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Addgene inc plko 1 shgfp control
Plko 1 Shgfp Control, supplied by Addgene inc, used in various techniques. Bioz Stars score: 95/100, based on 170 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Average 95 stars, based on 170 article reviews

plko 1 shgfp control - by Bioz Stars, 2026-05

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(A-C) IMR90 normal human fibro-blasts were transduced with retrovirus expressing HRASG12V (RAS) with or without a lentivirus expressing a short hairpin targeting p16 (shp16). (A) Western blot of RAS and p16. Vinculin was used as a loading control. One of >10 independent experiments shown. (B) DNA damage foci were assessed by immunofluorescence. One of three independent experiments shown. (C) Senescence-associated beta-galactosidase (SA-β-Gal) activity. One of >10 independent experiments shown. (D-E) Ovcar8 ovarian cancer cells were transduced with a lentivirus expressing a short hairpin targeting GFP (shCont) or p16 (shp16). (D) p16 was assessed by western blotting. β-actin was used as a loading control. One of 3 independent experiments. (E) DNA damage foci were assessed by immunofluorescence. One of three independent experiments shown. (F-G) Yumm5.2 melanoma cells were transduced with a lentivirus expressing a short hairpin targeting GFP (shCont) or two independent hairpins targeting Cdkn2a (sh Cdkn2a #1 and sh Cdkn2a #2). (F) The indicated proteins were assessed by western blotting. β-actin was used as a loading control. One of 10 independent experiments. (G) and DNA damage foci were assessed by immunofluorescence. One of three independent experiments shown. Graphs represent mean ± SD. T-test or one-way ANOVA. *p<0.05, **p<0.01, ***p<0.005, ****p<0.001

Journal: bioRxiv

Article Title: ATR promotes mTORC1 activation via de novo cholesterol synthesis in p16-low cancer cells

doi: 10.1101/2023.10.27.564195

Figure Lengend Snippet: (A-C) IMR90 normal human fibro-blasts were transduced with retrovirus expressing HRASG12V (RAS) with or without a lentivirus expressing a short hairpin targeting p16 (shp16). (A) Western blot of RAS and p16. Vinculin was used as a loading control. One of >10 independent experiments shown. (B) DNA damage foci were assessed by immunofluorescence. One of three independent experiments shown. (C) Senescence-associated beta-galactosidase (SA-β-Gal) activity. One of >10 independent experiments shown. (D-E) Ovcar8 ovarian cancer cells were transduced with a lentivirus expressing a short hairpin targeting GFP (shCont) or p16 (shp16). (D) p16 was assessed by western blotting. β-actin was used as a loading control. One of 3 independent experiments. (E) DNA damage foci were assessed by immunofluorescence. One of three independent experiments shown. (F-G) Yumm5.2 melanoma cells were transduced with a lentivirus expressing a short hairpin targeting GFP (shCont) or two independent hairpins targeting Cdkn2a (sh Cdkn2a #1 and sh Cdkn2a #2). (F) The indicated proteins were assessed by western blotting. β-actin was used as a loading control. One of 10 independent experiments. (G) and DNA damage foci were assessed by immunofluorescence. One of three independent experiments shown. Graphs represent mean ± SD. T-test or one-way ANOVA. *p<0.05, **p<0.01, ***p<0.005, ****p<0.001

Article Snippet: The plasmids are the following: pBABE-puro-H-RASG12V (Addgene, 39526); pBabe-puro (Addgene, 1764); pLKO.1-shp16 (TRCN0000010482); pLKO.1-shGFP control (Addgene, cat#30323); pLKO.1-shATR (TCRN0000039615, TCRN0000039616); pLKO.1-shATM (TCRN0000038658); pLKO.1-shChk1 (TRCN0000000499, TRCN0000000502); pLKO.1-shRPTOR (TRCN0000039772); pLKO.1-LSS (TRCN0000045481, TRCN0000045482); pLKO.1-shCdkn2a (TRCN0000077816, TRCN0000362595).

Techniques: Transduction, Expressing, Western Blot, Control, Immunofluorescence, Activity Assay

(A) SKMEL28 melanoma cells were transduced with a lentivirus expressing a short hairpin targeting GFP (shCont) or p16 (shp16), and DNA damage foci were assessed by immunofluorescence. Representative data from 8 independent experiments. (B) SKMEL28 melanoma cells were transduced with a guide RNA targeting p16 (p16 KO). Details on controls in Methods. DNA damage foci were assessed by immunofluorescence. Representative data from 3 independent experiments. (C) The indicated western blots were performed in SKMEL28 and RPMI-7951 melanoma cells. β-actin or vinculin were used as loading controls. Representative data from 2-3 independent experiments. (D) Gene Set Enrichment Analysis of RNA-Seq from SKMEL28 melanoma cells in (A) show positive enrichment of G2M checkpoint signature in shp16 cells. (E) RPPA results from Melanoma patient samples show upregulation of proteins related to the DNA damage response and repair in tumors with homozygous deletion of the CDKN2A locus compared to intact CDKN2A locus (all proteins p<0.05). Graphs represent mean ± SD. T-test. *p<0.05

Journal: bioRxiv

Article Title: ATR promotes mTORC1 activation via de novo cholesterol synthesis in p16-low cancer cells

doi: 10.1101/2023.10.27.564195

Figure Lengend Snippet: (A) SKMEL28 melanoma cells were transduced with a lentivirus expressing a short hairpin targeting GFP (shCont) or p16 (shp16), and DNA damage foci were assessed by immunofluorescence. Representative data from 8 independent experiments. (B) SKMEL28 melanoma cells were transduced with a guide RNA targeting p16 (p16 KO). Details on controls in Methods. DNA damage foci were assessed by immunofluorescence. Representative data from 3 independent experiments. (C) The indicated western blots were performed in SKMEL28 and RPMI-7951 melanoma cells. β-actin or vinculin were used as loading controls. Representative data from 2-3 independent experiments. (D) Gene Set Enrichment Analysis of RNA-Seq from SKMEL28 melanoma cells in (A) show positive enrichment of G2M checkpoint signature in shp16 cells. (E) RPPA results from Melanoma patient samples show upregulation of proteins related to the DNA damage response and repair in tumors with homozygous deletion of the CDKN2A locus compared to intact CDKN2A locus (all proteins p<0.05). Graphs represent mean ± SD. T-test. *p<0.05

Techniques: Transduction, Expressing, Immunofluorescence, Western Blot, RNA Sequencing

$SKMEL28 melanoma cells were transduced with a lentivirus expressing a short hairpin targeting GFP or p16 (shp16). (A) shp16 cells were transduced with a lentivirus expressing a short hairpin targeting GFP or ATR (shATR), and pS6K was assessed by western blotting. Vinculin was used as a loading control. (B) Cells were treated for 30min with 63nM VE822 ATR inhibitor (ATRi), and the indicated proteins were assessed by western blotting. Vinculin was used as a loading control. (C) Cells were treated for 10min with 250nM of the mTORC1 inhibitor Torin1, and the indicated proteins were assessed by western blotting. Vinculin was used as a loading control. (D) Analysis of heavy ribosome fraction vs. total mRNA in the indicated cells.$

Journal: bioRxiv

Article Title: ATR promotes mTORC1 activation via de novo cholesterol synthesis in p16-low cancer cells

doi: 10.1101/2023.10.27.564195

Figure Lengend Snippet: SKMEL28 melanoma cells were transduced with a lentivirus expressing a short hairpin targeting GFP or p16 (shp16). (A) shp16 cells were transduced with a lentivirus expressing a short hairpin targeting GFP or ATR (shATR), and pS6K was assessed by western blotting. Vinculin was used as a loading control. (B) Cells were treated for 30min with 63nM VE822 ATR inhibitor (ATRi), and the indicated proteins were assessed by western blotting. Vinculin was used as a loading control. (C) Cells were treated for 10min with 250nM of the mTORC1 inhibitor Torin1, and the indicated proteins were assessed by western blotting. Vinculin was used as a loading control. (D) Analysis of heavy ribosome fraction vs. total mRNA in the indicated cells.

Techniques: Transduction, Expressing, Western Blot, Control

(A-D) SKMEL28 cells were transduced with lentivirus expressing shRNA targeting p16 (shp16) or control (shCont). (A) Cells were transduced with lentivirus expressing shRNA targeting ATM (shATM) or control, and the indicated proteins were assessed by western blotting. Vinculin was used as a loading control. One of three independent experiments is shown. (B) Cells were treated with the ATM inhibitor 313nM (ATMi) KU60019 for 30min, and the indicated proteins were assessed by western blotting. Vinculin was used as a loading control. One of three independent experiments is shown. (C) Cells were transduced with lentivirus expressing shRNA targeting Chk1 (shChk1) or control, and the indicated proteins were assessed by western blotting. Vinculin was used as a loading control. One of three independent experiments is shown. (D) Cells were treated with the Chk1 inhibitor 313nM (Chk1i) LY2603618 for 30min, and the indicated proteins were assessed by western blotting. Vinculin was used as a loading control. One of three independent experiments is shown.

Journal: bioRxiv

Article Title: ATR promotes mTORC1 activation via de novo cholesterol synthesis in p16-low cancer cells

doi: 10.1101/2023.10.27.564195

Figure Lengend Snippet: (A-D) SKMEL28 cells were transduced with lentivirus expressing shRNA targeting p16 (shp16) or control (shCont). (A) Cells were transduced with lentivirus expressing shRNA targeting ATM (shATM) or control, and the indicated proteins were assessed by western blotting. Vinculin was used as a loading control. One of three independent experiments is shown. (B) Cells were treated with the ATM inhibitor 313nM (ATMi) KU60019 for 30min, and the indicated proteins were assessed by western blotting. Vinculin was used as a loading control. One of three independent experiments is shown. (C) Cells were transduced with lentivirus expressing shRNA targeting Chk1 (shChk1) or control, and the indicated proteins were assessed by western blotting. Vinculin was used as a loading control. One of three independent experiments is shown. (D) Cells were treated with the Chk1 inhibitor 313nM (Chk1i) LY2603618 for 30min, and the indicated proteins were assessed by western blotting. Vinculin was used as a loading control. One of three independent experiments is shown.

Techniques: Transduction, Expressing, shRNA, Control, Western Blot

(A-I) SKMEL28 cells were transduced with lentivirus expressing shRNA targeting p16 (shp16) or control (shCont). (A) Venn diagram comparing proteomics data with publicly available dataset of mTORC1 regulators. (B) Nine hits were found in the cross-comparison from (A) . (C) LSS protein abundance in the indicated groups. (D) Simplified pathway of de novo cholesterol synthesis. (E) Cholesterol abundance was assessed by filipin staining and analysis of intensity using ImageJ. One of two independent experiments. (F) Representative images from filipin staining. (G) shp16 SKMEL28 cells were transduced with lentivirus expressing shRNA targeting LSS (shLSS #1 and #2), and p-S6K/S6K was assessed by western blotting. Vinculin was used as a loading control. One of four independent experiments. (H) shp16 SKMEL28 cells were treated with 2.5μM LSS inhibitor (LSSi) Ro 48-8071 for 24h, and p-S6K/S6K was assessed by western blotting. Vinculin was used as a loading control. One of three independent experiments. (I) shp16 SKMEL28 cells were transduced with lentivirus expressing shRNA targeting ATR (shATR) with or without treatment with 50μM cholesterol for 24h, and p-S6K/S6K was assessed by western blotting. Vinculin was used as a loading control. One of two independent experiments. Graphs represent mean ± SD. T-test or one-way ANOVA. ***p<0.005, ****p<0.001, ns = not significant

Journal: bioRxiv

Article Title: ATR promotes mTORC1 activation via de novo cholesterol synthesis in p16-low cancer cells

doi: 10.1101/2023.10.27.564195

Figure Lengend Snippet: (A-I) SKMEL28 cells were transduced with lentivirus expressing shRNA targeting p16 (shp16) or control (shCont). (A) Venn diagram comparing proteomics data with publicly available dataset of mTORC1 regulators. (B) Nine hits were found in the cross-comparison from (A) . (C) LSS protein abundance in the indicated groups. (D) Simplified pathway of de novo cholesterol synthesis. (E) Cholesterol abundance was assessed by filipin staining and analysis of intensity using ImageJ. One of two independent experiments. (F) Representative images from filipin staining. (G) shp16 SKMEL28 cells were transduced with lentivirus expressing shRNA targeting LSS (shLSS #1 and #2), and p-S6K/S6K was assessed by western blotting. Vinculin was used as a loading control. One of four independent experiments. (H) shp16 SKMEL28 cells were treated with 2.5μM LSS inhibitor (LSSi) Ro 48-8071 for 24h, and p-S6K/S6K was assessed by western blotting. Vinculin was used as a loading control. One of three independent experiments. (I) shp16 SKMEL28 cells were transduced with lentivirus expressing shRNA targeting ATR (shATR) with or without treatment with 50μM cholesterol for 24h, and p-S6K/S6K was assessed by western blotting. Vinculin was used as a loading control. One of two independent experiments. Graphs represent mean ± SD. T-test or one-way ANOVA. ***p<0.005, ****p<0.001, ns = not significant

Techniques: Transduction, Expressing, shRNA, Control, Comparison, Quantitative Proteomics, Staining, Western Blot

(A) Gene Set Enrichment Analysis (GSEA) of SKMEL28 cells with p16 knockdown (left) and Melanoma samples with low CDKN2A expression (right) show enrichment of signatures related to cholesterol. (B) Ovcar8 cells were transduced with lentivirus expressing shRNA targeting p16 (shp16) or control (shCont). Cholesterol abundance was assessed by filipin staining and analysis of intensity using ImageJ (left). Representative images from filipin staining (right). (C) Yumm5.2 cells were transduced with lentivirus expressing shRNA targeting Cdkn2a (sh Cdkn2a ) or control (shCont). Cholesterol abundance was assessed by filipin staining and analysis of intensity using ImageJ (left). Representative images from filipin staining (right). (D) SKMEL28 cells were transduced with lentivirus expression hairpins targeting LSS (shLSS), and LSS expression was assessed by RT-qPCR. Knockdown was normalized to respective controls. (E) shp16 Ovcar8 cells were treated with 5μM LSS inhibitor (LSSi) Ro 48-8071 for 24h, and p-S6K/S6K was assessed by western blotting. Vinculin was used as a loading control. One of two independent experiments. Graphs represent mean ± SD. T-test. *p<0.05

Journal: bioRxiv

Article Title: ATR promotes mTORC1 activation via de novo cholesterol synthesis in p16-low cancer cells

doi: 10.1101/2023.10.27.564195

Figure Lengend Snippet: (A) Gene Set Enrichment Analysis (GSEA) of SKMEL28 cells with p16 knockdown (left) and Melanoma samples with low CDKN2A expression (right) show enrichment of signatures related to cholesterol. (B) Ovcar8 cells were transduced with lentivirus expressing shRNA targeting p16 (shp16) or control (shCont). Cholesterol abundance was assessed by filipin staining and analysis of intensity using ImageJ (left). Representative images from filipin staining (right). (C) Yumm5.2 cells were transduced with lentivirus expressing shRNA targeting Cdkn2a (sh Cdkn2a ) or control (shCont). Cholesterol abundance was assessed by filipin staining and analysis of intensity using ImageJ (left). Representative images from filipin staining (right). (D) SKMEL28 cells were transduced with lentivirus expression hairpins targeting LSS (shLSS), and LSS expression was assessed by RT-qPCR. Knockdown was normalized to respective controls. (E) shp16 Ovcar8 cells were treated with 5μM LSS inhibitor (LSSi) Ro 48-8071 for 24h, and p-S6K/S6K was assessed by western blotting. Vinculin was used as a loading control. One of two independent experiments. Graphs represent mean ± SD. T-test. *p<0.05

Techniques: Knockdown, Expressing, Transduction, shRNA, Control, Staining, Quantitative RT-PCR, Western Blot

Journal: bioRxiv

Article Title: ATR promotes mTORC1 activation via de novo cholesterol synthesis in p16-low cancer cells

doi: 10.1101/2023.10.27.564195

Figure Lengend Snippet: (A-D) SKMEL28 cells were transduced with lentivirus expressing shRNA targeting p16 (shp16) or control (shCont). (A) Cells were transduced with lentivirus expressing shRNA targeting LSS (shLSS), and cytotoxicity was assessed using Incucyte Cytotox reagent. One of three independent experiments is shown. (B) Same as (A) but % death and endpoint is shown. One of three independent experiments is shown. Graph represents mean ± SD. One-way ANOVA. ****p<0.001 (C) Cells were treated with the LSS inhibitor (LSSi) Ro 48-8071, and proliferation was assessed by crystal violet staining. One of three independent experiments is shown. (D) Cells were treated with the de novo cholesterol synthesis inhibitors atorvastatin, mevastatin, and lovastatin, and proliferation was assessed by crystal violet staining. One of three independent experiments is shown.

Techniques: Transduction, Expressing, shRNA, Control, Staining

(A) Ovcar8 (left) or Yumm5.2 (right) cells were transduced with lentivirus expressing shRNA targeting p16 (shp16) or Cdkn2a (sh Cdkn2a ). Cells were treated for with the LSS inhibitor (LSSi) Ro 48-8071, and proliferation was assessed by crystal violet staining. One of at least 2 independent experiments is shown. (B) Ovcar8 cells were treated with the de novo cholesterol synthesis inhibitors atorvastatin, mevastatin, and lovastatin, and proliferation was assessed by crystal violet staining. One of at least two independent experiments is shown. (C) Increased drug sensitivity from DepMap data of cutaneous melanoma cell lines with high or low CDKN2A expression. Data are mean ± SD. T-test. *p<0.05, **p<0.01

Journal: bioRxiv

Article Title: ATR promotes mTORC1 activation via de novo cholesterol synthesis in p16-low cancer cells

doi: 10.1101/2023.10.27.564195

Figure Lengend Snippet: (A) Ovcar8 (left) or Yumm5.2 (right) cells were transduced with lentivirus expressing shRNA targeting p16 (shp16) or Cdkn2a (sh Cdkn2a ). Cells were treated for with the LSS inhibitor (LSSi) Ro 48-8071, and proliferation was assessed by crystal violet staining. One of at least 2 independent experiments is shown. (B) Ovcar8 cells were treated with the de novo cholesterol synthesis inhibitors atorvastatin, mevastatin, and lovastatin, and proliferation was assessed by crystal violet staining. One of at least two independent experiments is shown. (C) Increased drug sensitivity from DepMap data of cutaneous melanoma cell lines with high or low CDKN2A expression. Data are mean ± SD. T-test. *p<0.05, **p<0.01

Techniques: Transduction, Expressing, shRNA, Staining

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