Journal: Redox Biology

Article Title: Fluorescent proteins such as eGFP lead to catalytic oxidative stress in cells ☆

doi: 10.1016/j.redox.2017.03.002

Figure Lengend Snippet: eGFP in cells induces gene expression associated with oxidative stress and other biological processes. Images of HeLa cells and stable eGFP-expressing HeLa cells (HeLa/eGFP) under a fluorescent microscope with a green filter can be seen in (a). eGFP was also detected by Western blotting, with GAPDH staining as the loading control. In (b) extracellular H 2 O 2 was determined in HeLa and HeLa/eGFP. Catalase was added to cultures at 500 U/mL. (c) Microarray-based experiments were designed to show the effect of GFP expression in the gene expression of two independent cell models. The two GSEA (Gene Set Enrichment Analyses ) were performed using the hallmark dataset (h.all.v5.0.symbols) and 1000 permutations. The contrasts were HeLa/eGFP versus HeLa, and Hela.tet.eGFP + doxycycline subtracted from HeLa.tet.eGFP versus HeLa.tet + doxycycline subtracted from HeLa.tet. The enriched gene sets with p -values lower than 0.001 were ranked, and matches between the two independent analyses are shown highlighted in yellow. The top genes contributing to the enrichment for the commonly enriched gene sets are depicted as heat maps in . (d) Intracellular levels of HIF1α were determined by Western blotting using whole cell lysates of HeLa cells and HeLa cells stably expressing eGFP. In (e), GAPDH densitometry was used as the loading control to normalize the densitometry of HIF1α. * is used to show statistically significant differences between HeLa and HeLa/eGFP.

Article Snippet: HeLa.tet and HeLa.tet.eGFP were purchased from GenTarget Inc. (San Diego, CA) and were also certified to be free of mycoplasma.

Techniques: Expressing, Microscopy, Western Blot, Staining, Microarray, Stable Transfection

Journal:

Article Title: The kinase haspin is required for mitotic histone H3 Thr 3 phosphorylation and normal metaphase chromosome alignment

doi: 10.1101/gad.1267105

Figure Lengend Snippet: Haspin is phosphorylated during mitosis, and its overexpression delays mitotic progression. (A) HeLa Tet-On/myc-haspin- or vector-alone-stably-transfected cells were induced with 1 μg/mL doxycycline for the times indicated, and myc-haspin expression was assessed by rabbit antihaspin immunoblotting. (B) HeLa Tet-On/myc-haspin- or vector-alone-stably-transfected cells were induced with 1 μg/mL doxycycline for 24 h before synchronization at G1/S and analysis as described for Figure 4B. (C) Synchronized HeLa Tet-On/myc-haspin cells as shown in B were lysed and analyzed by immunoblotting and haspin in vitro kinase assay with γ32P-ATP using H3–GST or H3-T3A–GST as a control. (D) Synchronized HeLa Tet-On/vector cells as shown in B were analyzed as described in C.(E) Induced HeLa Tet-On/myc-haspin cells were fractionated into looselyadherent mitotic (mitosis) and adherent interphase-enriched (interphase) populations or treated with colcemid prior to lysis. Rabbit antihaspin or rabbit IgG control immunoprecipitates were analyzed by rabbit antihaspin immunoblotting. (F) Induced HeLa Tet-On/myc-haspin cells were treated colcemid for 12 h prior to release. Whole-cell lysates made at the times indicated were analyzed by antihaspin immunoblotting. (G) Induced HeLa Tet-On/myc-haspin cells were treated colcemid for 12 h or untreated prior to cell lysis. Antihaspin immunoprecipitates were incubated with or without λ phosphatase as described in Materials and Methods and analyzed by antihaspin immunoblotting.

Article Snippet: Stable transfection of HeLa Tet-On (BD Clontech) cells was carried out using Lipofectin with Plus Reagent (Invitrogen).

Techniques: Over Expression, Plasmid Preparation, Stable Transfection, Transfection, Expressing, Western Blot, In Vitro, Kinase Assay, Lysis, Incubation

Journal:

Article Title: The kinase haspin is required for mitotic histone H3 Thr 3 phosphorylation and normal metaphase chromosome alignment

doi: 10.1101/gad.1267105

Figure Lengend Snippet: Endogenous haspin is required for phosphorylation of histone H3 on Thr 3. (A) Immunoprecipitates with rabbit antihaspin or rabbit IgG negative control (neg) antibodies from HeLa cell lysates were subjected to in vitro kinase assays with γ32P-ATP and H3–GST or H3-T3A–GST as substrates. Coomassie blue staining was used to confirm equivalent levels of GST proteins. (B) Antihaspin and anti-aurora B immunoprecipitates from HeLa cell lysates were subjected to in vitro kinase reactions with γ32P-ATP and H3–GST, H3-T3A–GST, or H3-S10A–GST proteins as substrates. Coomassie blue staining was used to confirm equivalent levels of GST proteins. (C) The synchronized HeLa Tet-On/vector cells shown in Figure 5B were analyzed by haspin in vitro kinase assay described for Figure 5C. Note that this is a longer exposure of the autoradiogram shown in Figure 5D, carried out in order to visualize endogenous haspin activity. Similar results were obtained with HeLa cells (data not shown). (D) HeLa cells were transfected with haspin siRNA (Ambion ID #1093), control siRNA (Ambion 4611), or no siRNA. Approximately 30 h after transfection, the cells were incubated with nocodazole for 16 h (mitotic) or left untreated (asynchronous) prior to lysis. Antihaspin and anti-aurora B immunoprecipitates were subjected to in vitro kinase reactions with γ32P-ATP and H3–GST, H3-T3A–GST, or H3-S10A–GST as substrates, and lysates were immunoblotted with anti-phospho-H3 (Thr-3) or anti-phospho-H3 (Ser-10) antibodies. The anti-phospho-H3 (Thr-3) blot was stripped and reprobed with antihistone H3 antibodies. The increased haspin activity in nocodazole-treated cells was not a reproducible finding, as shown in B and C. (E) NIH3T3 cells were transfected with mouse haspin (Ambion ID 67120) or negative control (Ambion 4611) siRNAs and analyzed as described for D.

Article Snippet: Stable transfection of HeLa Tet-On (BD Clontech) cells was carried out using Lipofectin with Plus Reagent (Invitrogen).

Techniques: Negative Control, In Vitro, Staining, Plasmid Preparation, Kinase Assay, Activity Assay, Transfection, Incubation, Lysis