Journal: Military Medical Research

Article Title: Elevated FBXL6 activates both wild-type KRAS and mutant KRAS G12D and drives HCC tumorigenesis via the ERK/mTOR/PRELID2/ROS axis in mice.

doi: 10.1186/s40779-023-00501-8

Figure Lengend Snippet: Fig. 5 mTOR and MEK inhibition significantly blocks hepatocarcinogenesis and lung metastasis triggered by Fbxl6 elevation and Kras mutation. a Nude mice bearing KLC orthotopic HCC tumors were randomized into three groups: the negative control (NC) group, everolimus (E) group and everolimus combined with trametinib (E + T) group. The NC group was treated with vehicle, the E group was treated with everolimus (diluted in DMSO and coil oil, 4 mg/kg, intraperitoneal injection) twice per week for six rounds, and the E + T group was treated with both everolimus (the everolimus dose was as in the E group) and trametinib (diluted in DMSO and coil oil, 0.5 mg/kg, intraperitoneal injection) twice per week for six rounds. The mice were sacrificed after the last injection, and representative images showing tumorigenesis are presented. n = 7. b Tumor weight, the tumor/liver weight ratio, and tumor volume were analyzed (n = 7). c Representative images showing the effect of mTOR and MEK/ERK inhibitors on lung metastases. The lung metastasis rate (d) and number of lung distant lung metastatic foci (e) were quantified. f Representative HE and IHC staining images for lipase C (LIPC) showing distinct lung metastatic foci expressing LIPC. Scale bars = 200 or 50 μm. g Representative images of HE and IHC staining for Prelid2, p-mTOR, p-4EBP1, and p-ERK in the NC, E and E + T groups. Representative consecutive IHC staining images are presented. Scale bars = 200 or 50 μm. One-way ANOVA with Tukey’s multiple comparisons test was used in (b, e). *P < 0.05; **P < 0.01; ***P < 0.001. KLC LSL-KrasG12D/+;LSL-Fbxl6KI/+;Alb-Cre, mTOR mammalian target of rapamycin, MEK mitogen-activated protein kinase kinase, Fbxl6 F-box and leucine-rich repeat 6, Kras kirsten rat sarcoma, NC negative control, E everolimus, T trametinib, DMSO dimethyl sulfoxide, ERK extracellular signal-regulated kinase, IHC immunohistochemistry

Article Snippet: Anti-p-mTOR (5536), anti-mTOR (2983), anti-p-Akt (4060), anti-Akt (4691), anti-p-ERK (4376), anti-ERK (4695), anti-p-S6 (2211), anti-S6 (2217), antip-70S6K (9234), anti-70S6K (9202), anti-p-eukaryotic translation initiation factor 4E binding protein 1 (4EBP1) (2855), and anti-4EBP1 (9644) antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Inhibition, Mutagenesis, Negative Control, Injection, Immunohistochemistry, Expressing

Journal: Breast Cancer Research

Article Title: The mTOR inhibitor rapamycin down-regulates the expression of the ubiquitin ligase subunit Skp2 in breast cancer cells

doi: 10.1186/bcr1533

Figure Lengend Snippet: The effect of rapamycin on cell cycle profile and mTOR activation. ( a ) The effect of rapamycin on cell cycle distribution. T47D cells were treated with rapamycin (20 nM) or DMSO (0.02%) for 24 h and subjected to FACS analysis to determine the distribution of the cell cycle. ( b ) The effect of rapamycin on the phosphorylation of the mTOR effectors p-S6K1 and p-4E-BP1. The breast cancer cell lines T47D and MDA-MB-231 were treated with rapamycin (20 nM) for different time periods and the phosphorylated forms of the proteins was determined by western blot analysis. Skp1 was used as a loading control. Arrows indicate the specific bands.

Article Snippet: To detect phosphorylated proteins in the mTOR pathway we used rabbit polyclonal antibodies against phospho-4E-BP1 (Thr37/46) or phospho-p70 S6 kinase (Thr389) (Cell Signaling Technology Inc., Beverly, MA, USA) diluted at 1:1000.

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

Journal:

Article Title: A role of the kinase mTOR in cellular transformation induced by the oncoproteins P3k and Akt

doi:

Figure Lengend Snippet: Constitutive phosphorylation of S6K and 4E-BP1 in CEF transformed with P3k and Akt. CEF and CEF infected with RCAS, RCAS-Myr-Akt, and RCAS-Myr-c-P3k were serum starved for 40 h and then stimulated with PDGF for 15 min. The cells were lysed, and the lysates were resolved in a 10% (A) or a 15% (B) SDS-polyacrylamide gel and then transferred to a polyvinylidene difluoride membrane. The blot was probed with anti-phospho-S6K (threonine 389), anti-S6K, anti-phospho-4E-BP1 (serine 65), anti-4E-BP1, or anti-phospho-Erk antibody.

Article Snippet: They were then probed with anti-p70 s6k (C-19, Santa Cruz Biotechnology) to detect S6K, anti-phospho-p70 s6k (Thr-389, Cell Signaling Technology) to detect phosphorylated S6K, anti-4E-BP1 (Santa Cruz Biotechnology) to detect 4E-BP1, anti-phospho-4E-BP1 (Ser-65, Cell Signaling Technology) to detect phosphorylated 4E-BP1, and anti-phospho-Erk (E10, Cell Signaling Technology) to detect phosphorylated extracellular signal-regulated kinase, Erk.

Techniques: Transformation Assay, Infection

Journal:

Article Title: A role of the kinase mTOR in cellular transformation induced by the oncoproteins P3k and Akt

doi:

Figure Lengend Snippet: Correlation between the ability to induce S6k activation and 4E-BP1 inactivation with the ability to induce oncogenic transformation. Cell lysates were prepared as described in the legend to Fig. ​Fig.1.1. The lysates were resolved in a 10% (A) or a 15% (B) SDS-polyacrylamide gel. The blot was probed with anti-phospho-S6K (threonine 389), anti-S6K, or anti-phospho-4E-BP1 (serine 65) antibody.

Article Snippet: They were then probed with anti-p70 s6k (C-19, Santa Cruz Biotechnology) to detect S6K, anti-phospho-p70 s6k (Thr-389, Cell Signaling Technology) to detect phosphorylated S6K, anti-4E-BP1 (Santa Cruz Biotechnology) to detect 4E-BP1, anti-phospho-4E-BP1 (Ser-65, Cell Signaling Technology) to detect phosphorylated 4E-BP1, and anti-phospho-Erk (E10, Cell Signaling Technology) to detect phosphorylated extracellular signal-regulated kinase, Erk.

Techniques: Activation Assay, Transformation Assay

Journal: The Journal of Biological Chemistry

Article Title: Role of Inositol Trisphosphate Receptors in Autophagy in DT40 Cells

doi: 10.1074/jbc.M110.114207

Figure Lengend Snippet: Possible sites of regulation of the canonical pathway of autophagy by IP3 receptors and Ca2+. The middle box shows several of the molecular complexes that participate in autophagy. The pathways by which physiological factors such as nutrient availability and growth factors (indicated in red) modulate autophagy are shown schematically. Possible sites at which IP3Rs or elevated cytosolic Ca2+ could also modulate this pathway are indicated in blue. The specific mechanisms are as follows: 1, transfer of Ca2+ from the ER to the mitochondria mediated by IP3Rs could contribute to the maintenance of an elevated “energy state” (high ATP/AMP ratio); 2, Ca2+ acting through calmodulin-dependent kinase kinase-β (CaMKK-β) can activate AMPK (15); 3, Vps34 has also been suggested to be a CaM-dependent enzyme (46); 4, Bcl-2, by binding IP3Rs, can influence the amount bound to the Beclin-1-Vps34 complex (37) ; and 5, activation of calpains has been proposed to inhibit autophagy (16). The only target of calpains in the autophagic pathway that has been described is Atg5 (63). Abbreviations used: FIP200, focal adhesion kinase-interacting protein; Ulk, Unc51-like kinase; TSC, tuberous sclerosis complex protein; Rheb, Ras homology enriched in brain protein; S6K-1, p70 ribosomal protein S6 kinase-1; 4E-BP1, eukaryotic translation initiation factor 4E-binding protein 1.

Article Snippet: The following antibodies were obtained from Cell Signaling (MA): LC3B rabbit polyclonal Ab; phospho-mTOR (Ser-2448) rabbit polyclonal Ab; phospho-mTOR (Ser-2481) rabbit polyclonal Ab; total mTOR rabbit polyclonal Ab; phospho-4E-BP1 (Thr-37/46) (236B4) rabbit monoclonal Ab; phospho-Akt (Ser-473) (587F11) mouse monoclonal Ab; total Akt (5G3) mouse monoclonal Ab; phospho-p70 S6 kinase (Thr-389) (108D2) rabbit monoclonal Ab; total p70 S6 kinase rabbit polyclonal Ab; Beclin-1 rabbit polyclonal Ab; phospho-AMPKα (Thr-172) rabbit polyclonal Ab; and total AMPKα rabbit polyclonal Ab.

Techniques: Binding Assay, Activation Assay

Journal: The Journal of Biological Chemistry

Article Title: Role of Inositol Trisphosphate Receptors in Autophagy in DT40 Cells

doi: 10.1074/jbc.M110.114207

Figure Lengend Snippet: Higher autophagic flux in IP3R TKO and pore-dead cell lines is associated with a reduced activity of mTOR. A, WT, IP3R TKO, and the D2550A pore-inactive (pore-dead) DT40 cells lines were incubated in nutrient-replete medium with serum in the presence or absence of staurosporine (1 μm) for 6 h. After treatment, the cells were lysed in a buffer containing protein phosphatase inhibitors and processed for immunoblotting with the indicated Abs against p70 phospho-S6 kinase (S6K) and 4E-BP1, two key substrates of the mTORC1 kinase complex. The data shown are representative of three experiments. B, data from three experiments were quantitated with densitometry and are shown as the means ± S.E. with the wild-type levels set to 100%. C, amount of phospho- and total S6 kinase was measured in lysates from wild-type and TKO DT40 cells as well as cells expressing the chicken (DKO-1) or rat type 1 IP3Rs.

Article Snippet: The following antibodies were obtained from Cell Signaling (MA): LC3B rabbit polyclonal Ab; phospho-mTOR (Ser-2448) rabbit polyclonal Ab; phospho-mTOR (Ser-2481) rabbit polyclonal Ab; total mTOR rabbit polyclonal Ab; phospho-4E-BP1 (Thr-37/46) (236B4) rabbit monoclonal Ab; phospho-Akt (Ser-473) (587F11) mouse monoclonal Ab; total Akt (5G3) mouse monoclonal Ab; phospho-p70 S6 kinase (Thr-389) (108D2) rabbit monoclonal Ab; total p70 S6 kinase rabbit polyclonal Ab; Beclin-1 rabbit polyclonal Ab; phospho-AMPKα (Thr-172) rabbit polyclonal Ab; and total AMPKα rabbit polyclonal Ab.

Techniques: Activity Assay, Incubation, Western Blot, Expressing

Journal: PLoS ONE

Article Title: Benzyl Isothiocyanate Causes FoxO1-Mediated Autophagic Death in Human Breast Cancer Cells

doi: 10.1371/journal.pone.0032597

Figure Lengend Snippet: (A) Immunoblotting for phospho-(S2448)-mTOR, phospho-(S65)-4E-BP1, and phospho-(T389)-P70s6k using lysates from MDA-MB-231 and MCF-7 cells treated with DMSO or BITC (2.5 or 5 µM BITC) for 6 or 12 h. Numbers on top of the bands represent change in protein level relative to corresponding DMSO-treated control. Immunoblotting for each protein was performed at least twice using independently prepared lysates and the results were comparable. (B) Immunoblotting for phospho-(S2448)-mTOR, total mTOR, and phospho-(T389)-P70s6k using supernatant from MDA-MB-231 xenografts harvested from control mice ( n = 5) and 7.5 µmol BITC-treated mice ( n = 4). Densitometric quantitation (mean arbitrary units ± SD) for control and BITC-treated groups is shown on top of the bands. Difference in expression of mTOR and phospho-P70s6k between control and BITC groups was statistically significant by two-tailed Student's t -test.

Article Snippet: Antibodies were purchased from the following sources- anti-LC3 antibody recognizing both full-length and cleaved forms was from MBL International (Woburn, MA); antibodies against p62 (for immunoblotting), phospho-(S2448)-mTOR, phospho-(T389)-P70s6k, phospho-(S65)-4E-BP1, FoxO1 (immunoblotting and immunohistochemistry), Atg7, and LC3 isoform B (LC3B) were from Cell Signaling Technology (Danvers, MA); anti-acetylated FoxO1 and anti-HA antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA); anti-actin antibody was purchased from Sigma-Aldrich; and anti-mTOR and anti-Mn-SOD antibodies were from EMD Chemicals-Calbiochem (Gibbstown, NJ).

Techniques: Western Blot, Control, Quantitation Assay, Expressing, Two Tailed Test