Journal: Thoracic Cancer

Article Title: Tofacitinib overcomes an IFNγ ‐induced decrease in NK cell‐mediated cytotoxicity via the regulation of immune‐related molecules in LC ‐2/ad

doi: 10.1111/1759-7714.13847

Figure Lengend Snippet: Expression of PD‐L1 in LC‐2/ad cells treated with IFNγ or EGF followed by each signal inhibitor. (a) The expression level of PD‐L1 in LC‐2/ad cells pretreated with each signaling inhibitor (gefitinib 1 μM; LY, LY294002 10 μM; PD, PD98059 10 μM; PF, PF4708671 10 μM; Tof, tofacitinib 1 μM) for 2 h followed by 100 ng/mL EGF or 0.5 ng/mL IFNγ for 24 h evaluated by flow cytometry. The results represent three independent experiments. The relative mean fluorescence intensity (rMFI) of PD‐L1 was calculated in cells pretreated with each signal inhibitor followed by (b) IFNγ and followed by (c) EGF, or (d) treated with each signal inhibitor alone based on three independent experiments and evaluated using Student's t ‐test. Bars indicate SD. * p < 0.05, ** p < 0.01, n.s., no significance; NT, no treatment control

Article Snippet: For cell culture, tofacitinib (#S5001, Selleck), gefitinib (#13166; Cayman), LY294002 (#70920; Cayman), PD98059 (#10006726; Cayman), and PF4708671 (#4032; Tocris) stock solutions were prepared in DMSO (Sigma‐Aldrich), whereas recombinant human IFNγ (#11500; PBL Assay Science) and epidermal growth factor (EGF) (#236‐EG; R&D Systems) stock solutions were prepared in PBS (−).

Techniques: Expressing, Flow Cytometry, Fluorescence, Control

Journal: Thoracic Cancer

Article Title: Tofacitinib overcomes an IFNγ ‐induced decrease in NK cell‐mediated cytotoxicity via the regulation of immune‐related molecules in LC ‐2/ad

doi: 10.1111/1759-7714.13847

Figure Lengend Snippet: IFNγ increases the expression of MHC class I molecules via the JAK‐STAT pathway, which is blocked by tofacitinib in LC‐2/ad cells. LC‐2/ad cells were (a) treated with 100 ng/mL EGF or 0.5 ng/mL IFNγ for 24 h, (b) pretreated with each signaling inhibitor for 2 h followed by 0.5 ng/mL IFNγ for 24 h, or (c) treated with each signaling inhibitor alone and evaluated by flow cytometry. The relative mean fluorescence intensity (rMFI) of MHC class I molecules was calculated based on three independent experiments and evaluated using Student's t ‐test. Bars indicate SD. * p < 0.05, ** p < 0.01, n.s., no significance; NT, no treatment control; IFN, IFNγ; LY, LY294002 (10 μM); PD, PD98059 (10 μM); PF, PF4708671 (10 μM); Tof, tofacitinib (1 μM)

Article Snippet: For cell culture, tofacitinib (#S5001, Selleck), gefitinib (#13166; Cayman), LY294002 (#70920; Cayman), PD98059 (#10006726; Cayman), and PF4708671 (#4032; Tocris) stock solutions were prepared in DMSO (Sigma‐Aldrich), whereas recombinant human IFNγ (#11500; PBL Assay Science) and epidermal growth factor (EGF) (#236‐EG; R&D Systems) stock solutions were prepared in PBS (−).

Techniques: Expressing, Flow Cytometry, Fluorescence, Control

Journal: Thoracic Cancer

Article Title: Tofacitinib overcomes an IFNγ ‐induced decrease in NK cell‐mediated cytotoxicity via the regulation of immune‐related molecules in LC ‐2/ad

doi: 10.1111/1759-7714.13847

Figure Lengend Snippet: IFNγ decreases the expression of NKG2D ligands MICA/B and ULBP2/5/6 in LC‐2/ad cells. The expression of MICA/B in LC‐2/ad cells (a) treated with 100 ng/mL EGF or 0.5 ng/mL IFNγ for 24 h, (b) pretreated with each signaling inhibitor for 2 h followed by 0.5 ng/mL IFNγ for 24 h, (c) followed by 100 ng/mL EGF for 24 h, or (d) treated with each signal inhibitor alone and evaluated by flow cytometry. The expression of ULBP2/5/6 in LC‐2/ad cells (e) treated with 100 ng/mL EGF or 0.5 ng/mL IFNγ for 24 h, (f) pretreated with each signaling inhibitor for 2 h followed by 0.5 ng/mL IFNγ for 24 h, (g) followed by 100 ng/mL EGF for 24 h, or (h) treated with each signal inhibitor alone and evaluated by flow cytometry. The relative mean fluorescence intensity (rMFI) of MICA/B or ULBP2/5/6 was calculated based on three independent experiments and evaluated using Student's t ‐test. Bars indicate SD. * p < 0.05, ** p < 0.01, n.s., no significance; NT, no treatment control; IFN, IFNγ; LY, LY294002 (10 μM); PD, PD98059 (10 μM); PF, PF4708671 (10 μM); Tof, tofacitinib (1 μM)

Article Snippet: For cell culture, tofacitinib (#S5001, Selleck), gefitinib (#13166; Cayman), LY294002 (#70920; Cayman), PD98059 (#10006726; Cayman), and PF4708671 (#4032; Tocris) stock solutions were prepared in DMSO (Sigma‐Aldrich), whereas recombinant human IFNγ (#11500; PBL Assay Science) and epidermal growth factor (EGF) (#236‐EG; R&D Systems) stock solutions were prepared in PBS (−).

Techniques: Expressing, Flow Cytometry, Fluorescence, Control

Journal: Biomedicines

Article Title: Targeting p70S6K1 Inhibits Glycated Albumin-Induced Triple-Negative Breast Cancer Cell Invasion and Overexpression of Galectin-3, a Potential Prognostic Marker in Diabetic Patients with Invasive Breast Cancer.

doi: 10.3390/biomedicines13030612

Figure Lengend Snippet: Figure 1. Bio-statistical analysis. (A) Kaplan–Meier plot of p70S6K1 overall survival (OS) in patients with invasive BC showing poor-survival patients with high p70S6K1 gene expression levels. Data were collected from the METABRIC dataset within cBioPortal. (B) The mRNA expression of p70S6K1 based on lymph node status reveals high p70S6K1 gene expression in patients with lymph node metastases (lymph node positive). Data were obtained from the SCAN-B dataset within MammOnc- DB. FPKM: Fragments per kilobase of transcript per million mapped reads. (C) The mRNA expression of p70S6K1 according to PAM50 subtypes indicates a higher gene expression of p70S6K1 among the basal-like BC subtype compared to other subtypes. Data were derived from the Creighton breast tumor compendium within MammOnc-DB. (D) The mRNA expression of p70S6K1 among DM and non-DM TNBC patients shows increased p70S6K1 gene expression among diabetic TNBC patients (data point outside the error bars is an outlier). Data were obtained from the study of Tamez-Peña et al. [34]. The log-rank test was employed for OS analysis. The Kaplan–Meier plot was created using GraphPad Prism 10.0. The middle line of the boxplots represents the median value; the error bars represent the maximum and minimum values.

Article Snippet: Prior to treatment, cells were exposed to the specific pharmacological p70S6K1 inhibitor PF-4708671 (#sc-361288, Santa Cruz Biotechnology, Heidelberg, Germany) at a concentration of 10 μM (the optimal concentration determined from preliminary studies) along with 0.04% dimethyl sulfoxide (DMSO, the solvent for the reconstitution of PF-4708671) for an incubation of 1 h. Throughout the experiment, cells were exposed to 100 μg/mL of GA.

Techniques: Gene Expression, Expressing, Derivative Assay

Journal: Biomedicines

Article Title: Targeting p70S6K1 Inhibits Glycated Albumin-Induced Triple-Negative Breast Cancer Cell Invasion and Overexpression of Galectin-3, a Potential Prognostic Marker in Diabetic Patients with Invasive Breast Cancer.

doi: 10.3390/biomedicines13030612

Figure Lengend Snippet: Figure 2. Impact of p70S6K1 downregulation using siRNA technology on glycated albumin (GA)-stimulated MDA-MB-231 cell invasion. (A) Representative Western blot showing p70S6K1 downregulation after 48 h of transfection using p70S6K1 siRNA, compared with the basal level of p70S6K1 detected in untransfected and negative control (NC) siRNA-transfected MDA-MB-231 cells. The bar graph shows the relative expression of p70S6K1 expressed in pixel density and related to GAPDH, the loading control. (B–E) Representative photomicrographs showing the stained and invaded untreated MDA-MB-231 cells (B), NC siRNA-transfected cells treated with GA (C), and p70S6K1 siRNA-transfected cells treated with GA (D) using the Boyden chamber system. Scale bar: 100 µm. (E) The bar graph shows the number of invaded MDA-MB-231 cells, NC siRNA, and p70S6K1 siRNA-transfected cells treated with or without GA, compared to the untreated cells, the control. The results are presented as the mean ± SD of three independent experiments. (***) and (****) signify a statistically significant difference (p < 0.001 and p < 0.0001) compared with the control.

Article Snippet: Prior to treatment, cells were exposed to the specific pharmacological p70S6K1 inhibitor PF-4708671 (#sc-361288, Santa Cruz Biotechnology, Heidelberg, Germany) at a concentration of 10 μM (the optimal concentration determined from preliminary studies) along with 0.04% dimethyl sulfoxide (DMSO, the solvent for the reconstitution of PF-4708671) for an incubation of 1 h. Throughout the experiment, cells were exposed to 100 μg/mL of GA.

Techniques: Western Blot, Transfection, Negative Control, Expressing, Control, Staining

Journal: Biomedicines

Article Title: Targeting p70S6K1 Inhibits Glycated Albumin-Induced Triple-Negative Breast Cancer Cell Invasion and Overexpression of Galectin-3, a Potential Prognostic Marker in Diabetic Patients with Invasive Breast Cancer.

doi: 10.3390/biomedicines13030612

Figure Lengend Snippet: Figure 3. Inhibition of glycated albumin (GA)-induced p70S6K1 and ERK1/2 phosphorylation in MDA-MB-231 cells after p70S6K1 downregulation. Representative Western blots showing the impact of p70S6K1 downregulation on GA-induced p70S6K1 and ERK1/2 phosphorylation compared with the negative control (NC) siRNA-transfected MDA-MB-231 cells. The results are presented as the mean ± SD of three independent experiments. (*) and (**) signify a statistically significant difference (p < 0.05 and p < 0.01) compared with the control.

Article Snippet: Prior to treatment, cells were exposed to the specific pharmacological p70S6K1 inhibitor PF-4708671 (#sc-361288, Santa Cruz Biotechnology, Heidelberg, Germany) at a concentration of 10 μM (the optimal concentration determined from preliminary studies) along with 0.04% dimethyl sulfoxide (DMSO, the solvent for the reconstitution of PF-4708671) for an incubation of 1 h. Throughout the experiment, cells were exposed to 100 μg/mL of GA.

Techniques: Inhibition, Phospho-proteomics, Western Blot, Negative Control, Transfection, Control

Journal: Biomedicines

Article Title: Targeting p70S6K1 Inhibits Glycated Albumin-Induced Triple-Negative Breast Cancer Cell Invasion and Overexpression of Galectin-3, a Potential Prognostic Marker in Diabetic Patients with Invasive Breast Cancer.

doi: 10.3390/biomedicines13030612

Figure Lengend Snippet: Figure 4. Decrease in glycated albumin (GA)-induced cancer-related protein expression in MDA- MB-231 cells after p70S6K1 downregulation. (A) Representative oncology array membranes of cancer-related proteins detected in untreated MDA-MB-231 cells, cells treated with GA, and negative control (NC) siRNA- and p70S6K1 siRNA-transfected MDA-MB-231 cells treated with GA, after 48 h of incubation. (B) The bar graphs show the relative expression of the relevant cancer-related proteins expressed in pixel density based on the similar brightness of spots considered references. The results are presented as the mean ± SD of three independent experiments. (*), (**), and (***) signify a statistically significant difference (p < 0.05, p < 0.01, and p < 0.001) compared with the control.

Article Snippet: Prior to treatment, cells were exposed to the specific pharmacological p70S6K1 inhibitor PF-4708671 (#sc-361288, Santa Cruz Biotechnology, Heidelberg, Germany) at a concentration of 10 μM (the optimal concentration determined from preliminary studies) along with 0.04% dimethyl sulfoxide (DMSO, the solvent for the reconstitution of PF-4708671) for an incubation of 1 h. Throughout the experiment, cells were exposed to 100 μg/mL of GA.

Techniques: Expressing, Negative Control, Transfection, Incubation, Control

Journal: Biomedicines

Article Title: Targeting p70S6K1 Inhibits Glycated Albumin-Induced Triple-Negative Breast Cancer Cell Invasion and Overexpression of Galectin-3, a Potential Prognostic Marker in Diabetic Patients with Invasive Breast Cancer.

doi: 10.3390/biomedicines13030612

Figure Lengend Snippet: Figure 5. Impact of p70S6K1 pathway blockade by PF-4708671 on glycated albumin (GA)-induced cell invasion, signaling pathways, and cancer-related protein expression in MDA-MB-231 cells. (A) Representative Western blot showing the decrease in the S6 ribosomal protein phosphorylation level in MDA-MB-231 cells after 1 h of treatment with the pharmacological p70S6K1 inhibitor PF- 4708671, compared with the S6 ribosomal protein phosphorylation level detected in untreated cells (control) and DMSO-treated cells. PF-4708671 did not affect the total S6 ribosomal protein expression. GAPDH was used as the loading control. (B) Representative Western blots showing the impact of the p70S6K1 blockade on GA-induced S6 ribosomal protein and ERK1/2 phosphorylation compared with the control and DMSO-pretreated MDA-MB-231 cells. (C–F) Representative photomicrographs showing the stained and invaded untreated MDA-MB-231 cells (C), DMSO-pretreated cells with GA (D), and PF-4708671-pretreated cells with GA (E) using the Boyden chamber system. Scale bar: 100 µm.

Article Snippet: Prior to treatment, cells were exposed to the specific pharmacological p70S6K1 inhibitor PF-4708671 (#sc-361288, Santa Cruz Biotechnology, Heidelberg, Germany) at a concentration of 10 μM (the optimal concentration determined from preliminary studies) along with 0.04% dimethyl sulfoxide (DMSO, the solvent for the reconstitution of PF-4708671) for an incubation of 1 h. Throughout the experiment, cells were exposed to 100 μg/mL of GA.

Techniques: Protein-Protein interactions, Expressing, Western Blot, Phospho-proteomics, Control, Staining

Journal: The Journal of Biological Chemistry

Article Title: p70S6K1 (S6K1)-mediated Phosphorylation Regulates Phosphatidylinositol 4-Phosphate 5-Kinase Type I γ Degradation and Cell Invasion *

doi: 10.1074/jbc.M116.742742

Figure Lengend Snippet: S6K1 phosphorylates PIPKIγ90 at Thr-553 and Ser-555. A, alignment of the Akt/S6K1 consensus sequences from different species. B, transfection of constitutively active Akt1 or S6K1 promoted PIPKIγ90 phosphorylation. FLAG-PIPKIγ90 was co-transfected with an empty vector, Myr-Akt1, and S6K1-F5A-E389-R3A into CHO-K1 cells. FLAG-PIPKIγ90 was immunoprecipitated, and PIPKIγ90 phosphorylation was detected with an anti-RXRXXpS/T motif antibody. a.u., arbitrary unit; *, p < 0.05. C, S6K1 phosphorylated PIPKIγ at Thr-553 and Ser-555 in vitro. Recombinant GST-PIPKIγ90501–668, -PIPKIγ90501–668T553A, -PIPKIγ90501–668S555A, and -PIPKIγ90501–668T553A,S555A were phosphorylated with constitutively active S6K1 that was immunoprecipitated from CHO-K1 cells. D, S6K1 phosphorylated PIPKIγ at Thr-553 and Ser-555 in CHO-K1 cells. HA-S6K1-F5A-E389-R3A was co-transfected with FLAG-PIPKIγ90, -PIPKIγ90T553A, -PIPKIγ90S555A, and -PIPKIγ90T553A,S555A into CHO-K1 cells. Data are presented as mean ± S.E. of three independent experiments. **, p < 0.01; ***, p < 0.001 versus WT. E, EGF and HGF stimulated PIPKIγ phosphorylation. MDA-MB-231 cells stably expressing FLAG-PIPKIγ90 were serum-starved and stimulated with EGF (20 ng/ml), HGF (50 ng/ml), SCF (20 ng/ml), and PDGF (20 ng/ml) for 20 min. FLAG-PIPKIγ90 was immunoprecipitated (IP), and phosphorylation was detected with an anti-RXRXXpS/T motif antibody. Data are presented as mean ± S.E. of three independent experiments. *, p < 0.05; **, p < 0.01 versus control (Ctrl). F, HGF-stimulated PIPKIγ phosphorylation was inhibited by Akt and S6K1 inhibitors. MDA-MB-231 cells stably expressing FLAG-PIPKIγ90 were serum-starved, treated with Akt inhibitor VIII and the S6K1 inhibitors DG2 (10 μm) or PF4708671 (10 μm), and then stimulated with HGF for 20 min. Data are presented as mean ± S.E. of four independent experiments. *, p < 0.05 versus HGF. G, HGF-stimulated PIPKIγ phosphorylation was suppressed by depletion of S6K1. S6K1 in MDA-MB-231 cells stably expressing FLAG-PIPKIγ90 was depleted using lentiviruses that express S6K1 shRNAs. Cell were serum-starved and then stimulated with HGF (20 ng/ml) for 20 min. Data are presented as mean ± S.E. of three independent experiments. *, p < 0.05.

Article Snippet: The S6K1 inhibitor PF4708671 was from ApexBio (Houston, TX).

Techniques: Transfection, Plasmid Preparation, Immunoprecipitation, In Vitro, Recombinant, Stable Transfection, Expressing

Journal: The Journal of Biological Chemistry

Article Title: p70S6K1 (S6K1)-mediated Phosphorylation Regulates Phosphatidylinositol 4-Phosphate 5-Kinase Type I γ Degradation and Cell Invasion *

doi: 10.1074/jbc.M116.742742

Figure Lengend Snippet: S6K1-mediated PIPKIγ90 phosphorylation is essential for the invasion. A, PIPKIγ90 and PIPKIγ90T553E,S555E restored the invasive capacity of PIPKIγ-depleted cells but PIPKIγT553A,S555A did not. PIPKIγ-depleted MDA-MB-231 cells were infected with retroviruses that express codon-modified ZZ-PIPKIγ90, -PIPKIγ90T553A,S555A, or PIPKIγ90T553E,S555E and then selected with neomycin. Cells that express shRNA control (Ctrl) were used as controls. w/o, without. B, quantification of the experiment in A. White column, without HGF; gray column, 20 ng/ml HGF. Data are presented as mean ± S.E., n = 3. *, p < 0.05; **, p < 0.01 versus shRNA A1. C, inhibition of the invasion of MDA-MB-231 cells in the absence (white columns) and presence (gray columns) of HGF by the S6K1 inhibitor DG2. Data are mean ± S.E. of three independent experiments. **, p < 0.01. D, S6K1 and Akt activation in MDA-MB-231 cells expressing a control shRNA or S6K1 shRNAs. E, depletion of S6K1 by shRNA inhibited the invasion of MDA-MB-231 cells. Data are presented as mean ± S.E. of three independent experiments. **, p < 0.01; ***, p < 0.001. F, S6K1 and ribosomal protein S6 phosphorylation in MDA-MB-231 cells expressing a control shRNA or Akt1 shRNAs. G, Akt1 knockdown did not significantly affect the invasion of MDA-MB-231 cells. White columns, without HGF; pink columns, with HGF. The data are expressed as mean ± S.E. of three independent experiments. H, effects of the S6K1 inhibitor DG2 on the invasion of PIPKI-depleted MDA-MB-231 cells that express ZZ-PIPKIγ90, -PIPKIγ90T553A,S555A, or -PIPKIγ90T553E,S555E. Cell invasion was performed in the presence of DG2 (black columns, 10 μm) or vehicle (white columns) with HGF (20 ng/ml) in the lower chambers. Data are mean ± S.E. of three independent experiments. *, p < 0.05; ***, p < 0.001.

Article Snippet: The S6K1 inhibitor PF4708671 was from ApexBio (Houston, TX).

Techniques: Infection, Modification, shRNA, Inhibition, Activation Assay, Expressing

Journal: The Journal of Biological Chemistry

Article Title: p70S6K1 (S6K1)-mediated Phosphorylation Regulates Phosphatidylinositol 4-Phosphate 5-Kinase Type I γ Degradation and Cell Invasion *

doi: 10.1074/jbc.M116.742742

Figure Lengend Snippet: S6K1-mediated PIPKIγ phosphorylation is crucial for matrix degradation. A, effect of PIPKIγT553A,S555A and PIPKIγT553E,S555E on gelatin degradation in PIPKIγ-depleted cells. PIPKIγ-depleted MDA-MB-231 cells that express FLAG-PIPKIγ90, -PIPKIγ90T553A,S555A, or -PIPKIγ90T553E,S555E were resuspended in DMEM containing 1% FBS and HGF, plated on Alexa 488 gelatin-coated glass-bottom dishes, and cultured for 10 h. Scale bar = 20 μm. B, quantification of the experiment in A. Data are presented as mean ± S.E. of three independent experiments. *, p < 0.05; **, p < 0.01 versus shRNA control (Ctrl). AU, arbitrary unit. C, inhibition of invadopodium formation in MDA-MB-231 cells by the S6K1 inhibitor DG2. Data are presented as mean ± S.E. of three independent experiments. *, p < 0.05; **, p < 0.01; ***, p < 0.001 versus control.

Article Snippet: The S6K1 inhibitor PF4708671 was from ApexBio (Houston, TX).

Techniques: Cell Culture, shRNA, Inhibition

Journal: The Journal of Biological Chemistry

Article Title: p70S6K1 (S6K1)-mediated Phosphorylation Regulates Phosphatidylinositol 4-Phosphate 5-Kinase Type I γ Degradation and Cell Invasion *

doi: 10.1074/jbc.M116.742742

Figure Lengend Snippet: S6K1 activation correlates with breast cancer metastasis in human clinical specimens. A, human breast cancer primary tumors and the matched metastatic tumors of lymph node tissue were stained with anti-phospho-S6 ribosome protein antibody. B, the intensities of phospho-S6 staining were scored from 0–4, with 4 as the strongest. AU, arbitrary unit.

Article Snippet: The S6K1 inhibitor PF4708671 was from ApexBio (Houston, TX).

Techniques: Activation Assay, Staining

Journal: The Journal of Biological Chemistry

Article Title: p70S6K1 (S6K1)-mediated Phosphorylation Regulates Phosphatidylinositol 4-Phosphate 5-Kinase Type I γ Degradation and Cell Invasion *

doi: 10.1074/jbc.M116.742742

Figure Lengend Snippet: S6K1-mediated phosphorylation regulates PIPKIγ degradation. A, the steady-state levels of PIPKIγWT, PIPKIγT553A,S555A, and PIPKIγT553E,S555E in CHO-K1 cells that were transiently transfected with FLAG-PIPKIγWT, -PIPKIγT553A,S555A and -PIPKIγT553E,S555E, respectively, and treated with DMSO or carfilzomib (1 μm). B, substitution of Thr-553 and Ser-555 with Ala, but not Glu, inhibited degradation of PIPKIγ. CHO-K1 cells expressing BirA were transfected with Avi-PIPKIγWT, -PIPKIγT553A,S555A, and -PIPKIγT553E,S555E and labeled with biotin. The levels of PIPKIγ were detected by Western blotting using Dylight 680-streptavidin. C, time course of degradation of PIPKIγWT, PIPKIγT553A,S555A, and PIPKIγT553E,S555E in CHO-K1 cells. Data represent mean ± S.E. of three experiments. **, p < 0.01; ***, p < 0.001. D, CHO-K1 cells were transiently transfected with Dendra2-PIPKIγ90WT, -PIPKIγ90T553A,S555A, and -PIPKIγ90T553E,S555E and plated on fibronectin. The cells were irradiated for 2 min by a 408-nm laser to convert the Dendra2 fusion protein into red Dendra2 fusion protein. The intensities of the red fluorescence were recorded using time-lapse imaging. Scale bar = 20 μm. E, quantification of the degradation of Dendra2-PIPKIγ90WT, -PIPKIγ90T553A,S555A, and -PIPKIγ90T553E,S555E. Data are presented as mean ± S.E. of four independent experiments. F, the S6K1 inhibitor DG2 or PF4708671 stabilizes PIPKIγ90WT. CHO-K1 cells were transfected with Dendra2-PIPKIγ90WT. 24 h post-transfection, cells were treated with DG2 (10 μm) or PF4708671 (10 μm) for 30 min and then irradiated for 2 min using a 408-nm laser. Data are presented as mean ± S.E. of three experiments. G, the S6K1 inhibitor DG2 (10 μm) had little effect on the degradation of Dendra2-PIPKIγ90T553A,S555E. Data are presented as mean ± S.E. of three experiments.

Article Snippet: The S6K1 inhibitor PF4708671 was from ApexBio (Houston, TX).

Techniques: Transfection, Expressing, Labeling, Western Blot, Irradiation, Fluorescence, Imaging

Journal: The Journal of Biological Chemistry

Article Title: p70S6K1 (S6K1)-mediated Phosphorylation Regulates Phosphatidylinositol 4-Phosphate 5-Kinase Type I γ Degradation and Cell Invasion *

doi: 10.1074/jbc.M116.742742

Figure Lengend Snippet: PIPKIγ90 degradation is required for cancer cell-mediated matrix degradation. A, ubiquitination of PIPKIγWT, -PIPKIγT553A,S555A, and -PIPKIγT553E,S555E. Avi-ubiquitin (Avi-Ub) was co-transfected with ZZ-PIPKIγWT, -PIPKIγT553A,S555A, and -PIPKIγT553E,S555E into CHO-K1 cells expressing BirA. The cells were labeled with biotin, and the ZZ-tagged proteins were immunoprecipitated with IgG-agarose. The ubiquitination was detected using Dylight 680-streptavidin. Data are representative of two independent experiments. B, the steady-state levels of PIPKIγ in MDA-MB-231 cells that express empty pLKO.1 vector or S6K1 shRNAs, treated with DMSO or carfilzomib (Carf, 5 μm). Data are presented as mean ± S.E. of three independent experiments. *, p < 0.05. Ctrl, control. C, the steady-state levels of PIPKIγ in MDA-MB-231 cells that express empty pLKO.1 vector or Akt1 shRNA, treated with DMSO or bortezomib/carfilzomib (B+C, 1 μm each). Data are presented as mean ± S.E. of three independent experiments. D, the expression levels of PIPKIγ in MDA-MB-231 cells expressing a control shRNA or PIPKIγ shRNA A1 and the PIPKIγ-depleted cells that stably express ZZ-PIPKIγ and -PIPKIγK97R. E, PIPKIγWT restored gelatin degradation in PIPKIγ-depleted MDA-MB-231 cells but PIPKIγK97R, a ubiquitination-deficient mutant, did not. Scale bar = 20 μm. F, quantification of the experiment in E. Data are mean ± S.E. of three independent experiments. *, p < 0.05. AU, arbitrary unit.

Article Snippet: The S6K1 inhibitor PF4708671 was from ApexBio (Houston, TX).

Techniques: Transfection, Expressing, Labeling, Immunoprecipitation, Plasmid Preparation, shRNA, Stable Transfection, Mutagenesis