nontargeting sgrna  (Addgene inc)

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: HEK TER Cas9 cells were transfected with sgRNA targeting PTPN14, LATS1 and LATS2, or nontargeting control (Synthego) ( ).

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

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: HEK TER Cas9 cells were transfected with sgRNA targeting PTPN14, LATS1 and LATS2, or nontargeting control (Synthego) ( ).

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

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: HEK TER Cas9 cells were transfected with sgRNA targeting PTPN14, LATS1 and LATS2, or nontargeting control (Synthego) ( ).

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

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: HEK TER Cas9 cells were transfected with sgRNA targeting PTPN14, LATS1 and LATS2, or nontargeting control (Synthego) ( ).

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

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: HEK TER Cas9 cells were transfected with sgRNA targeting PTPN14, LATS1 and LATS2, or nontargeting control (Synthego) ( ).

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

Journal: Cancer Discovery

Article Title: A Functional Survey of the Regulatory Landscape of Estrogen Receptor–Positive Breast Cancer Evolution

doi: 10.1158/2159-8290.CD-23-1157

Figure Lengend Snippet: Targeted CRE perturbations facilitate or disturb the adaptive processes. A, Overview of the experimental design. A, Cell growth dynamics of MCF7 cells under estrogen deprivation (−E2) were monitored by tracking the total number of GFP-positive nuclei with continuous live imaging over the course of 21 days. Cells carrying sgRNA for MYD88 , TLR5 , and UNC93B1 have a significant higher chance of avoiding therapy induced dormancy B and C, Retrospective patient stratification based on RNA expression ( B ) or CNVs ( C ) for MYD88 and TLR5 . Log-rank P values calculated with a Mantel–Cox test. D, Cell growth dynamics for a panel of estrogen dependent (MCF7, T47D, CAMA1, and EFM-19) and estrogen independent (MDA-MB231 and MCF7 Y537S) breast cancer cell lines under estrogen deprivation (−E2) were monitored with continuous live imaging over the course of 60 days in presence of a low dose of MYD88 inhibitor (MyD88-IN-1). Chemical MYD88 perturbation increased the number of dormant persister and in turn the chances of early awakening. The same concentration did not have any significant effect in +E2 condition. E, Same as A but targeting the USP8 gene promoter. Cell growth dynamics of MCF7 cells under estrogen deprivation (−E2) were monitored by tracking the total number of GFP-positive nuclei with continuous live imaging over the course of 21 days. Cells carrying sgRNA for USP8 have a lower chance of adapting to therapy. F, CRISPR-Cas9 knockout of USP8 . FACS sorting was used to quantify green ( USP8 sgRNAs carrying cells) and red (nontargeting sgRNAs). FACS analyses were carried out at three specific timepoints. G, Cell growth dynamics for a panel of estrogen dependent (MCF7, T47D, CAMA1, and EFM-19) and estrogen independent (MDA-MB231 and MCF7 Y537S) breast cancer cell lines under estrogen deprivation (−E2) were monitored with continuous live imaging over the course of 60 days in presence of low dose of USP8 inhibitor (DUB-IN-2). Area under the curve during the entire length of experiment was compared with the average of the controls to quantify the overall impact of USP8 inhibition. Chemical inhibition of USP8 significantly impact the survival of cells adapting to long term −E2 conditions. *, P < 0.01; **, P < 0.001; ***, P <10 −5 (Mann–Whitney test).

Article Snippet: RNA was extracted from dcas9-KRAB-MCF7 cells transduced with targeting and nontargeting sgRNA (Qiagen, cat no. 74016).

Techniques: Imaging, Clinical Proteomics, RNA Expression, Concentration Assay, CRISPR, Knock-Out, Inhibition, MANN-WHITNEY