mitophagy-associated execution of colorectal cancer cells upon drug-induced activation of mutant KRAS

doi: 10.1080/15548627.2024.2307224

Figure Lengend Snippet: ROS production downstream of MTORC2 affects cell viability. (A and B) Flow cytometric analysis of cellular and mitochondrial ROS upon inhibiting MTORC2 or upon silencing RPTOR or RICTOR . HCT116 cells were pre-treated for 2 h with rapamycin (250 nM) or torin 1 (250 nM) before exposure to 50 μg/mL of C1 for 1 h. Cells were then loaded with (A) CM-H 2 DCFDA for detecting intracellular ROS or (B) MitoSOX TM Red for detecting mitochondrial O 2 . − . (C and D) HCT116 cells were transfected with si RPTOR or si RICTOR or non-targeting si RNA control (50 nM for 48 h) and exposed to 50 µg/mL of C1 for 1 h. Cells were stained with (C) CM-H 2 DCFDA for detecting intracellular ROS or (D) MitoSOX TM Red for detecting mitochondrial O 2 . − . at least 10,000 cells were analyzed. Histogram data shown are representative of at least 3 independent experiments. MFI = mean fluorescence intensity. (E) HCT116 cells were preincubated with CAT for 6 h before the addition of C1 (25 µg/mL) for 18 h and long-term 3D spheroid formation was carried out, as described in materials and methods. Scale bar: 200 µm. (F and G) spheroids in (E) were measured in diameter and area respectively via Zeiss Zen 3.7 software. Prism data shown are representative of at least 3 independent experiments.

Article Snippet: Primary Antibodies: LC3B (Cell Signaling Technology, 3868), GAPDH (Cell Signaling Technology, 2118), anti-phospho-AKT (S473; Cell Signaling Technology, 4060), anti-phospho-AKT (T450; Cell Signaling Technology, 9267), AKT (Cell Signaling Technology, 9272), anti-phospho-MAPKAP1/SIN1 (T86; Cell Signaling Technology 14716), MAPKAP1/SIN1 (Cell Signaling Technology 12860), anti-phospho-DNM1L/DRP1 (S616; Cell Signaling Technology, 4494), anti-phospho-DNM1L/DRP1 (S637; Cell Signaling Technology, 6319), DNM1L/DRP1 (Cell Signaling Technology, 5391), anti-phospho-MAPK/ERK (Y202/Y204; Cell Signaling Technology, 9101), MAPK/ERK (Cell Signaling Technology, 9102), SOD1 (Cell Signaling Technology, 2770), VDAC1 (Cell Signaling Technology, 4661), VDAC2 (Cell Signaling Technology, 9412), HSPA1A/HSP70–1 (Cell Signaling Technology, 4872), BAK1 (Cell Signaling Technology, 3814), TOMM20 (Cell Signaling Technology 13929), OMA1 (Cell Signaling Technology 95,473), human IGF1 (Cell Signaling Technology, 8917), TUBA1A/α-tubulin (Cell Signaling Technology, 2125), KRAS (Bio-Rad Laboratories, MCA-3223Z), anti-phospho-AKT (T308; BD Biosciences 558316), SOD2 (BD Biosciences 611580), OPA1 (Thermo Fisher Scientific, MA5–16149), SQSTM1/p62 (Santa Cruz Biotechnology, sc-28359), RICTOR (Santa Cruz Biotechnology, sc-270181), RPTOR/raptor (Santa Cruz Biotechnology, sc-81537), TIMM23 (Santa Cruz Biotechnology, sc-514463), HSP90AA1A/HSP90AA1B (Santa Cruz Biotechnology, sc-13110) and DNM1L/DRP1 (Santa Cruz Biotechnology, sc-271583).

Techniques: Transfection, Control, Staining, Fluorescence, Software

Journal: Molecular Biology of the Cell

Article Title: CRISPR-mediated reversion of oncogenic KRAS mutation results in increased proliferation and reveals independent roles of Ras and mTORC2 in the migration of A549 lung cancer cells

doi: 10.1091/mbc.E23-05-0152

Figure Lengend Snippet: Cell growth and proliferation are increased in the K-Ras revertant A549 cells. (A) 2D colony-formation assay performed with the A549 NT , A549 REV1 , and A549 REV2 cells as described in Materials and Methods . Representative images of three independent experiments are shown. Data on the graph represent the average number of colonies of nine replicates from three independent experiments ± SD ( n = 9). (B) 3D soft agar tumorgenicity assay performed as described in Materials and Methods . Representative images of three independent experiments are shown. Data on the graph represent the measured diameter of every colony in a field of view from three independent experiments ± SD ( n = 27−37 colonies). (C) MTT assay comparing cell growth in the different strains, performed as described in Materials and Methods . Data on the graph represent measurements from four independent experiments normalized to the control A549 cells ± SD ( n = 4). (D) MTT assay performed with cells pretreated with 10 μM rapamycin or 0.1% DMSO as control (Ctrl). Data on the graph represent measurements from three independent experiments normalized to the A549 NT DMSO control ± SD ( n = 3). (E) MTT assay performed on cells treated with NT, K-Ras, or Pan-Ras siRNAs. Data on the graph represent measurements from three independent experiments normalized to the control NT siRNA condition ± SD ( n = 3). Adjusted p values: * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001. ns, nonstatistically significant.

Article Snippet: Dharmacon ON-TARGET plus Non-targeting siRNA Control Pool and SMARTPool of Human H-Ras (3265), Human N-Ras (4893), and Human K-Ras (3845) were obtained from Horizon Discovery (Waterbeach, UK), and SignalSilence Control siRNA (unconjugated) and SignalSilence Rictor siRNA were purchased from Cell Signaling Technology (Danvers, MA).

Techniques: Colony Assay, MTT Assay, Control

Journal: Molecular Biology of the Cell

Article Title: CRISPR-mediated reversion of oncogenic KRAS mutation results in increased proliferation and reveals independent roles of Ras and mTORC2 in the migration of A549 lung cancer cells

doi: 10.1091/mbc.E23-05-0152

Figure Lengend Snippet: K-Ras promotes A549 cell migration independently of its mutation. (A) Wound closure cell-migration assays were performed with A549 NT , A549 REV1 , and A549 REV2 cells as described in Materials and Methods . Representative images of three independent experiments are shown, taken immediately after wounding (0 h) and after 24 h. Data on the graph represent measured wound closure (migration distances) in five areas of each wound from three independent experiments ± SD ( n = 15). (B) Wound-healing migration assays were performed with A549 NT , A549 REV1 , and A549 REV2 subjected to siRNA-mediated knockdown of K-Ras alone, of K-, H-, and N-Ras (Pan-Ras siRNA) or treated with NT siRNA control. The data were analyzed and graphed as described in (A). Adjusted p values for the differences between siRNA-mediated knockdowns and the NT siRNA control for each strain: ## p < 0.01; ### p < 0.001; #### p < 0.0001. ns, nonstatistically significant.

Article Snippet: Dharmacon ON-TARGET plus Non-targeting siRNA Control Pool and SMARTPool of Human H-Ras (3265), Human N-Ras (4893), and Human K-Ras (3845) were obtained from Horizon Discovery (Waterbeach, UK), and SignalSilence Control siRNA (unconjugated) and SignalSilence Rictor siRNA were purchased from Cell Signaling Technology (Danvers, MA).

Techniques: Migration, Mutagenesis, Knockdown, Control

Journal: Molecular Biology of the Cell

Article Title: CRISPR-mediated reversion of oncogenic KRAS mutation results in increased proliferation and reveals independent roles of Ras and mTORC2 in the migration of A549 lung cancer cells

doi: 10.1091/mbc.E23-05-0152

Figure Lengend Snippet: mTORC2 mediates the migration of A549 cells independently of K-Ras’s mutational status. (A and B) Wound closure migration assays were performed with the A549 NT , A549 REV1 , and A549 REV2 cells treated with 10 μM rapamycin (mTORC1 inhibitor), 10 nM PP242 (mTORC1/mTORC2 inhibitor), or 0.1% DMSO control (A), or subjected to siRNA-mediated Rictor knockdown or treated with NT siRNA control (B), as described in Materials and Methods . Rictor immunoblots were performed to verify its siRNA-mediated knockdown and the data shown are representative of three independent experiments. Data on graphs represent measured migration distances in five areas of each wound from three independent experiments ± SD ( n = 15). (C) Transwell 3D invasion assays were performed with the cells and conditions as described in (A). Representative images of three independent experiments are shown. Data on graph represent the average number of cells that have invaded per area measured from three separate experiments ± SD ( n = 3). **** adjusted p value < 0.0001. Adjusted p values for the differences between treatment conditions and their respective control for each strain: #, p < 0.05; ##, p < 0.01; ###, p < 0.001; ####, p < 0.0001. ns, nonstatistically significant.

Article Snippet: Dharmacon ON-TARGET plus Non-targeting siRNA Control Pool and SMARTPool of Human H-Ras (3265), Human N-Ras (4893), and Human K-Ras (3845) were obtained from Horizon Discovery (Waterbeach, UK), and SignalSilence Control siRNA (unconjugated) and SignalSilence Rictor siRNA were purchased from Cell Signaling Technology (Danvers, MA).

Techniques: Migration, Control, Knockdown, Western Blot

Journal: Molecular Biology of the Cell

Article Title: CRISPR-mediated reversion of oncogenic KRAS mutation results in increased proliferation and reveals independent roles of Ras and mTORC2 in the migration of A549 lung cancer cells

doi: 10.1091/mbc.E23-05-0152

Figure Lengend Snippet: EGF stimulates mTORC2 activity independently of Ras in A549 cells. pAKT(S473) and pERK were assessed in cells pretreated with K-Ras siRNA, Pan-Ras siRNAs, or NT siRNA control, and stimulated or not with 100 ng/mL EGF as described in Materials and Methods . Ras knockdowns were verified by immunoblot. Representative immunoblots of three independent experiments are shown. Bands were quantified by densitometry and the data on the graph represent the ratio of pAKT(S473)/AKT expressed as percentage of A549 NT control stimulation measured from three separate experiments ± SD ( n = 3). Adjusted p value: **, p < 0.01. ns, nonstatistically significant.

Article Snippet: Dharmacon ON-TARGET plus Non-targeting siRNA Control Pool and SMARTPool of Human H-Ras (3265), Human N-Ras (4893), and Human K-Ras (3845) were obtained from Horizon Discovery (Waterbeach, UK), and SignalSilence Control siRNA (unconjugated) and SignalSilence Rictor siRNA were purchased from Cell Signaling Technology (Danvers, MA).

Techniques: Activity Assay, Control, Western Blot