epithelial cell line female  (ATCC)

Journal: iScience

Article Title: M-Motif, a potential non-conventional NLS in YAP/TAZ and other cellular and viral proteins that inhibits classic protein import

doi: 10.1016/j.isci.2025.112105

Figure Lengend Snippet: Characterization of structural requirements and functional properties of TAZ and YAP nuclear import (A) Domain organization of TAZ with regions important for nucleocytoplasmic shuttling highlighted. Lats-phosphorylation sites are shown as yellow circles with labels, hydrophobic motifs FLxx[I,V,L,M] as blue lines. CC: coiled-coil region; NES: nuclear export signal; NLS: nuclear localization signal; PBM: PDZ domain binding motif; TAD: transactivation domain; TBD: TEAD-binding domain; WW: WW domain; WW-NLS: globular NLS composed of the WW domain. (B) Localization of diffusion-limited 5C constructs in pig proximal tubular epithelial LLC-PK1 cells, also see Figure S1 . 5C-R5A-NES: shuttling control comprising a variant of the SV40 NLS and the HIV Ref NES. WT: 5C-TAZ 4SA or 5C-YAP 5SA; ΔNLS: constructs with deletion of residues 321–345 (TAZ) or 413–427 (YAP); ΔPBM: constructs missing the last 5 aa (the PBM); control: 5C control. Left: Representative fluorescence microscopy images of transfected cells, without (top) and with 4h LMB treatment (bottom). The scale bar represents 50 μm. Right: Quantification of the nuclear accumulation of the 5C-constructs upon LMB treatment for the indicated times. Median nucleocytoplasmic fluorescence ratios (median N/C of >100 cells) were determined for individual constructs as described previously and data were fitted to mono-exponential growth curves with plateau. Number of repeats: 4. (C) Localization of 5C-NLS constructs. Left: Representative fluorescence microscopy images of transfected LLC-PK1 cells. The scale bar represents 50 μm. Right: Quantification of the nuclear accumulation of 5C, 5C-TAZ NLS, 5C-YAP-NLS, and 5C-R5A in LLC-PK1 and HEK cells. (D) The TAZ NLS inhibits the nuclear uptake of full-length TAZ. Cells were transfected with constant amounts of 5C or 5C-TAZ 4SA in combination with mCherry-TAZ NLS at ratios 1:0, 1:1, 1:2, 1:3, and 1:4. mCherry encoding vector was added to keep the total amounts of DNA constant. The increase in nuclear accumulation of 5C constructs after 6h LMB treatment, relative to no LMB addition is shown as ΔN/C (LMB). Number of repeats: 8. Also see Figure S2 A. (E) Localization of 1C constructs in LLC-PK1 and RPE-1 cells. (F and G) Interaction between TAZ and IPO7. mCitrine (1C) and 1C-TAZ constructs were expressed in HEK cells. IPO7 (F) or Citrine-constructs (G) were immunoprecipitated and analyzed by western blotting using GFP- and IPO7 specific antibodies. Red asterisks indicate the position of relevant bands. (H) Effect of ivermectin (iver; 25 μM) on the nuclear accumulation of endogenous TAZ/YAP and MRTF upon treatment with low calcium medium (LCM). Response is given as the percentage of uninhibited N/C increase at 60 min (TAZ/YAP) or 20 min (MRTF) LCM treatment. (I) Nuclear accumulation of 5C-TAZ NLS and 5C-SV40 NLS in the presence of indicated concentrations of ivermectin. Also see Figure S2 B. (J) Effect of ivermectin on the nuclear accumulation of shuttling 5C-constructs induced by LMB treatment. ΔN/C (LMB) are calculated as in (D). (K) Nuclear import of TAZ 4SA is Ran-independent. Cells were co-transfected with indicated constructs and non-related (siNR) or Ran-specific (siRan) siRNA and treated with LMB for 4h. (L) Nuclear import of TAZ 4SA is ATP-independent. Left: Representative fluorescence microscopy images showing the effect of 2-deoxyglucose and azide on the cellular distribution of KPNA2. The scale bar represents 50 μm. Right: cells transfected with indicated constructs and treated for 4h with or without LMB, in the presence or absence of 2-deoxyglucose and azide. Means ± SD are depicted, and number of repeats are indicated. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.005, ∗∗∗∗ p < 0.001; one-way ANOVA and Tukey-Kramer test.

Article Snippet: These cells were cultured in high-glucose DMEM. hTERT RPE cells, a Telomerase immortalized human retina pigmented epithelial cell line (female) were obtained from the American Type Culture Collection (ATCC Cat# CRL-4000, RRID:CVCL_4388).

Techniques: Functional Assay, Phospho-proteomics, Binding Assay, Diffusion-based Assay, Construct, Control, Variant Assay, Fluorescence, Microscopy, Transfection, Plasmid Preparation, Immunoprecipitation, Western Blot

Journal: iScience

Article Title: M-Motif, a potential non-conventional NLS in YAP/TAZ and other cellular and viral proteins that inhibits classic protein import

doi: 10.1016/j.isci.2025.112105

Figure Lengend Snippet:

Article Snippet: These cells were cultured in high-glucose DMEM. hTERT RPE cells, a Telomerase immortalized human retina pigmented epithelial cell line (female) were obtained from the American Type Culture Collection (ATCC Cat# CRL-4000, RRID:CVCL_4388).

Techniques: Recombinant, Transfection, Luciferase, Reporter Assay, Control, Plasmid Preparation, Software

Journal: The Journal of Neuroscience

Article Title: PINK1 Content in Mitochondria is Regulated by ER-Associated Degradation

doi: 10.1523/JNEUROSCI.1691-18.2019

Figure Lengend Snippet: Ub-PINK1 interacts with VCP. A, Endogenous PINK1 interacting proteins revealed by Coomassie blue-stained SDS-PAGE gel. Endogenous PINK1 and its associated complexes were enriched by IP with anti-PINK1 antibody in HeLa cells. Anti-HA antibody and preimmune IgG (Ctr IgG) were used as controls. Bands labeled 1, 2, and 3 were identified as Ufd2A, VCP, and Npl4, respectively. Individual LC-MS/MS data can be found in Figure 1-1. B, PINK1 and VCP interaction in HEK293T cells cotransfected with WT PINK1 and VCP evidenced by co-IP using either an anti-PINK1 or anti-VCP antibody. *Denotes IgG heavy chain. C, Endogenous PINK1 and VCP interaction in SH-SY5Y cells evidenced by co-IP using anti-VCP-antibody. D, As in B, except that cells were transfected either with WT PINK1 or mutant PINK1K137R. *Denotes IgG heavy chain. E, PINK1 and Ufd2A interaction in HEK293T cells stably-expressing WT PINK1 and transfected with Ufd2A-flag evidenced by IP using an anti-PINK1 antibody. F, Ub-PINK1 IB in WT PINK1 stably-transfected HEK293T cells. Endogenous VCP was efficiently knocked down by VCP shRNAs. G, Ub-PINK1 IB in WT PINK1 stably-transfected HEK293T cells. Endogenous Ufd2A was efficiently knocked down by Ufd2A shRNAs. H, Ub-PINK1 IB in PINK1 stably-transfected HEK293T cells. Endogenous Ufd1 was efficiently knocked down by Ufd1 shRNAs. Data are representative of three independent experiments performed under identical conditions.

Article Snippet: Human female embryonic kidney epithelial HEK293T, human female cervical epithelial HeLa, monkey kidney fibroblast COS-7 (unknown sex), and human female bone marrow epithelial SH-SY5Y cell lines were from ATCC and maintained in medium suggested by the provider.

Techniques: Staining, SDS Page, Labeling, Liquid Chromatography with Mass Spectroscopy, Co-Immunoprecipitation Assay, Transfection, Mutagenesis, Stable Transfection, Expressing

Journal: The Journal of Neuroscience

Article Title: PINK1 Content in Mitochondria is Regulated by ER-Associated Degradation

doi: 10.1523/JNEUROSCI.1691-18.2019

Figure Lengend Snippet: 52 kDa PINK1 interacts with the ERAD machinery. A, PINK1 and Sec61β interaction in WT PINK1 stably-expressing HEK293T cells transfected with Sec61β evidenced by IP using either an anti-PINK1 or anti-Sec61β antibody. *Denotes IgG heavy chain. B, Endogenous PINK1 and Sec61β interaction in SH-SY5Y cells evidenced by IP using an anti-Sec61β antibody. C, WT PINK1 and endogenous gp78 interaction in stably PINK1 expressing HEK293T cells evidenced by co-IP using either an anti-PINK1 or anti-gp78 antibody. D, Ub-PINK1 IB in PINK1 stably-transfected HEK293T cells. Endogenous Sec61β was efficiently knocked down by Sec61β shRNAs. E, Ub-PINK1 IB in PINK1 stably-transfected HEK293T cells. Endogenous gp78 was efficiently knocked down by gp78 shRNA validated by IB. F, Ub-PINK1 IB in Hrd1 deficient HEK293T cells generated by CRISPR that were transfected with WT PINK1. Experiment shown in F was repeated once independently with similar results while those shown in A–E are representative of 3–4 independent experiments performed under identical conditions.

Article Snippet: Human female embryonic kidney epithelial HEK293T, human female cervical epithelial HeLa, monkey kidney fibroblast COS-7 (unknown sex), and human female bone marrow epithelial SH-SY5Y cell lines were from ATCC and maintained in medium suggested by the provider.

Techniques: Stable Transfection, Expressing, Transfection, Co-Immunoprecipitation Assay, shRNA, Generated, CRISPR