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    Addgene inc type ptpn14 coding sequence
    Phosphorylation on YAP1 S127 is reduced in <t>PTPN14</t> knockout keratinocytes. N/Tert-1 keratinocytes were transduced with LentiCRISPRv2 vectors encoding spCas9 and a sgRNA sequence targeting PTPN14 or a nontargeting control sequence. sgRNA sequences were chosen from the Broad Institute Brunello Library. Four cell lines were generated using two different non-targeting control (sgNT-1 and sgNT-2) and two different PTPN14 (sgPTPN14-1 and sgPTPN14-3) sgRNAs. Cells were treated with cytochalasin D for up to 90 minutes and whole-cell lysates were harvested at 30-minute intervals. ( A ) Whole-cell protein lysates were separated by SDS-PAGE and proteins were detected by immunoblotting for YAP1, YAP1 pS127, PTPN14, and actin. ( B ) Western blot band intensity for three independent experiments was measured using ImageJ. The graph displays the ratio of YAP1 pS127/total YAP1 band intensity from three biological replicate experiments, plotted as mean ± standard deviation. Significance was determined by a two-way ANOVA with Holm-Šidak’s multiple comparisons test (* P < 0.05).
    Type Ptpn14 Coding Sequence, supplied by Addgene inc, used in various techniques. Bioz Stars score: 91/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/38944+coding+for+ptpn14/PTPN14+(Plasmid+%2338944)/pmc11481495-189-11-25
    Average 91 stars, based on 3 article reviews
    type ptpn14 coding sequence - by Bioz Stars, 2026-09
    91/100 stars

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    1) Product Images from "HPV18 E7 inhibits LATS1 kinase and activates YAP1 by degrading PTPN14"

    Article Title: HPV18 E7 inhibits LATS1 kinase and activates YAP1 by degrading PTPN14

    Journal: mBio

    doi: 10.1128/mbio.01811-24

    Phosphorylation on YAP1 S127 is reduced in PTPN14 knockout keratinocytes. N/Tert-1 keratinocytes were transduced with LentiCRISPRv2 vectors encoding spCas9 and a sgRNA sequence targeting PTPN14 or a nontargeting control sequence. sgRNA sequences were chosen from the Broad Institute Brunello Library. Four cell lines were generated using two different non-targeting control (sgNT-1 and sgNT-2) and two different PTPN14 (sgPTPN14-1 and sgPTPN14-3) sgRNAs. Cells were treated with cytochalasin D for up to 90 minutes and whole-cell lysates were harvested at 30-minute intervals. ( A ) Whole-cell protein lysates were separated by SDS-PAGE and proteins were detected by immunoblotting for YAP1, YAP1 pS127, PTPN14, and actin. ( B ) Western blot band intensity for three independent experiments was measured using ImageJ. The graph displays the ratio of YAP1 pS127/total YAP1 band intensity from three biological replicate experiments, plotted as mean ± standard deviation. Significance was determined by a two-way ANOVA with Holm-Šidak’s multiple comparisons test (* P < 0.05).
    Figure Legend Snippet: Phosphorylation on YAP1 S127 is reduced in PTPN14 knockout keratinocytes. N/Tert-1 keratinocytes were transduced with LentiCRISPRv2 vectors encoding spCas9 and a sgRNA sequence targeting PTPN14 or a nontargeting control sequence. sgRNA sequences were chosen from the Broad Institute Brunello Library. Four cell lines were generated using two different non-targeting control (sgNT-1 and sgNT-2) and two different PTPN14 (sgPTPN14-1 and sgPTPN14-3) sgRNAs. Cells were treated with cytochalasin D for up to 90 minutes and whole-cell lysates were harvested at 30-minute intervals. ( A ) Whole-cell protein lysates were separated by SDS-PAGE and proteins were detected by immunoblotting for YAP1, YAP1 pS127, PTPN14, and actin. ( B ) Western blot band intensity for three independent experiments was measured using ImageJ. The graph displays the ratio of YAP1 pS127/total YAP1 band intensity from three biological replicate experiments, plotted as mean ± standard deviation. Significance was determined by a two-way ANOVA with Holm-Šidak’s multiple comparisons test (* P < 0.05).

    Techniques Used: Phospho-proteomics, Knock-Out, Transduction, Sequencing, Control, Generated, SDS Page, Western Blot, Standard Deviation

    Phosphorylation on LATS1 T1079 is reduced in PTPN14 knockout keratinocytes. Control and PTPN14 knockout N/Tert-1 cells generated using LentiCRISPRv2 vectors were trypsinized and kept in suspension for 10 minutes. Whole-cell protein lysates were separated by SDS-PAGE and proteins were detected by immunoblotting for YAP1, YAP1 pS127, LATS1, LATS1 pT1079, NF2, NF2 pS518, PTPN14, and actin. Panels ( A and B ) show replicate experiments using the same four distinct cell lines as in . ( C ) Western blot band intensity for three independent experiments was measured using ImageJ. The graph displays the ratio of YAP1 pS127/total YAP1, LATS1 pT1079/total LATS1, or NF2 pS518/total NF2 band intensity from three biological replicate experiments, plotted as mean ± standard deviation. Significance was determined by a two-way ANOVA with Holm-Šidak’s multiple comparisons test (* P < 0.05; ** P < 0.01; *** P < 0.001).
    Figure Legend Snippet: Phosphorylation on LATS1 T1079 is reduced in PTPN14 knockout keratinocytes. Control and PTPN14 knockout N/Tert-1 cells generated using LentiCRISPRv2 vectors were trypsinized and kept in suspension for 10 minutes. Whole-cell protein lysates were separated by SDS-PAGE and proteins were detected by immunoblotting for YAP1, YAP1 pS127, LATS1, LATS1 pT1079, NF2, NF2 pS518, PTPN14, and actin. Panels ( A and B ) show replicate experiments using the same four distinct cell lines as in . ( C ) Western blot band intensity for three independent experiments was measured using ImageJ. The graph displays the ratio of YAP1 pS127/total YAP1, LATS1 pT1079/total LATS1, or NF2 pS518/total NF2 band intensity from three biological replicate experiments, plotted as mean ± standard deviation. Significance was determined by a two-way ANOVA with Holm-Šidak’s multiple comparisons test (* P < 0.05; ** P < 0.01; *** P < 0.001).

    Techniques Used: Phospho-proteomics, Knock-Out, Control, Generated, Suspension, SDS Page, Western Blot, Standard Deviation

    HPV18 E7-mediated PTPN14 degradation reduces phosphorylation on YAP1 S127 and LATS1 T1079. N/Tert-1 keratinocytes that stably express HA-tagged HPV18 E7 or HA-tagged HPV18 E7 R84S, which is unable to bind or degrade PTPN14, were used in assays of Hippo pathway activity. Cells transduced with empty vectors were included as a control. ( A ) Cells were treated with cytochalasin D for up to 90 minutes and whole-cell lysates were harvested at 30-minute intervals. Whole-cell protein lysates were separated by SDS-PAGE and proteins were detected by immunoblotting for YAP1, YAP1 pS127, PTPN14, RB1, HA, and actin. ( B ) Western blot band intensity for three independent experiments was measured using ImageJ. The graph displays the ratio of YAP1 pS127/total YAP1 band intensity from three biological replicate cytochalasin D experiments, plotted as mean ± standard deviation. Significance was determined by a two-way ANOVA with Holm-Šidak’s multiple comparisons test (* P < 0.05). ( C ) Cells were trypsinized and kept in suspension for 10 minutes. Whole-cell protein lysates were separated by SDS-PAGE and proteins were detected by immunoblotting for YAP1, YAP1 pS127, LATS1, LATS1 pT1079, HA, PTPN14, and actin. ( D ) Western blot band intensity for three independent experiments was measured using ImageJ. The graph displays the ratio of YAP1 pS127/total YAP1 or LATS1 pT1079/total LATS1 band intensity from three biological replicate detachment experiments, plotted as mean ± standard deviation. Significance was determined by a two-way ANOVA with Holm-Šidak’s multiple comparisons test (* P < 0.05; ** P < 0.01).
    Figure Legend Snippet: HPV18 E7-mediated PTPN14 degradation reduces phosphorylation on YAP1 S127 and LATS1 T1079. N/Tert-1 keratinocytes that stably express HA-tagged HPV18 E7 or HA-tagged HPV18 E7 R84S, which is unable to bind or degrade PTPN14, were used in assays of Hippo pathway activity. Cells transduced with empty vectors were included as a control. ( A ) Cells were treated with cytochalasin D for up to 90 minutes and whole-cell lysates were harvested at 30-minute intervals. Whole-cell protein lysates were separated by SDS-PAGE and proteins were detected by immunoblotting for YAP1, YAP1 pS127, PTPN14, RB1, HA, and actin. ( B ) Western blot band intensity for three independent experiments was measured using ImageJ. The graph displays the ratio of YAP1 pS127/total YAP1 band intensity from three biological replicate cytochalasin D experiments, plotted as mean ± standard deviation. Significance was determined by a two-way ANOVA with Holm-Šidak’s multiple comparisons test (* P < 0.05). ( C ) Cells were trypsinized and kept in suspension for 10 minutes. Whole-cell protein lysates were separated by SDS-PAGE and proteins were detected by immunoblotting for YAP1, YAP1 pS127, LATS1, LATS1 pT1079, HA, PTPN14, and actin. ( D ) Western blot band intensity for three independent experiments was measured using ImageJ. The graph displays the ratio of YAP1 pS127/total YAP1 or LATS1 pT1079/total LATS1 band intensity from three biological replicate detachment experiments, plotted as mean ± standard deviation. Significance was determined by a two-way ANOVA with Holm-Šidak’s multiple comparisons test (* P < 0.05; ** P < 0.01).

    Techniques Used: Phospho-proteomics, Stable Transfection, Activity Assay, Transduction, Control, SDS Page, Western Blot, Standard Deviation, Suspension

    PTPN14 knockout impairs keratinocyte differentiation. ( A ) N/Tert-1 cells stably expressing Cas9 were transfected with sgRNA targeting PTPN14 (Synthego) or mock transfected and PTPN14 knockout was validated by immunoblot. ( B ) Mock transfected and PTPN14 knockout N/Tert-1 cells were cultured in media containing 1.5 mM calcium and harvested for RNA analysis at indicated times. KRT10 and IVL RNA levels were analyzed by qRT-PCR and normalized to G6PD. Graphs show values for technical duplicate experiments. Error bars display mean ± range.
    Figure Legend Snippet: PTPN14 knockout impairs keratinocyte differentiation. ( A ) N/Tert-1 cells stably expressing Cas9 were transfected with sgRNA targeting PTPN14 (Synthego) or mock transfected and PTPN14 knockout was validated by immunoblot. ( B ) Mock transfected and PTPN14 knockout N/Tert-1 cells were cultured in media containing 1.5 mM calcium and harvested for RNA analysis at indicated times. KRT10 and IVL RNA levels were analyzed by qRT-PCR and normalized to G6PD. Graphs show values for technical duplicate experiments. Error bars display mean ± range.

    Techniques Used: Knock-Out, Stable Transfection, Expressing, Transfection, Western Blot, Cell Culture, Quantitative RT-PCR

    PTPN14 promotes keratinocyte differentiation and Hippo pathway activity. N/Tert-1 keratinocytes that express endogenous PTPN14 were transduced with a lentiviral vector encoding doxycycline-inducible PTPN14 (pLIX-PTPN14). Cells were treated with 1 µg/mL doxycycline for 24 hours or left untreated. ( A ) KRT10 and IVL RNA levels were measured by qRT-PCR and normalized to GAPDH. Graphs show data points for two technical replicate experiments. Error bars display mean ± range. ( B ) Whole-cell protein lysates were separated by SDS-PAGE and proteins analyzed by immunoblotting for YAP1, YAP1 pS127, LATS1, LATS1 pT1079, NF2, NF2 pS518, PTPN14, and actin. Bands for YAP1, YAP1 pS127, LATS1, LATS1 pT1079, NF2, and NF2 pS518 were quantified by densitometry. Values reflect the ratio of phosphoprotein/total protein band density.
    Figure Legend Snippet: PTPN14 promotes keratinocyte differentiation and Hippo pathway activity. N/Tert-1 keratinocytes that express endogenous PTPN14 were transduced with a lentiviral vector encoding doxycycline-inducible PTPN14 (pLIX-PTPN14). Cells were treated with 1 µg/mL doxycycline for 24 hours or left untreated. ( A ) KRT10 and IVL RNA levels were measured by qRT-PCR and normalized to GAPDH. Graphs show data points for two technical replicate experiments. Error bars display mean ± range. ( B ) Whole-cell protein lysates were separated by SDS-PAGE and proteins analyzed by immunoblotting for YAP1, YAP1 pS127, LATS1, LATS1 pT1079, NF2, NF2 pS518, PTPN14, and actin. Bands for YAP1, YAP1 pS127, LATS1, LATS1 pT1079, NF2, and NF2 pS518 were quantified by densitometry. Values reflect the ratio of phosphoprotein/total protein band density.

    Techniques Used: Activity Assay, Transduction, Plasmid Preparation, Quantitative RT-PCR, SDS Page, Western Blot

    PTPN14 PY1/2 motifs are required to induce differentiation. ( A ) Schematic of PTPN14 domains and location of PPxY motifs. ( B ) HFK were transfected with lentiviral constructs encoding GFP, WT PTPN14, or PTPN14 mutants as indicated. Total cellular RNA was collected and KRT1 transcript levels were analyzed by qRT-PCR and normalized to GAPDH. Graph shows individual data points for three independent experiments. Data are plotted as mean ± standard deviation. Significance was determined by ANOVA with Holm-Šidak’s multiple comparisons test (ns, not significant; **** P < 0.0001). ( C ) N/Tert-1 immortalized keratinocytes transduced with pLIX-PTPN14 were treated with 1 µg/mL doxycycline for 48 hours to induce HA-tagged PTPN14 expression. Whole-cell protein lysates were collected and PTPN14 immunoprecipitated with anti-HA agarose beads. Protein lysates from input and elution fractions were separated by SDS-PAGE and proteins were analyzed by immunoblotting for WWC1, PTPN14, and actin. Bands for WWC1 and PTPN14 in the IP fraction were quantified by densitometry. Values reflect the ratio of WWC1/PTPN14 band intensity.
    Figure Legend Snippet: PTPN14 PY1/2 motifs are required to induce differentiation. ( A ) Schematic of PTPN14 domains and location of PPxY motifs. ( B ) HFK were transfected with lentiviral constructs encoding GFP, WT PTPN14, or PTPN14 mutants as indicated. Total cellular RNA was collected and KRT1 transcript levels were analyzed by qRT-PCR and normalized to GAPDH. Graph shows individual data points for three independent experiments. Data are plotted as mean ± standard deviation. Significance was determined by ANOVA with Holm-Šidak’s multiple comparisons test (ns, not significant; **** P < 0.0001). ( C ) N/Tert-1 immortalized keratinocytes transduced with pLIX-PTPN14 were treated with 1 µg/mL doxycycline for 48 hours to induce HA-tagged PTPN14 expression. Whole-cell protein lysates were collected and PTPN14 immunoprecipitated with anti-HA agarose beads. Protein lysates from input and elution fractions were separated by SDS-PAGE and proteins were analyzed by immunoblotting for WWC1, PTPN14, and actin. Bands for WWC1 and PTPN14 in the IP fraction were quantified by densitometry. Values reflect the ratio of WWC1/PTPN14 band intensity.

    Techniques Used: Transfection, Construct, Quantitative RT-PCR, Standard Deviation, Transduction, Expressing, Immunoprecipitation, SDS Page, Western Blot

    LATS kinases and NF2 are required for PTPN14 to induce KRT10 expression. HFK were transfected with siRNA and then transduced with lentiviruses encoding GFP or PTPN14 at 24 hours post-transfection. Total cellular RNA was collected 72 hours post-knockdown and 48 hours post-transduction. RNA transcripts for KRT10 were measured by qRT-PCR and normalized to GAPDH. Six individual experiments were conducted, each in technical duplicate. Each experiment included a siControl condition, a YAP1 and TAZ siRNA-treated condition, and siRNAs targeting additional component(s) of the Hippo pathway. Each component of the Hippo pathway was targeted with two different siRNAs per gene, denoted as A and B. Panels display data from knockdowns as follows: ( A ) LATS1 and LATS2, ( B ) MST1 and MST2, ( C ) NF2, or ( D ) WWC1, WWC2, and WWC3. Data are graphed as mean ± standard deviation of combined replicate data. PTPN14/GFP denotes the ratio of KRT10 level in PTPN14 transduced cells vs GFP transduced cells in each siRNA-treated condition. Since Experiment 3 included several siRNAs (Control, YAP1/TAZ, MST1/2, NF2, WWC1/2/3), the same siControl and siYAP1/TAZ data from experiment 3 is included in panels B, C, and D.
    Figure Legend Snippet: LATS kinases and NF2 are required for PTPN14 to induce KRT10 expression. HFK were transfected with siRNA and then transduced with lentiviruses encoding GFP or PTPN14 at 24 hours post-transfection. Total cellular RNA was collected 72 hours post-knockdown and 48 hours post-transduction. RNA transcripts for KRT10 were measured by qRT-PCR and normalized to GAPDH. Six individual experiments were conducted, each in technical duplicate. Each experiment included a siControl condition, a YAP1 and TAZ siRNA-treated condition, and siRNAs targeting additional component(s) of the Hippo pathway. Each component of the Hippo pathway was targeted with two different siRNAs per gene, denoted as A and B. Panels display data from knockdowns as follows: ( A ) LATS1 and LATS2, ( B ) MST1 and MST2, ( C ) NF2, or ( D ) WWC1, WWC2, and WWC3. Data are graphed as mean ± standard deviation of combined replicate data. PTPN14/GFP denotes the ratio of KRT10 level in PTPN14 transduced cells vs GFP transduced cells in each siRNA-treated condition. Since Experiment 3 included several siRNAs (Control, YAP1/TAZ, MST1/2, NF2, WWC1/2/3), the same siControl and siYAP1/TAZ data from experiment 3 is included in panels B, C, and D.

    Techniques Used: Expressing, Transfection, Transduction, Knockdown, Quantitative RT-PCR, Standard Deviation, Control

    PTPN14 knockout promotes anchorage-independent growth and reduces YAP1 phosphorylation in HEK TER cells. ( A ) HEK TER cells expressing spCas9 were transfected with sgRNA targeting PTPN14 or LATS1 and LATS2. HEK TER cells expressing SV40 ST were used as a positive control for colony formation. Cells were plated in soft agar in technical triplicate and incubated at 37°C for 18 days, then photographed. Colonies were counted and quantified using ImageJ software. Graphs show individual data points for each plate and indicate mean ± standard deviation. Statistical significance of the nontargeting control condition compared to experimental conditions was determined by ANOVA with Dunnett’s multiple comparisons test (* P < 0.05; *** P < 0.001). ( B ) Cells were treated with cytochalasin D for up to 90 minutes and whole-cell lysates were harvested at 30-minute intervals. Whole-cell protein lysates were separated by SDS-PAGE and proteins were detected by immunoblotting for YAP1, YAP1 pS127, PTPN14, LATS1, and actin. ( C ) Band intensity for blots of two independent experiments was measured by ImageJ. The graph displays the ratio of YAP1 pS127/total YAP1 band intensity from two biological replicate cytochalasin D experiments as mean ± range. The blots shown in panel B are from experiment 1.
    Figure Legend Snippet: PTPN14 knockout promotes anchorage-independent growth and reduces YAP1 phosphorylation in HEK TER cells. ( A ) HEK TER cells expressing spCas9 were transfected with sgRNA targeting PTPN14 or LATS1 and LATS2. HEK TER cells expressing SV40 ST were used as a positive control for colony formation. Cells were plated in soft agar in technical triplicate and incubated at 37°C for 18 days, then photographed. Colonies were counted and quantified using ImageJ software. Graphs show individual data points for each plate and indicate mean ± standard deviation. Statistical significance of the nontargeting control condition compared to experimental conditions was determined by ANOVA with Dunnett’s multiple comparisons test (* P < 0.05; *** P < 0.001). ( B ) Cells were treated with cytochalasin D for up to 90 minutes and whole-cell lysates were harvested at 30-minute intervals. Whole-cell protein lysates were separated by SDS-PAGE and proteins were detected by immunoblotting for YAP1, YAP1 pS127, PTPN14, LATS1, and actin. ( C ) Band intensity for blots of two independent experiments was measured by ImageJ. The graph displays the ratio of YAP1 pS127/total YAP1 band intensity from two biological replicate cytochalasin D experiments as mean ± range. The blots shown in panel B are from experiment 1.

    Techniques Used: Knock-Out, Phospho-proteomics, Expressing, Transfection, Positive Control, Incubation, Software, Standard Deviation, Control, SDS Page, Western Blot

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    Article Snippet: Plasmids 38891 coding for PTPRN2 (715- 1010) and 38944 coding for PTPN14 (886- 1187) were obtained from Addgene (Cambridge, MA).



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    Phosphorylation on YAP1 S127 is reduced in <t>PTPN14</t> knockout keratinocytes. N/Tert-1 keratinocytes were transduced with LentiCRISPRv2 vectors encoding spCas9 and a sgRNA sequence targeting PTPN14 or a nontargeting control sequence. sgRNA sequences were chosen from the Broad Institute Brunello Library. Four cell lines were generated using two different non-targeting control (sgNT-1 and sgNT-2) and two different PTPN14 (sgPTPN14-1 and sgPTPN14-3) sgRNAs. Cells were treated with cytochalasin D for up to 90 minutes and whole-cell lysates were harvested at 30-minute intervals. ( A ) Whole-cell protein lysates were separated by SDS-PAGE and proteins were detected by immunoblotting for YAP1, YAP1 pS127, PTPN14, and actin. ( B ) Western blot band intensity for three independent experiments was measured using ImageJ. The graph displays the ratio of YAP1 pS127/total YAP1 band intensity from three biological replicate experiments, plotted as mean ± standard deviation. Significance was determined by a two-way ANOVA with Holm-Šidak’s multiple comparisons test (* P < 0.05).
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    Phosphorylation on YAP1 S127 is reduced in PTPN14 knockout keratinocytes. N/Tert-1 keratinocytes were transduced with LentiCRISPRv2 vectors encoding spCas9 and a sgRNA sequence targeting PTPN14 or a nontargeting control sequence. sgRNA sequences were chosen from the Broad Institute Brunello Library. Four cell lines were generated using two different non-targeting control (sgNT-1 and sgNT-2) and two different PTPN14 (sgPTPN14-1 and sgPTPN14-3) sgRNAs. Cells were treated with cytochalasin D for up to 90 minutes and whole-cell lysates were harvested at 30-minute intervals. ( A ) Whole-cell protein lysates were separated by SDS-PAGE and proteins were detected by immunoblotting for YAP1, YAP1 pS127, PTPN14, and actin. ( B ) Western blot band intensity for three independent experiments was measured using ImageJ. The graph displays the ratio of YAP1 pS127/total YAP1 band intensity from three biological replicate experiments, plotted as mean ± standard deviation. Significance was determined by a two-way ANOVA with Holm-Šidak’s multiple comparisons test (* P < 0.05).

    Journal: mBio

    Article Title: HPV18 E7 inhibits LATS1 kinase and activates YAP1 by degrading PTPN14

    doi: 10.1128/mbio.01811-24

    Figure Lengend Snippet: Phosphorylation on YAP1 S127 is reduced in PTPN14 knockout keratinocytes. N/Tert-1 keratinocytes were transduced with LentiCRISPRv2 vectors encoding spCas9 and a sgRNA sequence targeting PTPN14 or a nontargeting control sequence. sgRNA sequences were chosen from the Broad Institute Brunello Library. Four cell lines were generated using two different non-targeting control (sgNT-1 and sgNT-2) and two different PTPN14 (sgPTPN14-1 and sgPTPN14-3) sgRNAs. Cells were treated with cytochalasin D for up to 90 minutes and whole-cell lysates were harvested at 30-minute intervals. ( A ) Whole-cell protein lysates were separated by SDS-PAGE and proteins were detected by immunoblotting for YAP1, YAP1 pS127, PTPN14, and actin. ( B ) Western blot band intensity for three independent experiments was measured using ImageJ. The graph displays the ratio of YAP1 pS127/total YAP1 band intensity from three biological replicate experiments, plotted as mean ± standard deviation. Significance was determined by a two-way ANOVA with Holm-Šidak’s multiple comparisons test (* P < 0.05).

    Article Snippet: A doxycycline-inducible, HA-tagged PTPN14 overexpression vector was developed by cloning the wild-type PTPN14 coding sequence into a lentiviral vector pLIX_402, a gift from David Root (Addgene #41394), using Gateway recombination.

    Techniques: Phospho-proteomics, Knock-Out, Transduction, Sequencing, Control, Generated, SDS Page, Western Blot, Standard Deviation

    Phosphorylation on LATS1 T1079 is reduced in PTPN14 knockout keratinocytes. Control and PTPN14 knockout N/Tert-1 cells generated using LentiCRISPRv2 vectors were trypsinized and kept in suspension for 10 minutes. Whole-cell protein lysates were separated by SDS-PAGE and proteins were detected by immunoblotting for YAP1, YAP1 pS127, LATS1, LATS1 pT1079, NF2, NF2 pS518, PTPN14, and actin. Panels ( A and B ) show replicate experiments using the same four distinct cell lines as in . ( C ) Western blot band intensity for three independent experiments was measured using ImageJ. The graph displays the ratio of YAP1 pS127/total YAP1, LATS1 pT1079/total LATS1, or NF2 pS518/total NF2 band intensity from three biological replicate experiments, plotted as mean ± standard deviation. Significance was determined by a two-way ANOVA with Holm-Šidak’s multiple comparisons test (* P < 0.05; ** P < 0.01; *** P < 0.001).

    Journal: mBio

    Article Title: HPV18 E7 inhibits LATS1 kinase and activates YAP1 by degrading PTPN14

    doi: 10.1128/mbio.01811-24

    Figure Lengend Snippet: Phosphorylation on LATS1 T1079 is reduced in PTPN14 knockout keratinocytes. Control and PTPN14 knockout N/Tert-1 cells generated using LentiCRISPRv2 vectors were trypsinized and kept in suspension for 10 minutes. Whole-cell protein lysates were separated by SDS-PAGE and proteins were detected by immunoblotting for YAP1, YAP1 pS127, LATS1, LATS1 pT1079, NF2, NF2 pS518, PTPN14, and actin. Panels ( A and B ) show replicate experiments using the same four distinct cell lines as in . ( C ) Western blot band intensity for three independent experiments was measured using ImageJ. The graph displays the ratio of YAP1 pS127/total YAP1, LATS1 pT1079/total LATS1, or NF2 pS518/total NF2 band intensity from three biological replicate experiments, plotted as mean ± standard deviation. Significance was determined by a two-way ANOVA with Holm-Šidak’s multiple comparisons test (* P < 0.05; ** P < 0.01; *** P < 0.001).

    Article Snippet: A doxycycline-inducible, HA-tagged PTPN14 overexpression vector was developed by cloning the wild-type PTPN14 coding sequence into a lentiviral vector pLIX_402, a gift from David Root (Addgene #41394), using Gateway recombination.

    Techniques: Phospho-proteomics, Knock-Out, Control, Generated, Suspension, SDS Page, Western Blot, Standard Deviation

    HPV18 E7-mediated PTPN14 degradation reduces phosphorylation on YAP1 S127 and LATS1 T1079. N/Tert-1 keratinocytes that stably express HA-tagged HPV18 E7 or HA-tagged HPV18 E7 R84S, which is unable to bind or degrade PTPN14, were used in assays of Hippo pathway activity. Cells transduced with empty vectors were included as a control. ( A ) Cells were treated with cytochalasin D for up to 90 minutes and whole-cell lysates were harvested at 30-minute intervals. Whole-cell protein lysates were separated by SDS-PAGE and proteins were detected by immunoblotting for YAP1, YAP1 pS127, PTPN14, RB1, HA, and actin. ( B ) Western blot band intensity for three independent experiments was measured using ImageJ. The graph displays the ratio of YAP1 pS127/total YAP1 band intensity from three biological replicate cytochalasin D experiments, plotted as mean ± standard deviation. Significance was determined by a two-way ANOVA with Holm-Šidak’s multiple comparisons test (* P < 0.05). ( C ) Cells were trypsinized and kept in suspension for 10 minutes. Whole-cell protein lysates were separated by SDS-PAGE and proteins were detected by immunoblotting for YAP1, YAP1 pS127, LATS1, LATS1 pT1079, HA, PTPN14, and actin. ( D ) Western blot band intensity for three independent experiments was measured using ImageJ. The graph displays the ratio of YAP1 pS127/total YAP1 or LATS1 pT1079/total LATS1 band intensity from three biological replicate detachment experiments, plotted as mean ± standard deviation. Significance was determined by a two-way ANOVA with Holm-Šidak’s multiple comparisons test (* P < 0.05; ** P < 0.01).

    Journal: mBio

    Article Title: HPV18 E7 inhibits LATS1 kinase and activates YAP1 by degrading PTPN14

    doi: 10.1128/mbio.01811-24

    Figure Lengend Snippet: HPV18 E7-mediated PTPN14 degradation reduces phosphorylation on YAP1 S127 and LATS1 T1079. N/Tert-1 keratinocytes that stably express HA-tagged HPV18 E7 or HA-tagged HPV18 E7 R84S, which is unable to bind or degrade PTPN14, were used in assays of Hippo pathway activity. Cells transduced with empty vectors were included as a control. ( A ) Cells were treated with cytochalasin D for up to 90 minutes and whole-cell lysates were harvested at 30-minute intervals. Whole-cell protein lysates were separated by SDS-PAGE and proteins were detected by immunoblotting for YAP1, YAP1 pS127, PTPN14, RB1, HA, and actin. ( B ) Western blot band intensity for three independent experiments was measured using ImageJ. The graph displays the ratio of YAP1 pS127/total YAP1 band intensity from three biological replicate cytochalasin D experiments, plotted as mean ± standard deviation. Significance was determined by a two-way ANOVA with Holm-Šidak’s multiple comparisons test (* P < 0.05). ( C ) Cells were trypsinized and kept in suspension for 10 minutes. Whole-cell protein lysates were separated by SDS-PAGE and proteins were detected by immunoblotting for YAP1, YAP1 pS127, LATS1, LATS1 pT1079, HA, PTPN14, and actin. ( D ) Western blot band intensity for three independent experiments was measured using ImageJ. The graph displays the ratio of YAP1 pS127/total YAP1 or LATS1 pT1079/total LATS1 band intensity from three biological replicate detachment experiments, plotted as mean ± standard deviation. Significance was determined by a two-way ANOVA with Holm-Šidak’s multiple comparisons test (* P < 0.05; ** P < 0.01).

    Article Snippet: A doxycycline-inducible, HA-tagged PTPN14 overexpression vector was developed by cloning the wild-type PTPN14 coding sequence into a lentiviral vector pLIX_402, a gift from David Root (Addgene #41394), using Gateway recombination.

    Techniques: Phospho-proteomics, Stable Transfection, Activity Assay, Transduction, Control, SDS Page, Western Blot, Standard Deviation, Suspension

    PTPN14 knockout impairs keratinocyte differentiation. ( A ) N/Tert-1 cells stably expressing Cas9 were transfected with sgRNA targeting PTPN14 (Synthego) or mock transfected and PTPN14 knockout was validated by immunoblot. ( B ) Mock transfected and PTPN14 knockout N/Tert-1 cells were cultured in media containing 1.5 mM calcium and harvested for RNA analysis at indicated times. KRT10 and IVL RNA levels were analyzed by qRT-PCR and normalized to G6PD. Graphs show values for technical duplicate experiments. Error bars display mean ± range.

    Journal: mBio

    Article Title: HPV18 E7 inhibits LATS1 kinase and activates YAP1 by degrading PTPN14

    doi: 10.1128/mbio.01811-24

    Figure Lengend Snippet: PTPN14 knockout impairs keratinocyte differentiation. ( A ) N/Tert-1 cells stably expressing Cas9 were transfected with sgRNA targeting PTPN14 (Synthego) or mock transfected and PTPN14 knockout was validated by immunoblot. ( B ) Mock transfected and PTPN14 knockout N/Tert-1 cells were cultured in media containing 1.5 mM calcium and harvested for RNA analysis at indicated times. KRT10 and IVL RNA levels were analyzed by qRT-PCR and normalized to G6PD. Graphs show values for technical duplicate experiments. Error bars display mean ± range.

    Article Snippet: A doxycycline-inducible, HA-tagged PTPN14 overexpression vector was developed by cloning the wild-type PTPN14 coding sequence into a lentiviral vector pLIX_402, a gift from David Root (Addgene #41394), using Gateway recombination.

    Techniques: Knock-Out, Stable Transfection, Expressing, Transfection, Western Blot, Cell Culture, Quantitative RT-PCR

    PTPN14 promotes keratinocyte differentiation and Hippo pathway activity. N/Tert-1 keratinocytes that express endogenous PTPN14 were transduced with a lentiviral vector encoding doxycycline-inducible PTPN14 (pLIX-PTPN14). Cells were treated with 1 µg/mL doxycycline for 24 hours or left untreated. ( A ) KRT10 and IVL RNA levels were measured by qRT-PCR and normalized to GAPDH. Graphs show data points for two technical replicate experiments. Error bars display mean ± range. ( B ) Whole-cell protein lysates were separated by SDS-PAGE and proteins analyzed by immunoblotting for YAP1, YAP1 pS127, LATS1, LATS1 pT1079, NF2, NF2 pS518, PTPN14, and actin. Bands for YAP1, YAP1 pS127, LATS1, LATS1 pT1079, NF2, and NF2 pS518 were quantified by densitometry. Values reflect the ratio of phosphoprotein/total protein band density.

    Journal: mBio

    Article Title: HPV18 E7 inhibits LATS1 kinase and activates YAP1 by degrading PTPN14

    doi: 10.1128/mbio.01811-24

    Figure Lengend Snippet: PTPN14 promotes keratinocyte differentiation and Hippo pathway activity. N/Tert-1 keratinocytes that express endogenous PTPN14 were transduced with a lentiviral vector encoding doxycycline-inducible PTPN14 (pLIX-PTPN14). Cells were treated with 1 µg/mL doxycycline for 24 hours or left untreated. ( A ) KRT10 and IVL RNA levels were measured by qRT-PCR and normalized to GAPDH. Graphs show data points for two technical replicate experiments. Error bars display mean ± range. ( B ) Whole-cell protein lysates were separated by SDS-PAGE and proteins analyzed by immunoblotting for YAP1, YAP1 pS127, LATS1, LATS1 pT1079, NF2, NF2 pS518, PTPN14, and actin. Bands for YAP1, YAP1 pS127, LATS1, LATS1 pT1079, NF2, and NF2 pS518 were quantified by densitometry. Values reflect the ratio of phosphoprotein/total protein band density.

    Article Snippet: A doxycycline-inducible, HA-tagged PTPN14 overexpression vector was developed by cloning the wild-type PTPN14 coding sequence into a lentiviral vector pLIX_402, a gift from David Root (Addgene #41394), using Gateway recombination.

    Techniques: Activity Assay, Transduction, Plasmid Preparation, Quantitative RT-PCR, SDS Page, Western Blot

    PTPN14 PY1/2 motifs are required to induce differentiation. ( A ) Schematic of PTPN14 domains and location of PPxY motifs. ( B ) HFK were transfected with lentiviral constructs encoding GFP, WT PTPN14, or PTPN14 mutants as indicated. Total cellular RNA was collected and KRT1 transcript levels were analyzed by qRT-PCR and normalized to GAPDH. Graph shows individual data points for three independent experiments. Data are plotted as mean ± standard deviation. Significance was determined by ANOVA with Holm-Šidak’s multiple comparisons test (ns, not significant; **** P < 0.0001). ( C ) N/Tert-1 immortalized keratinocytes transduced with pLIX-PTPN14 were treated with 1 µg/mL doxycycline for 48 hours to induce HA-tagged PTPN14 expression. Whole-cell protein lysates were collected and PTPN14 immunoprecipitated with anti-HA agarose beads. Protein lysates from input and elution fractions were separated by SDS-PAGE and proteins were analyzed by immunoblotting for WWC1, PTPN14, and actin. Bands for WWC1 and PTPN14 in the IP fraction were quantified by densitometry. Values reflect the ratio of WWC1/PTPN14 band intensity.

    Journal: mBio

    Article Title: HPV18 E7 inhibits LATS1 kinase and activates YAP1 by degrading PTPN14

    doi: 10.1128/mbio.01811-24

    Figure Lengend Snippet: PTPN14 PY1/2 motifs are required to induce differentiation. ( A ) Schematic of PTPN14 domains and location of PPxY motifs. ( B ) HFK were transfected with lentiviral constructs encoding GFP, WT PTPN14, or PTPN14 mutants as indicated. Total cellular RNA was collected and KRT1 transcript levels were analyzed by qRT-PCR and normalized to GAPDH. Graph shows individual data points for three independent experiments. Data are plotted as mean ± standard deviation. Significance was determined by ANOVA with Holm-Šidak’s multiple comparisons test (ns, not significant; **** P < 0.0001). ( C ) N/Tert-1 immortalized keratinocytes transduced with pLIX-PTPN14 were treated with 1 µg/mL doxycycline for 48 hours to induce HA-tagged PTPN14 expression. Whole-cell protein lysates were collected and PTPN14 immunoprecipitated with anti-HA agarose beads. Protein lysates from input and elution fractions were separated by SDS-PAGE and proteins were analyzed by immunoblotting for WWC1, PTPN14, and actin. Bands for WWC1 and PTPN14 in the IP fraction were quantified by densitometry. Values reflect the ratio of WWC1/PTPN14 band intensity.

    Article Snippet: A doxycycline-inducible, HA-tagged PTPN14 overexpression vector was developed by cloning the wild-type PTPN14 coding sequence into a lentiviral vector pLIX_402, a gift from David Root (Addgene #41394), using Gateway recombination.

    Techniques: Transfection, Construct, Quantitative RT-PCR, Standard Deviation, Transduction, Expressing, Immunoprecipitation, SDS Page, Western Blot

    LATS kinases and NF2 are required for PTPN14 to induce KRT10 expression. HFK were transfected with siRNA and then transduced with lentiviruses encoding GFP or PTPN14 at 24 hours post-transfection. Total cellular RNA was collected 72 hours post-knockdown and 48 hours post-transduction. RNA transcripts for KRT10 were measured by qRT-PCR and normalized to GAPDH. Six individual experiments were conducted, each in technical duplicate. Each experiment included a siControl condition, a YAP1 and TAZ siRNA-treated condition, and siRNAs targeting additional component(s) of the Hippo pathway. Each component of the Hippo pathway was targeted with two different siRNAs per gene, denoted as A and B. Panels display data from knockdowns as follows: ( A ) LATS1 and LATS2, ( B ) MST1 and MST2, ( C ) NF2, or ( D ) WWC1, WWC2, and WWC3. Data are graphed as mean ± standard deviation of combined replicate data. PTPN14/GFP denotes the ratio of KRT10 level in PTPN14 transduced cells vs GFP transduced cells in each siRNA-treated condition. Since Experiment 3 included several siRNAs (Control, YAP1/TAZ, MST1/2, NF2, WWC1/2/3), the same siControl and siYAP1/TAZ data from experiment 3 is included in panels B, C, and D.

    Journal: mBio

    Article Title: HPV18 E7 inhibits LATS1 kinase and activates YAP1 by degrading PTPN14

    doi: 10.1128/mbio.01811-24

    Figure Lengend Snippet: LATS kinases and NF2 are required for PTPN14 to induce KRT10 expression. HFK were transfected with siRNA and then transduced with lentiviruses encoding GFP or PTPN14 at 24 hours post-transfection. Total cellular RNA was collected 72 hours post-knockdown and 48 hours post-transduction. RNA transcripts for KRT10 were measured by qRT-PCR and normalized to GAPDH. Six individual experiments were conducted, each in technical duplicate. Each experiment included a siControl condition, a YAP1 and TAZ siRNA-treated condition, and siRNAs targeting additional component(s) of the Hippo pathway. Each component of the Hippo pathway was targeted with two different siRNAs per gene, denoted as A and B. Panels display data from knockdowns as follows: ( A ) LATS1 and LATS2, ( B ) MST1 and MST2, ( C ) NF2, or ( D ) WWC1, WWC2, and WWC3. Data are graphed as mean ± standard deviation of combined replicate data. PTPN14/GFP denotes the ratio of KRT10 level in PTPN14 transduced cells vs GFP transduced cells in each siRNA-treated condition. Since Experiment 3 included several siRNAs (Control, YAP1/TAZ, MST1/2, NF2, WWC1/2/3), the same siControl and siYAP1/TAZ data from experiment 3 is included in panels B, C, and D.

    Article Snippet: A doxycycline-inducible, HA-tagged PTPN14 overexpression vector was developed by cloning the wild-type PTPN14 coding sequence into a lentiviral vector pLIX_402, a gift from David Root (Addgene #41394), using Gateway recombination.

    Techniques: Expressing, Transfection, Transduction, Knockdown, Quantitative RT-PCR, Standard Deviation, Control

    PTPN14 knockout promotes anchorage-independent growth and reduces YAP1 phosphorylation in HEK TER cells. ( A ) HEK TER cells expressing spCas9 were transfected with sgRNA targeting PTPN14 or LATS1 and LATS2. HEK TER cells expressing SV40 ST were used as a positive control for colony formation. Cells were plated in soft agar in technical triplicate and incubated at 37°C for 18 days, then photographed. Colonies were counted and quantified using ImageJ software. Graphs show individual data points for each plate and indicate mean ± standard deviation. Statistical significance of the nontargeting control condition compared to experimental conditions was determined by ANOVA with Dunnett’s multiple comparisons test (* P < 0.05; *** P < 0.001). ( B ) Cells were treated with cytochalasin D for up to 90 minutes and whole-cell lysates were harvested at 30-minute intervals. Whole-cell protein lysates were separated by SDS-PAGE and proteins were detected by immunoblotting for YAP1, YAP1 pS127, PTPN14, LATS1, and actin. ( C ) Band intensity for blots of two independent experiments was measured by ImageJ. The graph displays the ratio of YAP1 pS127/total YAP1 band intensity from two biological replicate cytochalasin D experiments as mean ± range. The blots shown in panel B are from experiment 1.

    Journal: mBio

    Article Title: HPV18 E7 inhibits LATS1 kinase and activates YAP1 by degrading PTPN14

    doi: 10.1128/mbio.01811-24

    Figure Lengend Snippet: PTPN14 knockout promotes anchorage-independent growth and reduces YAP1 phosphorylation in HEK TER cells. ( A ) HEK TER cells expressing spCas9 were transfected with sgRNA targeting PTPN14 or LATS1 and LATS2. HEK TER cells expressing SV40 ST were used as a positive control for colony formation. Cells were plated in soft agar in technical triplicate and incubated at 37°C for 18 days, then photographed. Colonies were counted and quantified using ImageJ software. Graphs show individual data points for each plate and indicate mean ± standard deviation. Statistical significance of the nontargeting control condition compared to experimental conditions was determined by ANOVA with Dunnett’s multiple comparisons test (* P < 0.05; *** P < 0.001). ( B ) Cells were treated with cytochalasin D for up to 90 minutes and whole-cell lysates were harvested at 30-minute intervals. Whole-cell protein lysates were separated by SDS-PAGE and proteins were detected by immunoblotting for YAP1, YAP1 pS127, PTPN14, LATS1, and actin. ( C ) Band intensity for blots of two independent experiments was measured by ImageJ. The graph displays the ratio of YAP1 pS127/total YAP1 band intensity from two biological replicate cytochalasin D experiments as mean ± range. The blots shown in panel B are from experiment 1.

    Article Snippet: A doxycycline-inducible, HA-tagged PTPN14 overexpression vector was developed by cloning the wild-type PTPN14 coding sequence into a lentiviral vector pLIX_402, a gift from David Root (Addgene #41394), using Gateway recombination.

    Techniques: Knock-Out, Phospho-proteomics, Expressing, Transfection, Positive Control, Incubation, Software, Standard Deviation, Control, SDS Page, Western Blot