ptk2 cell  (ATCC)

Journal: PLoS ONE

Article Title: Acute Toxicity of the Antifouling Compound Butenolide in Non-Target Organisms

doi: 10.1371/journal.pone.0023803

Figure Lengend Snippet: Source and culture methods of organisms used in the toxicity tests.

Article Snippet: Ptk2 cell , 2 , Seeded at 50000 cells well −1 , 24 h in 5% CO 2 at 37°C , ATCC, Manassas, VA.

Techniques: Cell Culture

Journal: PLoS ONE

Article Title: Acute Toxicity of the Antifouling Compound Butenolide in Non-Target Organisms

doi: 10.1371/journal.pone.0023803

Figure Lengend Snippet: Toxicity test methods. 1

Article Snippet: Ptk2 cell , 2 , Seeded at 50000 cells well −1 , 24 h in 5% CO 2 at 37°C , ATCC, Manassas, VA.

Techniques:

Journal: Journal of Microbiology & Biology Education

Article Title: The Impact of a Semester-Long, Cell Culture and Fluorescence Microscopy CURE on Learning and Attitudes in an Underrepresented STEM Student Population

doi: 10.1128/jmbe.v21i1.2001

Figure Lengend Snippet: A semester-long CURE, using cell culture and fluorescence microscopy techniques.

Article Snippet: PtK2 cells (ATCC; Catalog# CCL-56) were cultured in DMEM/F12K media (Corning; Catalog# MT10092CM) supplemented with 10% heat-inactivated fetal bovine serum (Gibco; Catalog# 16140071), 1% antibiotic/antimycotic solution (Hyclone Catalog# SV3007901) and cultured at 37°C in a CO 2 water jacket incubator.

Techniques: Cell Culture, Fluorescence, Microscopy, Labeling

Journal: BMC Biochemistry

Article Title: Archazolid and apicularen: Novel specific V-ATPase inhibitors

doi: 10.1186/1471-2091-6-13

Figure Lengend Snippet: Inhibition of lysosomal acidification by the novel inhibitors . Potoroo kidney cells (PtK 2 ) were treated with V-ATPase inhibitors for 4 hours and stained for lysosomes (red) with the acidotropic reagent LysoTracker, for mitochondria with MitoTracker (A, green) or for nuclei with Hoechst 33258 (B, blue). The control cells show many red vesicles indicating acidic lysosomes whereas in cells treated with the inhibitors only few red spots can be observed.

Article Snippet: PtK 2 (ATCC CCL-56) or KB-3-1 cells were grown on glass coverslips (13 mm diameter) in four-well-plates.

Techniques: Inhibition, Staining

Journal: The Journal of Cell Biology

Article Title: Stress-induced phosphorylation of CLIP-170 by JNK promotes microtubule rescue

doi: 10.1083/jcb.201909093

Figure Lengend Snippet: JNK-mediated phosphorylation of CLIP-170 at T25 and S147 in various cell lines (related to and ). (A and B) CLIP-170 expression in HeLa cells subjected to acute stresses and to MAPK inhibition. (A) Western blots of cell lysates obtained with the acute stress conditions depicted in . (B) Western blots of cell lysates obtained after acute MAPK inhibition coupled with anisomycin stress, as described in . The histograms (A and B) show the mean ratio between CLIP-170 and β-actin expression levels ± SD, with individual values shown as black dots. Statistics were done using Kruskal-Wallis nonparametric analysis of variance. (C) Western blot analyses of endogenous CLIP-170 migration in lysates of MEF, HuH7, and PtK2 cells subjected to the indicated acute stresses. CLIP-170 motility was increased upon JNK inhibition, except for PtK2 cells (note the absence of T25 in rat-kangaroo CLIP-170 sequence shown in ). (D) Western blots of GFP T186A -H1 from HeLa and PtK2 cell lysates to show its expression levels in experiments corresponding to , , and E of this figure. In the 3 h–DMSO condition, “25” means that the GFP-H1 mutant expressed is suitable for the detection of T25 phosphorylation (therefore, it stands for T147A-S312A). Accordingly, “147” stands for T25A-S312A, “312” for T25A-S147A, and “AAA” for T25A-S147A-S312A GFP T186A -H1 mutants. For cotransfected Flag-MKK7-JNK1APF, “25” stands for T45A-S147A-S312A, “45” for T25A-S147A-S312A, “147” for T25A-T45A-S312A, “312” for T25A-T45A-S147A, and AAAA for T25A-T45A-S147A-S312A GFP T186A -H1 mutants. (E) Phos-tag Western blots of GFP T186A -H1 showing the phosphorylations at T25, S147, and S312 (P = phosphorylated ; np = nonphosphorylated) in PtK2 and HeLa cells, stressed as indicated. The percentages of each phospho-form to total-GFP T186A -H1 are indicated below each gel (P%). (F) Phosphorylation levels were measured as in E in RPE-1, HuH7, and MDA-MB-231 cells. GFP T186A -H1 proteins were probed using the anti–CLIP-170 antibody. The histograms (E and F) show the mean phosphorylation levels ± SD, with individual values shown as black dots. Statistical analyses were done using Kruskal-Wallis nonparametric analysis of variance followed by Mann-Whitney nonparametric comparisons with controls. n indicates the number of independent experiments. *, P < 0.05. n.s., not significant. Ani., anisomycin; P-JNK, phospho-JNK; inh., inhibition.

Article Snippet: Initial PtK2 cells are from (American Type Culture Collection [ATCC]; CCL-56).

Techniques: Expressing, Inhibition, Western Blot, Migration, Sequencing, Mutagenesis, MANN-WHITNEY

Journal: The Journal of Cell Biology

Article Title: Stress-induced phosphorylation of CLIP-170 by JNK promotes microtubule rescue

doi: 10.1083/jcb.201909093

Figure Lengend Snippet: Full-length CLIP-170 phosphomimetics increase rescue frequency in cells. MT dynamics parameters were determined from time-lapse imaging of living PtK2 cells stably expressing GFP–α-tubulin and transiently expressing WT, nonphosphorylatable (A), or phosphomimetic (E) mCherry–CLIP-170 transgenes. (A) Diamond graphs represent mean values of MT dynamic instability parameters after normalization relative to the WT. (B) The values (from the same experiments as in A) of rescue frequencies in each of the depicted classes are reported in box plots showing representative percentiles and outliers. (C) Sample images of the MT network of living PtK2 GFP–α-tubulin cells expressing mCherry–CLIP-170 WT (inset zooms on a comet). Note that the CLIP-170 patches are due to its Zn finger domains and did not interfere with MT dynamics. Scale bars correspond to 5 µm. The numerical mean values ± SD of each parameter are shown in A and B, but also the numbers of cells, MTs, and rescues are detailed in . The statistical comparisons were performed using one-factor ANOVA followed by Fisher's protected t tests for pairwise comparisons . n indicates the number of independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

Article Snippet: Initial PtK2 cells are from (American Type Culture Collection [ATCC]; CCL-56).

Techniques: Imaging, Stable Transfection, Expressing

Journal: The Journal of Cell Biology

Article Title: Stress-induced phosphorylation of CLIP-170 by JNK promotes microtubule rescue

doi: 10.1083/jcb.201909093

Figure Lengend Snippet: mCherry–CLIP-170 expression levels used to record MT dynamics and CLIP-170 comet/remnant parameters (related to and ) . (A) Western blots of CLIP-170 were performed to compare the expression levels of the endogenous CLIP-170 with that of the mCherry–CLIP-170 transgenes used in and and in B–D. Note that the endogenous PtK2 CLIP-170 is not well recognized by the human antibody. Since acrylamide SDS-PAGE was migrated for a long time to separate endogenous and transgenic CLIP-170, low-molecular mass proteins were lost from the gel, and this precluded the use of β-actin as loading control. (B) Sample images of the MT network (GFP–α-tubulin) and mCherry–CLIP-170 comets in living PtK2 (left panels) and MEF (right panels) cells (insets zoom on comets). mCherry–CLIP-170 patches/aggregates are due to the presence of the C-terminal Zn-finger domain of CLIP-170 and do not interfere with MT dynamics. Scale bars correspond to 5 µm. (C) MT dynamics parameters were determined from time-lapse imaging of living PtK2 cells stably expressing GFP–α-tubulin and transiently expressing or not the WT mCherry–CLIP-170 transgene. The same analysis was done in WT MEF cells transfected with GFP–α-tubulin. Diamond graphs represent mean values of MT dynamic instability parameters after normalization relative to the mCherry–CLIP-170 WT. The values of the overall rescue frequencies are reported in box plots showing representative percentiles and outliers. (D) Full-length CLIP-170 phosphomimetics increase the frequency of rescues with immediate or delayed regrowth in cells. MT dynamics parameters were determined from time-lapse imaging of living PtK2 cells stably expressing GFP-α-tubulin and transiently expressing the indicated mCherry–CLIP-170 transgenes (related to ). The values of the rescue frequencies in each class are reported in box plots showing representative percentiles and outliers. The numerical mean values ± SD of each parameter are shown in C and D, but also the numbers of cells, MTs, and transitions are detailed in . The statistical comparisons were performed using one-factor ANOVA followed by Fisher's protected t tests for pairwise comparisons . n indicates the number of independent experiments. ****, P < 0.0001. n.s., not significant.

Article Snippet: Initial PtK2 cells are from (American Type Culture Collection [ATCC]; CCL-56).

Techniques: Expressing, Western Blot, SDS Page, Transgenic Assay, Imaging, Stable Transfection, Transfection

Journal: The Journal of Cell Biology

Article Title: Stress-induced phosphorylation of CLIP-170 by JNK promotes microtubule rescue

doi: 10.1083/jcb.201909093

Figure Lengend Snippet: CLIP-170 H1 phosphomimetics increase rescue frequency in cells. MT dynamics parameters were determined from time-lapse imaging of living PtK2, HeLa, or Clip1/Clip2 KO MEF cells expressing GFP T186A -H1 T25-S147 WT, AA, or EE transgenes. (A) Diamond graphs represent mean values of MT dynamic instability parameters after normalization relative to WT in PtK2 and HeLa cells. (B) The values (from the same experiments as in A) of rescue frequencies in each of the depicted classes are reported in box plots showing representative percentiles and outliers. (C) The overall rescue frequency values for KO MEF cells treated or not with anisomycin 1.2 µM (for ∼30 min) are reported in box plots showing representative percentiles and outliers. (D) Sample images of the MT network of living PtK2 cells expressing GFP T186A -H1 WT (inset zooms on comets). This type of GFP fluorescent signal was used in A, B, and C to follow MT dynamics. Scale bars correspond to 5 µm. The numerical mean values ± SD of each parameter are shown in A, B, and C, but also the numbers of cells, MTs, and rescues are detailed in . The statistical comparisons were performed using one-factor ANOVA followed by Fisher's protected t tests for pairwise comparisons . n indicates the numbers of independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. n.s., not significant. Ani., anisomycin.

Article Snippet: Initial PtK2 cells are from (American Type Culture Collection [ATCC]; CCL-56).

Techniques: Imaging, Expressing

Journal: The Journal of Cell Biology

Article Title: Stress-induced phosphorylation of CLIP-170 by JNK promotes microtubule rescue

doi: 10.1083/jcb.201909093

Figure Lengend Snippet: GFP T186A -H1 expression levels used to record MT dynamic parameters (related to ) . (A) Western blots of CLIP-170 were performed to compare the expression levels of the endogenous CLIP-170 with that of the GFP T186A -H1 transgenes used in and in panels B and C. Due to the low detection of endogenous CLIP-170 in PtK2 and the absence of endogenous CLIP in Clip1/Clip2 KO MEF cells, we could not estimate precisely the level of expression of H1 in these lines. Instead, we show normalized ratios to β-actin. n indicates the number of independent experiments. (B) Sample images of the MT network and comets highlighted by GFP T186A -H1 in living PtK2, HeLa, and MEF cells (insets zoom on comets). Scale bars correspond to 5 µm. (C) Sample event showing a GFP T186A -H1 remnant (cyan arrowheads) left behind a comet before an MT rescue occurs at the same site (magenta arrowheads) in HeLa cells. The recorded sequence is shown both in the form of relevant frames and in a kymograph. The length scale bar is indicated both for the images and for the kymograph.

Article Snippet: Initial PtK2 cells are from (American Type Culture Collection [ATCC]; CCL-56).

Techniques: Expressing, Western Blot, Sequencing

Journal: The Journal of Cell Biology

Article Title: Stress-induced phosphorylation of CLIP-170 by JNK promotes microtubule rescue

doi: 10.1083/jcb.201909093

Figure Lengend Snippet: Full-length CLIP-170 phosphomimetics frequently form short-lived remnants, highlighting potential future rescue sites. Parameters of comets and remnants were measured from time-lapse imaging of PtK2 cells stably expressing GFP–α-tubulin and transiently expressing WT, nonphosphorylatable (A), or phosphomimetic (E) mCherry–CLIP-170 transgenes. (A) Sample image sequences showing the progression of mCherry–CLIP-170 comets (perpendicular dashed lines at the front tips) in cells treated or not with 1.2 µM anisomycin or 20 µM SP600125 (30 min to 1 h). CLIP-170 remnants are left behind comets (white arrows). (B) From the same experiments as in A, the values of comet and remnant parameters are reported in box plots showing representative percentiles and outliers. The histogram on the right shows the ratios (means normalized to the WT values ± SD) between the frequencies of MT regrowth and those of remnant occurrence. The mean values ± SD of each parameter are shown, but also the numbers of cells, comets, and remnants are detailed in . The statistical comparisons were performed using one-factor ANOVA followed by Fisher's protected t tests for pairwise comparisons . (C) Sample event showing an mCherry–CLIP-170 remnant (cyan arrowheads) left behind a comet before an MT rescue occurred at the same site (magenta arrowheads). The recorded sequence is shown in both the form of relevant frames and in a kymograph. n indicates the number of independent experiments. ***, P < 0.001; ****, P < 0.0001. Scale bars are indicated for images and kymographs.

Article Snippet: Initial PtK2 cells are from (American Type Culture Collection [ATCC]; CCL-56).

Techniques: Imaging, Stable Transfection, Expressing, Sequencing