Journal: Molecular Cancer Research

Article Title: Hierarchical Phosphorylation of HOXB13 by mTOR Dictates Its Activity and Oncogenic Function in Prostate Cancer

doi: 10.1158/1541-7786.MCR-23-0086

Figure Lengend Snippet: mTOR negatively regulates HOXB13 protein stability. A, Time course treatment of LNCaP and PC3 cells with the mTOR inhibitor Torin1 (100 nmol/L). B, Detection of HOXB13 protein levels in LNCaP and PC3 cells with mTOR knockdown using two inducible shRNAs (induced by 1 μg/mL Doxycycline for 3 days). C, Assessment of HOXB13 protein levels in LNCaP cells that were androgen deprived for 48 hours prior to treatment with the synthetic androgen R1881 (10 nmol/L) and/or Torin1 (100 nmol/L) for another 24 hours. D, PC3 cells were serum-starved for 24 hours and then stimulated by 10% serum ± 100 nmol/L Torin1 for another 24 hours. E, HOXB13 immunoblot analysis in LNCaP cells with genetic overexpression of the mTOR upstream activator RHEB ± pharmacologic inhibition of mTOR with Torin1 (250 nmol/L, 8 hours). F, HOXB13 immunoblot analysis in LNCaP cells with genetic knockdown of mTOR upstream inhibitor TSC1 or TSC2 ± pharmacologic inhibition of mTOR with Torin1 (250 nmol/L, 8 hours). G, HOXB13 polyubiquitination was examined in response to the mTOR inhibitor Torin1 (100 nmol/L) for 24 hours in LNCaP cells. H, HOXB13 polyubiquitination following inducible shRNA-mediated mTOR knockdown (1 μg/mL Dox, 3 days) in LNCaP cells. Relative HOXB13 protein levels were quantified and normalized over Tubulin using Image J software. Data in A–F represent means ± SEM of three independent experiments. Statistics were calculated by one-way ANOVA. *, P < 0.05; **, P < 0.01; ns, not significant.

Article Snippet: Inducible shRNAs of mTOR and HOXB13 were self-cloned by inserting the short hairpin sequence into the inducible shRNA pLKO-TetON backbone (Addgene, catalog no. 21915).

Techniques: Knockdown, Western Blot, Over Expression, Inhibition, shRNA, Software

Journal: Molecular Cancer Research

Article Title: Hierarchical Phosphorylation of HOXB13 by mTOR Dictates Its Activity and Oncogenic Function in Prostate Cancer

doi: 10.1158/1541-7786.MCR-23-0086

Figure Lengend Snippet: HOXB13 phosphorylation promotes its degradation by SKP2. A, Protein synthesis inhibitor Cycloheximide was used to evaluate the protein stability of HOXB13 WT, phospho-deficient mutant (3A) and phospho-mimicking mutant (3D) expressed in 293T cells. B, Polyubiquitination of HOXB13 mutants were examined using 293T whole cell lysates. C, Exogenous HOXB13 interacted with E3 ligase SKP2 in 293T cells. D, HOXB13 and E3 ligase SKP2 interact endogenously in LNCaP cells ± R1881 (10 nmol/L, 24 hours). E, SKP2 overexpression decreased HOXB13 protein levels which were rescued by proteasome inhibitor MG132 (20 μmol/L, 8 hours) in LNCaP cells. F, Genetic knockdown of SKP2 increased HOXB13 protein levels in LNCaP cells. G, Inducible shRNA-mediated knockdown of mTOR (1 μg/mL Dox, 3 days) decreased HOXB13 interaction with SKP2 in LNCaP cells. H, Pharmacologic inhibition of mTOR (100 nmol/L Torin1, 24 hours) impaired HOXB13 interaction with SKP2 in LNCaP cells. I, HOXB13 phosphomimic mutant 3D interacted more strongly with SKP2 in 293T cells compared with WT or phospho-deficient mutant 3A. J, Diminished HOXB13 protein levels provoked by SKP2 overexpression was rescued by Torin1 treatment (250 nmol/L, 8 hours) in LNCaP cells. K, Schematic of mTOR primed and SKP2 licensed HOXB13 degradation. Relative HOXB13 protein levels were quantified and normalized over Tubulin using Image J software. Data in A, E, F, and J represent means ± SEM of three independent experiments. Statistics were calculated by one-way ANOVA. *, P < 0.05; **, P < 0.01; ns, not significant.

Article Snippet: Inducible shRNAs of mTOR and HOXB13 were self-cloned by inserting the short hairpin sequence into the inducible shRNA pLKO-TetON backbone (Addgene, catalog no. 21915).

Techniques: Phospho-proteomics, Mutagenesis, Over Expression, Knockdown, shRNA, Inhibition, Software

Journal: Oncogene

Article Title: MutT Homolog 1 (MTH1) maintains multiple KRAS-driven pro-malignant pathways

doi: 10.1038/onc.2014.195

Figure Lengend Snippet: (A) MTH1 suppression via lentiviral shRNA. Immunoblotting for MTH1 expression was carried out against approximately 25 μg of protein lysates derived from the indicated NSCLC cell lines transduced with either plko.shGFP or plko.shMTH1 to confirm MTH1 knockdown. GAPDH was used to normalize for protein loading. (B) Proliferation rates in the indicated samples transduced with either shGFP or shMTH1. **p<0.01, *p<0.05. (C) SA-beta-gal activity. As described in the Methods section, SA-beta-gal staining was assayed and quantitated in the indicated samples. (D) MTH1 suppression reduces xenograft tumor formation by KRAS-mutant NSCLC cells. Tumor formation kinetics are shown for the indicated samples. For A549 cells, which were transduced with either the Tet-on shLuc or shMTH1 constructs, doxycycline was administered to the animals at day 14 post-injection (indicated by an arrow on the tumor growth curves). (E) Representative images of animals used in the xenograft studies. Animals from each group are shown to the right of the respective tumor curves in (D). White arrows indicate sites of subcutaneous tumors. Tumor incidences (tumors formed/sites injected) were as follows: A549 shLuc and shMTH1: 12/12; H358 shGFP: 13/13, H358 shMTH1: 12/13; H23 shGFP: 7/12, H23 shMTH1: 4/12. **p < 0.01. (F) In vivo correlation between MTH1 and proliferation. Quantification of immunohistochemical staining for Ki67 and MTH1 is shown for the indicated samples as number of positively stained cells/high-powered field (hpf) counted.

Article Snippet: The retroviral pBABE.puro.KRASV12 construct (Addgene plasmid 9052), the pBABE.neo.Myr-AKT1 (15266) and the inducible lentiviral plko-TET-on backbone construct were obtained from Addgene.

Techniques: shRNA, Western Blot, Expressing, Derivative Assay, Transduction, Activity Assay, Staining, Mutagenesis, Construct, Injection, In Vivo, Immunohistochemical staining

Journal: Oncogene

Article Title: MutT Homolog 1 (MTH1) maintains multiple KRAS-driven pro-malignant pathways

doi: 10.1038/onc.2014.195

Figure Lengend Snippet: (A) High KRAS levels in human NSCLC tumors correlate with high MTH1 levels. A qPCR analysis of MTH1 and KRAS mRNA levels in matched tumor/adjacent normal NSCLC samples from untreated patients is shown. ActinB was used for normalization. All matched normal tissue values were set at 1 (indicated by a black horizontal line). Linear regression analysis is shown below the qPCR analysis and was carried out using Microsoft Excel macro. (B) MTH1 levels promote maintenance of KRAS expression. BEAS2B cells were transformed with KRASV12 as described in the Results section and then transduced with either an MTH1 overexpression construct, shMTH1 construct or counterpart control vectors. Immunoblotting was carried out on 15 μg of lysates from the indicated samples, expressing either a control vector (pBp), KRASV12, KRASV12 in conjunction with pBh.MTH1 (KRASV12/MTH1) or KRASV12 in the background of either lentiviral shGFP or shMTH1 (KRASV12/shGFP, KRASV12/shMTH1). Blots were probed with the indicated antibodies. (C) MTH1 suppression reduces KRAS mRNA and protein levels. qPCR analysis is shown from the indicated samples. The black horizontal line indicates a baseline of 1. Two independent Western blots (representative blots shown in (B)) were quantitated via densitometry using the ImageJ gel analysis module. Normalized fold-changes in protein expression are shown. (D) MTH1 enhances soft agar colony formation by KRASV12-transformed BEAS2B cells. Representative images, three weeks post-seeding, are shown from the indicated samples to depict extent of colony formation. (E) Quantitation of results from the soft agar assay depicted in (D), ***p<0.005.

Article Snippet: The retroviral pBABE.puro.KRASV12 construct (Addgene plasmid 9052), the pBABE.neo.Myr-AKT1 (15266) and the inducible lentiviral plko-TET-on backbone construct were obtained from Addgene.

Techniques: Expressing, Transformation Assay, Transduction, Over Expression, Construct, Western Blot, Plasmid Preparation, Quantitation Assay, Soft Agar Assay

Journal: Molecular cell

Article Title: KAP1 is a Chromatin Reader that Couples Steps of RNA Polymerase II Transcription to Sustain Oncogenic Programs

doi: 10.1016/j.molcel.2020.04.024

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Plasmid: pLKO.1/Doxycycline Inducible Addgene Cat# 21915 Plasmid: pLKO.1/Doxycycline Inducible-shNT Addgene Cat# 47541 Plasmid: pLKO.1/Doxycycline Inducible-shCDK9 This Paper N.A.

Techniques: Mutagenesis, Recombinant, Protease Inhibitor, SYBR Green Assay, Western Blot, Staining, Plasmid Preparation, RNA HS Assay, Bicinchoninic Acid Protein Assay, Clone Assay, shRNA, Software

Journal: American Journal of Physiology - Gastrointestinal and Liver Physiology

Article Title: Krüppel-like factor 5 is essential for maintenance of barrier function in mouse colon

doi: 10.1152/ajpgi.00172.2017

Figure Lengend Snippet: Epithelial barrier function is impaired in KLF5 knockdown Caco-2 BBe cells. Caco-2 BBe pLKO.1 cells and Caco-2 BBe KLF5ΔIND cells were seeded in Transwell plates. On day 3 of the culture, cells were treated with water or doxycycline to induce expression of short-hairpin RNA (shRNA). Cells were maintained for a total 28 days. A: Western blot analysis of the KLF5 protein levels in Caco-2 BBe KLF5ΔIND –DOX and Caco-2 BBe KLF5ΔIND +DOX cells (bottom) and Caco-2 BBe pLKO.1 –DOX and Caco-2 BBe pLKO.1 +DOX cells (top) on day 28. A result from 3 independent experiments is shown. B: quantitative representation of KLF5 protein levels in 3 independent experiments, normalized to GAPDH. Data represent means ± SE (n = 3), ***P < 0.001, Student’s t-test. C: transepithelial electrical resistances of Caco-2 BBe KLF5ΔIND (gray lines) and Caco-2 BBe wild-type (WT, black lines) monolayers grown in absence (–DOX, solid line) and presence (+DOX, broken line) of doxycycline were measured during 28 days of culture. Data represent means ± SE (n = 3), *P < 0.05, **P < 0.01, and ***P < 0.001, Student’s t-test. D: permeability to FITC-4 kDa dextran measured on day 28. Data are shown as pass-through concentrations of FITC-dextran. Empty boxes, –DOX; filled boxes, +DOX. Data represent means ± SE (n = 3), **P < 0.01 by Student’s t-test.

Article Snippet: The inducible short-hairpin RNA (shRNA) expression plasmid pLKO.1-tet-on was obtained from Addgene (Cambridge, MA) ( 42 ).

Techniques: Expressing, shRNA, Western Blot, Permeability

Journal: American Journal of Physiology - Gastrointestinal and Liver Physiology

Article Title: Krüppel-like factor 5 is essential for maintenance of barrier function in mouse colon

doi: 10.1152/ajpgi.00172.2017

Figure Lengend Snippet: Expression of DSG2 but not DSC2 is reduced and desmosomal morphology is changed upon KLF5 knockdown in Caco-2 BBe cells. Caco-2 BBe pLKO.1 and KLF5ΔIND cells were seeded in Transwell plates, and, on day 3 of the culture, cells were treated with water or doxycycline to induce expression of shRNA. A: Western blot analysis of the DSG2 and DSC2 protein levels in Caco-2 BBe pLKO.1 –DOX and +DOX and Caco-2 Bbe KLF5ΔIND –DOX and +DOX cells (bottom) on day 28. A result from 3 independent experiments is shown. B: quantitative representation of DSG2 and DSC2 protein levels in 3 independent experiments, normalized to GAPDH. Data represent means ± SE (n = 3), ***P < 0.001, Student’s t-test. C: immunofluorescence staining of DSG2 (a–d) and DSC2 (e–h) in Caco-2 BBe pLKO.1 –DOX/+DOX and Caco-2 BBe KLF5ΔIND –DOX/+DOX cells collected on day 28 of culture. The top part of each panel shows X–Y projections obtained from Z-stacks, and companion Z-plane cross sections are shown on bottom. DNA was counterstained with TO-PRO3. Images were taken at ×400 magnification. Scale bars indicate 20 µm. Relative mean fluorescence intensity was quantified (i and j). Data represent means ± SE (n = 6), ***P < 0.001 by Student’s t-test. D: transmission electron microscopy images showing different morphologies involving decreased microvilli numbers and altered desmosome structures between Caco-2 BBe pLKO.1 –DOX/+DOX, Caco-2 BBe KLF5ΔIND –DOX (a–c, ×30,000 magnification), and Caco-2 BBe KLF5ΔIND +DOX cells (d, ×30,000 magnification). White arrows, apical junctional complexes; yellow arrowheads, desmosomes. Scale bars indicate 400 nm. E: quantitative analysis of the number of desmosomes in Caco-2 BBe pLKO.1 –DOX/+DOX, Caco-2 BBe KLF5ΔIND –DOX, and Caco-2 BBe KLF5ΔIND +DOX cells. Data represent means ± SE (n = 4 for pLKO.1 –DOX, n = 8 for pLKO.1 +DOX, n = 4 for Caco-2 BBe KLF5ΔIND –DOX, and n = 5 for Caco-2 BBe KLF5ΔIND +DOX), **P < 0.01, Student’s t-test. F: quantitative analysis of the width of the intercellular spaces between desmosomes in Caco-2 BBe KLF5ΔIND –DOX and Caco-2 BBe KLF5ΔIND +DOX cells. Data represent means ± SE (n = 26 for pLKO.1 –DOX, n = 42 for pLKO.1 +DOX, n = 12 for Caco-2 BBe KLF5ΔIND –DOX, and n = 8 for Caco-2 BBe KLF5ΔIND +DOX), **P < 0.01, Student’s t-test.

Article Snippet: The inducible short-hairpin RNA (shRNA) expression plasmid pLKO.1-tet-on was obtained from Addgene (Cambridge, MA) ( 42 ).

Techniques: Expressing, shRNA, Western Blot, Immunofluorescence, Staining, Fluorescence, Transmission Assay, Electron Microscopy