Journal: Nucleic Acids Research

Article Title: BTG3-dependent VCP/p97 nuclear translocation is required for efficient repair of UV-induced DNA lesions

doi: 10.1093/nar/gkaf626

Figure Lengend Snippet: Loss of BTG3 leads to reduced mobility of XPC at damage sites. ( A – C ) Colocalization of BTG3 and XPC at damage sites after UV. PLA was conducted with HaCaT cells using anti-BTG3 and anti-XPC (A). Mean fluorescence intensity and PLA foci number were quantified and shown in panels (B) and (C), respectively, with n ≧ 100. Data were analyzed using unpaired two-tailed t -test and presented as mean ± SEM Scale bar, 10 μm. ( D ) Co-immunoprecipitation of endogenous BTG3 and XPC from HaCaT cells. ( E, F ) Retention of XPC at localized UV damage in BTG3 KO cells. Localized UVC-irradiation (100 J/m 2 ) was performed with 5 μM isopore membrane filter and XPC loading at localized UV damage sites was assessed using confocal microscopy (E). Scale bar, 10 μm. Percent cells with XPC-positive foci were counted and presented as mean ± SEM in panel (F). Data were analyzed using unpaired two-tailed t -test. n ≧ 50. ( G, H ) Mobility of XPC was reduced in BTG3 KO cells. The mobility of XPC-GFP in mock-treated or global UV-irradiated (20 J/m 2 ) parental and BTG3 KO HEK293T cells was assessed using FRAP. Fluorescence recovery was monitored over 120 s and normalized to prebleach intensity ( n = 36 from 3 independent experiments) (G). Data were analyzed using unpaired two-tailed t -tests and shown as mean ± SEM in panel (H).

Article Snippet: After incubation with PLA blocking solution, HaCaT cells were incubated with anti-XPC (Santa Cruz, 1:200 dilution) and anti-BTG3 (Lab raised, 1:100 dilution) antibodies overnight at 4°C with gentle rocking.

Techniques: Fluorescence, Two Tailed Test, Immunoprecipitation, Irradiation, Membrane, Confocal Microscopy

Journal: Nature Communications

Article Title: Fortilin binds IRE1α and prevents ER stress from signaling apoptotic cell death

doi: 10.1038/s41467-017-00029-1

Figure Lengend Snippet: Fortilin interacts with the cytosolic domain of IRE1α and inhibits its protein kinase and RNase activities. a Proximity ligation assay (PLA) shows a specific interaction between fortilin and P-IRE1α in EGF-SubA-treated PC3 cells. The cells were treated with 2 nM EGF-SubA for 24 h and subjected to PLA, using anti-IREα and anti-P-IRE1α antibodies to evaluate fortilin-IRE1α and fortilin-P-IRE1α interaction, respectively. PLA interaction indices were calculated by dividing the number of red dots by the number of nuclei, expressed as means ± s.d. ( n = 3), and analyzed by two-tailed unpaired t -test. NS not statistically significant; *** P < 0.005. Scale bar = 10 µm. b Fortilin co-immunoprecipitates P-IRE1α. PC3 cells were treated with 2 nM EGF-SubA for 24 h, lysed and subjected to immunoprecipitation (IP). c Domain structure of human IRE1α. Human IRE1α consists of the ER luminal domain (aa 1–443), transmembrane domain (aa 444–464), linker region (aa 465–567), kinase domain (aa 568–833), and endoribonuclease (RNase) domain (aa 836–997). The following recombinant proteins were used for biolayer interferometry: full-length IRE1α (aa 1-977), IRE1α-Myc-DDK (aa 1–977); IRE1α-L, GST-IRE1α (aa 1–70); IRE1α-TM, GST-IRE1α (aa 401–500); and IRE1α-C, GST-IRE1α (aa 468–977). d – h Fortilin binds to P-IRE1α through its cytosolic domain. Biotinylated fortilin was immobilized to the streptavidin biosensor. Recombinant IRE1α, either full-length or fragment, was applied to the biosensor at various concentrations, and dissociation constants (Kds, expressed as mean ± s.d., n = 3) were derived. i Lowest energy binding pose of fortilin ( blue ) with cytosolic domain of IRE1α ( green ) (the right panel ) presented with that of a fortilin-fortilin dimer (the left panel ). j Intermolecular interactions between phosphorylated serine724 (pS 724 ) and serine726 (pS 726 ) of the cytosolic domain of IRE1α with lysine residues (K 19 and K 34 ) of fortilin. k Fortilin inhibits the RNase activity of IRE1α. An in vitro IRE1α RNase activity assay was performed by incubating IRE1α with human recombinant fortilin and the substrate fluorescently tagged XBP1 RNA stem loop, the cleavage of which would allow the fluorescein amidite (FAM) to fluoresce. Data were expressed as means ± s.d. ( n = 4) and analyzed by two-tailed unpaired t -test. *** P < 0.005. l Fortilin inhibits the kinase activity of IRE1α. An in vitro IRE1α kinase activity assay was performed by incubating IRE1α with [γ- 33 P]ATP, recombinant fortilin, and myelin basic protein (MBP) as a substrate of the kinase in the kinase reaction buffer. The phosphorylation index was calculated by dividing the radioactivity of MBP for a given fortilin concentration by that of the vehicle control and expressed as means ± s.d. ( n = 2) from which half maximal inhibitory concentration (IC 50 ) was calculated

Article Snippet: Briefly, recombinant human IRE1α (10 µg, SignalChem) was pre-incubated with various concentrations of recombinant human fortilin (0–4 µM) in RNase Assay Buffer (50 mM HEPES, pH 7.2, 100 mM KOAc, 0.005% TritonX-100, 5 mM DTT, 5% glycerol) for 30 min at room temperature.

Techniques: Proximity Ligation Assay, Two Tailed Test, Immunoprecipitation, Recombinant, Derivative Assay, Binding Assay, Activity Assay, In Vitro, Kinase Assay, Radioactivity, Concentration Assay

Journal: Nature Communications

Article Title: Fortilin binds IRE1α and prevents ER stress from signaling apoptotic cell death

doi: 10.1038/s41467-017-00029-1

Figure Lengend Snippet: Protection by fortilin against EGF-SubA-induced liver damage is mediated by its ability to block the activation of the IRE1α-JNK apoptosis pathway. a Experimental protocol. Five-week-old male fortilin WT-liver or fortilin KO-liver mice ( n = 6) were pretreated by vehicle or the IRE1α kinase inhibiting RNAse attenuator-6 (KIRA6) for 3 days, challenged by EGF-SubA once on the third day, and treated by vehicle or KIRA6 for an additional 6 days. b KIRA6 protects both fortilin WT-liver and fortilin KO-liver mice against EGF-SubA-induced liver damage. The sera from EGF-SubA-challenged fortilin WT-liver and fortilin KO-liver mice, treated with either vehicle or KIRA6, were assayed for ALT. Data were expressed as means ± s.d. ( n = 6) and analyzed by two-tailed unpaired t -test. NS not statistically significant; *** P < 0.005. c The livers of EGF-SubA-challenged fortilin WT-liver and fortilin KO-liver mice exhibit similar gross appearance when treated with KIRA6. Scale bar = 10 mm. d Lack of change in expression and phosphorylation patterns of the PERK and ATF6 pathway proteins in the EGF-SubA-challenged livers, regardless of the status of fortilin or of KIRA6 treatment. The total lysates from the livers of EGF-SubA-challenged fortilin WT-liver and fortilin KO-liver mice, treated with either KIRA6 or vehicle, were subjected to quantitative IB using the indicated antibodies. Data were expressed as means ± s.d. ( n = 4) and analyzed by two-tailed unpaired t -test. e The livers of EGF-SubA-challenged fortilin WT-liver and fortilin KO-liver mice exhibit a similar degree of IRE1α and JNK phosphorylation when treated with KIRA6. Data were expressed as means ± s.d. ( n = 4) and analyzed by two-tailed unpaired t -test. * P < 0.05; *** P < 0.005. f The livers of EGF-SubA-challenged, KIRA6-treated, fortilin WT-liver and fortilin KO-liver mice exhibit a similar degree of XBP1 splicing. The total RNA from the livers of EGF-SubA-challenged fortilin WT-liver and fortilin KO-liver mice were assayed for the amounts of XBP1s and XBP1u . Data were expressed as means ± s.d. ( n = 4) and analyzed by two-tailed unpaired t -test. *** P < 0.005. g The livers of EGF-SubA-challenged fortilin WT-liver and fortilin KO-liver mice exhibit equal amounts of apoptosis when treated with KIRA6. Paraffin sections from the livers of EGF-SubA-treated fortilin WT-liver and fortilin KO-liver mice ( n = 6 each) for cleaved lamin. Data were expressed as means ± s.d. ( n = 6) and analyzed by two-tailed unpaired t -test. ** P < 0.01; F.C . fold change. Scale bar = 50 µm. h , i The immunostaining of the livers of EGF-SubA-challenged fortilin WT-liver and fortilin KO-liver mice exhibit the same degree of IRE1α pathway activation when treated with KIRA6. Data were expressed as means ± s.d. ( n = 6) and analyzed by two-tailed unpaired t -test. *** P < 0.005. Scale bar = 50 µm. j Proposed model of the role of fortilin in ER stress-induced apoptosis

Article Snippet: Briefly, recombinant human IRE1α (10 µg, SignalChem) was pre-incubated with various concentrations of recombinant human fortilin (0–4 µM) in RNase Assay Buffer (50 mM HEPES, pH 7.2, 100 mM KOAc, 0.005% TritonX-100, 5 mM DTT, 5% glycerol) for 30 min at room temperature.

Techniques: Blocking Assay, Activation Assay, Two Tailed Test, Expressing, Immunostaining

Journal: Oncotarget

Article Title: Role of creatine shuttle in colorectal cancer cells

doi: 10.18632/oncotarget.28436

Figure Lengend Snippet: ( A ) Effect of DNFB (5 μM) and knockdown of MTCK or CKB on EGFR phosphorylation. Lower panels, effect of siMTCK and siCKB on expression of MTCK and CKB, respectively. ( B ) Semi-quantification of EGFR phosphorylation levels. ( C ) Effect of ATP on suppressed EGFR phosphorylation by DNFB (5 μM). ( D ) Effect of oligomycin on EGFR phosphorylation. ( E ) Effect of CyCr on creatine phosphorylation. ( F ) Effect of CyCr on EGFR phosphorylation. ( G ) Co-immunoprecipitation using anti-CKB antibody or anti-EGFR antibody to examine binding of CKB and EGFR. ( H ) Duolink ® proximity ligation assay. Red, proximity signal of EGFR and CKB. Blue, DAPI. Scale bar, 10 μm. Error bars: standard deviation of three independent trials. Statistical significance was calculated using a two-tailed ordinary analysis of variance. Abbreviations: DNFB: dinitrofluorobenzene; EGFR: epithelial growth factor receptor; pEGFR: phosphorylated EGFR; EGF: epithelial growth factor; CKB: creatine kinase B; MTCK: mitochondrial creatine kinase; siC: short interfering RNA (siRNA) used as control; siMTCK: siRNA for MTCK; siCKB: siRNA for CKB; GAPDH: glyceraldehyde 3-phosphate dehydrogenase; OM: oligomycin; CyCr: cyclocreatine; pCr: phosphocreatine; IP: immunoprecipitation; CB: Coomassie blue; WCL: whole cell lysate; DAPI: 4′,6-diamidino-2-phenylindole.

Article Snippet: The following is a brief description: HT29 cells (1 × 10 5 ) were seeded and incubated for 24 h on Nunc chamber slides (Thermo Fisher), and then incubated with Duolink ® Blocking Solution at 37°C for 60 min. Anti-EGFR antibody (mouse monoclonal, 0.5 μg/mL, Santa Cruz) and anti-CKB antibody (rabbit polyclonal, 0.5 μg/mL, Boster) were incubated with the Duolink ® Antibody Diluent.

Techniques: Knockdown, Phospho-proteomics, Expressing, Immunoprecipitation, Binding Assay, Proximity Ligation Assay, Standard Deviation, Two Tailed Test, Small Interfering RNA, Control

Journal: Cancers

Article Title: Metallothioneins and Megalin Expression Profiling in Premalignant and Malignant Oral Squamous Epithelial Lesions

doi: 10.3390/cancers13184530

Figure Lengend Snippet: Basic demographic and clinicopathological features of involved subjects, and variables measured in the study.

Article Snippet: Tissues were subsequently incubated with mouse monoclonal anti-MT I + II antibody (clone E9; Dako Cytomation, Santa Clara, CA, USA; diluted 1:50 with blocking solution) and rabbit polyclonal anti-megalin antibody (H-245, Santa Cruz Biotechnology, Dallas, TX, USA; diluted 1:50 with blocking solution) for 12 h at 4 °C in a humid chamber.

Techniques: Biomarker Discovery

Journal: Cancers

Article Title: Metallothioneins and Megalin Expression Profiling in Premalignant and Malignant Oral Squamous Epithelial Lesions

doi: 10.3390/cancers13184530

Figure Lengend Snippet: Megalin expression in premalignant and malignant oral squamous epithelial lesions. Representative photomicrographs show immunohistochemical staining with the anti-megalin antibody on paraffin-embedded sections of tissue samples obtained from subjects with healthy oral mucosa ( A ), patients with oral lichen planus ( B ), oral leukoplakia ( C ), grade I OSCC ( D – F ), grade II OSCC ( G – I ), and grade III OSCC ( J – L ). Arrows on I and L indicate chromosomal megalin immunopositivity in mitotic cells. Magnifications: ( D , G , J ) × 100; ( A – C , E , F , H , K ) × 400; ( I , L ) × 1000.

Article Snippet: Tissues were subsequently incubated with mouse monoclonal anti-MT I + II antibody (clone E9; Dako Cytomation, Santa Clara, CA, USA; diluted 1:50 with blocking solution) and rabbit polyclonal anti-megalin antibody (H-245, Santa Cruz Biotechnology, Dallas, TX, USA; diluted 1:50 with blocking solution) for 12 h at 4 °C in a humid chamber.

Techniques: Expressing, Immunohistochemical staining, Staining

Journal: Cancers

Article Title: Metallothioneins and Megalin Expression Profiling in Premalignant and Malignant Oral Squamous Epithelial Lesions

doi: 10.3390/cancers13184530

Figure Lengend Snippet: Quantified megalin staining intensities in premalignant and malignant oral squamous epithelial lesions. The measurements were made by ImageJ software and data are expressed in arbitrary units (AU) as the median of average gray values with range. HOM, healthy oral mucosa; OLP, oral lichen planus; OSCC, oral squamous cell carcinoma. * p < 0.000001 when each group is compared to the other.

Article Snippet: Tissues were subsequently incubated with mouse monoclonal anti-MT I + II antibody (clone E9; Dako Cytomation, Santa Clara, CA, USA; diluted 1:50 with blocking solution) and rabbit polyclonal anti-megalin antibody (H-245, Santa Cruz Biotechnology, Dallas, TX, USA; diluted 1:50 with blocking solution) for 12 h at 4 °C in a humid chamber.

Techniques: Staining, Software

Journal: Cancers

Article Title: Metallothioneins and Megalin Expression Profiling in Premalignant and Malignant Oral Squamous Epithelial Lesions

doi: 10.3390/cancers13184530

Figure Lengend Snippet: Co-expression and interaction of metallothionein I/II and megalin in oral squamous cell carcinoma. ( A ) Representative photomicrographs of double immunofluorescence staining with anti-MT I/II (red staining) and anti-megalin (green staining) antibodies on paraffin-embedded sections of the OSCC tissue. Blue marks DAPI staining of nuclei. Arrows indicate sites of membrane colocalization. Magnification: × 1000. ( B ) Representative photomicrographs obtained by proximity ligation assay (PLA) on paraffin-embedded sections of the OSCC tissue using anti-MT I/II and anti-megalin antibodies. Red fluorescent signals represent interaction sites. Blue marks DAPI staining of nuclei. Magnification: × 1000.

Article Snippet: Tissues were subsequently incubated with mouse monoclonal anti-MT I + II antibody (clone E9; Dako Cytomation, Santa Clara, CA, USA; diluted 1:50 with blocking solution) and rabbit polyclonal anti-megalin antibody (H-245, Santa Cruz Biotechnology, Dallas, TX, USA; diluted 1:50 with blocking solution) for 12 h at 4 °C in a humid chamber.

Techniques: Expressing, Double Immunofluorescence Staining, Staining, Membrane, Proximity Ligation Assay

Journal: Cancers

Article Title: Metallothioneins and Megalin Expression Profiling in Premalignant and Malignant Oral Squamous Epithelial Lesions

doi: 10.3390/cancers13184530

Figure Lengend Snippet: Correlation of metallothionein I/II and megalin staining intensities. Pearson correlation analysis included data obtained from the overall study group. Correlation coefficient (r) = 0.8 ( p < 0.001).

Article Snippet: Tissues were subsequently incubated with mouse monoclonal anti-MT I + II antibody (clone E9; Dako Cytomation, Santa Clara, CA, USA; diluted 1:50 with blocking solution) and rabbit polyclonal anti-megalin antibody (H-245, Santa Cruz Biotechnology, Dallas, TX, USA; diluted 1:50 with blocking solution) for 12 h at 4 °C in a humid chamber.

Techniques: Staining

Journal: Cancers

Article Title: Metallothioneins and Megalin Expression Profiling in Premalignant and Malignant Oral Squamous Epithelial Lesions

doi: 10.3390/cancers13184530

Figure Lengend Snippet: Impact of smoking habit on metallothionein I/II ( A ) and megalin ( B ) expression. The measurements were made by ImageJ software and data are expressed in arbitrary units (AU) as the median of average gray values with range. HOM, healthy oral mucosa; OLP, oral lichen planus; OSCC, oral squamous cell carcinoma.

Article Snippet: Tissues were subsequently incubated with mouse monoclonal anti-MT I + II antibody (clone E9; Dako Cytomation, Santa Clara, CA, USA; diluted 1:50 with blocking solution) and rabbit polyclonal anti-megalin antibody (H-245, Santa Cruz Biotechnology, Dallas, TX, USA; diluted 1:50 with blocking solution) for 12 h at 4 °C in a humid chamber.

Techniques: Expressing, Software