nontargeting control sgrnas  (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: bioRxiv

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

doi: 10.1101/2024.03.07.583953

Figure Lengend Snippet: N/Tert-1 keratinocytes were transduced with LentiCRISPRv2 vectors encoding spCas9 and an 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, sgNT-2) and two different PTPN14 (sgPTPN14-1, sgPTPN14-3) sgRNAs. 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, and actin.

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

Techniques: Transduction, Sequencing, Generated, SDS Page, Western Blot

Journal: bioRxiv

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

doi: 10.1101/2024.03.07.583953

Figure Lengend Snippet: HFK transfected with siRNA for 72 hours were also transduced for 48 hours with lentiviral vectors for GFP or PTPN14 overexpression. Cells were transfected with siRNA: nontargeting control, YAP1, TAZ, and two separate pairs for LATS1 and LATS2 denoted (A) and (B). Total cellular RNA was measured by qRT-PCR for KRT1 and IVL for two biological replicate experiments, each performed in technical duplicate. Transcript levels were normalized to GAPDH. Graphs show mean ± standard deviation. Samples are the same as those analyzed in .

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

Techniques: Transfection, Over Expression, Quantitative RT-PCR, Standard Deviation

Journal: bioRxiv

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

doi: 10.1101/2024.03.07.583953

Figure Lengend Snippet: (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.

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

Techniques: Expressing, Transfection, Positive Control, Incubation, Software, Standard Deviation, SDS Page, Western Blot

Journal: bioRxiv

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

doi: 10.1101/2024.03.07.583953

Figure Lengend Snippet: (A) HEK TER cells expressing Cas9 were transfected with a second set of sgRNA compared to those used in . HEK TER cells expressing SV40 ST were used as a positive control for colony formation. Cells were plated in soft agar in technical triplicates 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 (ns, not significant; ****, P < 0.0001). (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.

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

Techniques: Expressing, Transfection, Positive Control, Incubation, Software, Standard Deviation, SDS Page, Western Blot

Journal: Cell Reports Medicine

Article Title: UCHL1 is a potential molecular indicator and therapeutic target for neuroendocrine carcinomas

doi: 10.1016/j.xcrm.2023.101381

Figure Lengend Snippet: UCHL1 regulates cancer cell growth in vitro and in vivo (A) UCHL1 and SYP levels in 22Rv1-RFP, 22Rv1-UCHL1-OV (WT UCHL1), and 22Rv1-UCHL1(C90S) cells were determined by western blot (WB) (left). SOX2, CD56, SYP, and UCHL1 levels in UCHL1 knockout pool cells were assessed by WB (right). (B and C) Colony formation assays of 22Rv1 with or without WT UCHL1 or UCHL1(C90S) overexpression (B) and colony formation assays of TD-NEPC parental (no transfection) cells, CTL (transfection with control non-targeting sgRNA), and UCHL1 knockout (transfection with multi-sgRNA targeting UCHL1) single-cell selection clones (C). Scale bar, 1 cm. The percentage of colony area per well was quantified using ImageJ. All experiments were performed in triplicate. Error bars, SD. (D) Subcutaneous tumor growth (left) and tumor weight (right) of 22Rv1-RFP (n = 10) and 22Rv1-UCHL1-OV (n = 10). Error bars represent standard error of the mean (SEM). (E) IHC staining of UCHL1, androgen receptor (AR) and SYP, CgA, and CD56 in 22Rv1 xenografts. Scale bar, 10 μm. (F) Subcutaneous tumor growth of TD-NEPC parental (no transduction), CTL 1 and 2, and UCHL1 knockout 1, 2, and 3 single-cell selection clone xenografts. Error bars represent SEM. (G) Harvested tumors (left) and tumor weights (right) at the endpoint (scale bar, 1 cm). (H) IHC staining for UCHL1, SYP, CgA, and CD56 in TD-NEPC parental, CTL, and UCHL1 knockout (KO) xenografts. Scale bars, 10 μm. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.005, ∗∗∗∗p < 0.001, determined by Student’s t test.

Article Snippet: Control nontargeting sgRNA , Synthego , sku: 063-1010-000-000.

Techniques: In Vitro, In Vivo, Western Blot, Knock-Out, Over Expression, Transfection, Control, Selection, Clone Assay, Immunohistochemistry, Transduction

Journal: Cell Reports Medicine

Article Title: UCHL1 is a potential molecular indicator and therapeutic target for neuroendocrine carcinomas

doi: 10.1016/j.xcrm.2023.101381

Figure Lengend Snippet:

Article Snippet: Control nontargeting sgRNA , Synthego , sku: 063-1010-000-000.

Techniques: Virus, Plasmid Preparation, Clinical Proteomics, Recombinant, Viability Assay, In Situ, Proximity Ligation Assay, Enzyme-linked Immunosorbent Assay, cDNA Synthesis, SYBR Green Assay, shRNA, Control, Ubiquitin Proteomics, Software

Journal: Cell Reports Medicine

Article Title: UCHL1 is a potential molecular indicator and therapeutic target for neuroendocrine carcinomas

doi: 10.1016/j.xcrm.2023.101381

Figure Lengend Snippet: UCHL1 regulates cancer cell growth in vitro and in vivo (A) UCHL1 and SYP levels in 22Rv1-RFP, 22Rv1-UCHL1-OV (WT UCHL1), and 22Rv1-UCHL1(C90S) cells were determined by western blot (WB) (left). SOX2, CD56, SYP, and UCHL1 levels in UCHL1 knockout pool cells were assessed by WB (right). (B and C) Colony formation assays of 22Rv1 with or without WT UCHL1 or UCHL1(C90S) overexpression (B) and colony formation assays of TD-NEPC parental (no transfection) cells, CTL (transfection with control non-targeting sgRNA), and UCHL1 knockout (transfection with multi-sgRNA targeting UCHL1) single-cell selection clones (C). Scale bar, 1 cm. The percentage of colony area per well was quantified using ImageJ. All experiments were performed in triplicate. Error bars, SD. (D) Subcutaneous tumor growth (left) and tumor weight (right) of 22Rv1-RFP (n = 10) and 22Rv1-UCHL1-OV (n = 10). Error bars represent standard error of the mean (SEM). (E) IHC staining of UCHL1, androgen receptor (AR) and SYP, CgA, and CD56 in 22Rv1 xenografts. Scale bar, 10 μm. (F) Subcutaneous tumor growth of TD-NEPC parental (no transduction), CTL 1 and 2, and UCHL1 knockout 1, 2, and 3 single-cell selection clone xenografts. Error bars represent SEM. (G) Harvested tumors (left) and tumor weights (right) at the endpoint (scale bar, 1 cm). (H) IHC staining for UCHL1, SYP, CgA, and CD56 in TD-NEPC parental, CTL, and UCHL1 knockout (KO) xenografts. Scale bars, 10 μm. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.005, ∗∗∗∗p < 0.001, determined by Student’s t test.

Article Snippet: Control nontargeting sgRNA (sku: 063-1010-000-000) and multi-sgRNA targeting UCHL1 (sku: GKO-HS1-000-0-1.5n-0-0) were purchased from Synthego (Synthego Corporation Biotechnology, Menlo Park, California, United States).

Techniques: In Vitro, In Vivo, Western Blot, Knock-Out, Over Expression, Transfection, Control, Selection, Clone Assay, Immunohistochemistry, Transduction

Journal: Cell Reports Medicine

Article Title: UCHL1 is a potential molecular indicator and therapeutic target for neuroendocrine carcinomas

doi: 10.1016/j.xcrm.2023.101381

Figure Lengend Snippet:

Article Snippet: Control nontargeting sgRNA (sku: 063-1010-000-000) and multi-sgRNA targeting UCHL1 (sku: GKO-HS1-000-0-1.5n-0-0) were purchased from Synthego (Synthego Corporation Biotechnology, Menlo Park, California, United States).

Techniques: Virus, Plasmid Preparation, Clinical Proteomics, Recombinant, Viability Assay, In Situ, Proximity Ligation Assay, Enzyme-linked Immunosorbent Assay, cDNA Synthesis, SYBR Green Assay, shRNA, Control, Ubiquitin Proteomics, Software