addgene plasmid  (Addgene inc)

Journal: eLife

Article Title: Dependency of human and murine LKB1-inactivated lung cancer on aberrant CRTC-CREB activation

doi: 10.7554/elife.66095

Figure Lengend Snippet: Figure 1. Three members of the CRTC co-activator family, CRTC1, CRTC2, and CRTC3, are expressed at varying levels in human lung epithelial and cancer cell lines. (A) The transcript levels of the three CRTC genes were determined by RT-qPCR assays. All three CRTC transcript levels were normalized against the level of the housekeeping gene GAPDH individually. The expression level of CRTC1 in BEAS-2B was then assigned as 1, and the expression levels for the three CRTCs in various cell lines were presented as relative values to that of CRTC1 in BEAS-2B cells. (B) The protein levels of three CRTCs and LKB1 were detected by western blotting. Blotting with anti-b-Tubulin was used as a loading control. The online version of this article includes the following source data and figure supplement(s) for figure 1:

Article Snippet: DOI: https://doi.org/10.7554/eLife.66095 18 of 30 Continued Reagent type (species) or resource Designation Source or reference Identifiers Additional information Recombinant DNA reagent pcDNA FLAG TORC1 (plasmid) Addgene Plasmid #25718 Recombinant DNA reagent pcDNA FLAG TORC2 (plasmid) Addgene Plasmid #22975 Recombinant DNA reagent pcDNA FLAG TORC3 (plasmid) Addgene Plasmid #22976 Recombinant DNA reagent lentiCas9Blast (plasmid) Addgene Plasmid #52962 Recombinant DNA reagent non-targeting control gRNA (plasmid) Addgene Plasmid #80180 Recombinant DNA reagent STK11 gRNA1 (plasmid) Addgene Plasmid #75912 Recombinant DNA reagent STK11 gRNA-2 (plasmid) Addgene Plasmid #75913 Recombinant DNA reagent pMD2.G (plasmid) Addgene Plasmid #12259 Lentiviral Envelope Recombinant DNA reagent psPAX2 (plasmid) Addgene Plasmid #12260 Lentiviral Packaging Sequencebased reagent CRTC1-qRT-F This paper qPCR primers TGTCTCTCTGACC CCCTTCCAATCC Sequencebased reagent CRTC1-qRT-R This paper qPCR primers GTCCGCGGGTGGT GAGAGGTA Sequencebased reagent CRTC2-qRT-F This paper qPCR primers AGCCCCCTGA GTTTGCTCGC Sequencebased reagent CRTC2-qRT-R This paper qPCR primers TGGGGGTAACCGC TGGTCAGT Sequencebased reagent CRTC3-qRT-F This paper qPCR primers TGACCAGCAGTC CATGAGGCCA Sequencebased reagent CRTC3-qRT-R This paper qPCR primers GGTCTTTGAACAG GCTGGTGCTGG Sequencebased reagent LINC00473-qRT-F This paper qPCR primers AAACGCGAACG TGAGCCCCG Sequencebased reagent LINC00473-qRT-R This paper qPCR primers CGCCATGCTCT GGCGCAGTT Sequencebased reagent FOS-qRT-F This paper qPCR primers CACTCCAAGC GGAGACAG Sequencebased reagent FOS-qRT-R This paper qPCR primers AGGTCATCAGG GATCTTGCAG Sequencebased reagent NR4A2-qRT-F This paper qPCR primers GCCGGAGAGGT CGTTTGCCC Sequencebased reagent NR4A2-qRT-R This paper qPCR primers AGGGTTCGCCT GGAACCTGGAA Sequencebased reagent INSL4-qRT-F This paper qPCR primers GATGTGGTCCC CGATTTGGA Sequencebased reagent INSL4-qRT-R This paper qPCR primers AGGTTGACACCA TTTCTTTGGG Sequencebased reagent CPS1-qRT-F This paper qPCR primers CTGATGCTGCC CACACAAAC Sequencebased reagent CPS1-qRT-R This paper qPCR primers AGGGGAAGGA TCGAGAAGCT Sequencebased reagent PDK4-qRT-F This paper qPCR primers ACAGACAGGAA ACCCAAGCC Continued on next page Zhou et al. eLife 2021;10:e66095.

Techniques: Quantitative RT-PCR, Expressing, Western Blot, Control

Journal: eLife

Article Title: Dependency of human and murine LKB1-inactivated lung cancer on aberrant CRTC-CREB activation

doi: 10.7554/elife.66095

Figure Lengend Snippet: Figure 2. Individual knockouts of the CRTC family members in human LKB1-null lung cancer cells inhibit the CREB-mediated target gene expression and moderately affect cell viability and anchorage-independent growth. (A) Western blot analysis of endogenous CRTC proteins in parental A549 cells, A549 cells stably transduced with non-targeting sgRNA, and two independent single knockout clones for each CRTC1, CRTC2, or CRTC3. The protein level of a CREB target gene, PDE4D was also detected. Blotting with anti-b-ACTIN was used as a loading control. (B) The transcript levels of CREB- mediated target genes (INSL4, LINC00473 and NR4A2) were determined by RT-qPCR assays (n = 2). The LKB1-wt cells, H522 parental (PA) cells, were also analyzed. (C,D) Individual CRTC knockout or control cells were cultured at 3 105 cells/well in the 6-well plates for 96 hr. The viable cells were quantified by trypan blue exclusion assay (C), and the number of apoptotic cells was determined by staining with annexin V/propidium iodide (PI) followed by flow cytometry (D). (E) Control and CRTC knockout cells were cultured in soft agar for 14 days, and the resulting colonies were stained by crystal violet and photographed under microscope. The number of colonies was counted using ImageJ. Assays were performed in triplicate. One-way ANOVA test was used to calculate the p values (*p<0.05, **p<0.01, ns p>0.05). The online version of this article includes the following source data and figure supplement(s) for figure 2:

Article Snippet: DOI: https://doi.org/10.7554/eLife.66095 18 of 30 Continued Reagent type (species) or resource Designation Source or reference Identifiers Additional information Recombinant DNA reagent pcDNA FLAG TORC1 (plasmid) Addgene Plasmid #25718 Recombinant DNA reagent pcDNA FLAG TORC2 (plasmid) Addgene Plasmid #22975 Recombinant DNA reagent pcDNA FLAG TORC3 (plasmid) Addgene Plasmid #22976 Recombinant DNA reagent lentiCas9Blast (plasmid) Addgene Plasmid #52962 Recombinant DNA reagent non-targeting control gRNA (plasmid) Addgene Plasmid #80180 Recombinant DNA reagent STK11 gRNA1 (plasmid) Addgene Plasmid #75912 Recombinant DNA reagent STK11 gRNA-2 (plasmid) Addgene Plasmid #75913 Recombinant DNA reagent pMD2.G (plasmid) Addgene Plasmid #12259 Lentiviral Envelope Recombinant DNA reagent psPAX2 (plasmid) Addgene Plasmid #12260 Lentiviral Packaging Sequencebased reagent CRTC1-qRT-F This paper qPCR primers TGTCTCTCTGACC CCCTTCCAATCC Sequencebased reagent CRTC1-qRT-R This paper qPCR primers GTCCGCGGGTGGT GAGAGGTA Sequencebased reagent CRTC2-qRT-F This paper qPCR primers AGCCCCCTGA GTTTGCTCGC Sequencebased reagent CRTC2-qRT-R This paper qPCR primers TGGGGGTAACCGC TGGTCAGT Sequencebased reagent CRTC3-qRT-F This paper qPCR primers TGACCAGCAGTC CATGAGGCCA Sequencebased reagent CRTC3-qRT-R This paper qPCR primers GGTCTTTGAACAG GCTGGTGCTGG Sequencebased reagent LINC00473-qRT-F This paper qPCR primers AAACGCGAACG TGAGCCCCG Sequencebased reagent LINC00473-qRT-R This paper qPCR primers CGCCATGCTCT GGCGCAGTT Sequencebased reagent FOS-qRT-F This paper qPCR primers CACTCCAAGC GGAGACAG Sequencebased reagent FOS-qRT-R This paper qPCR primers AGGTCATCAGG GATCTTGCAG Sequencebased reagent NR4A2-qRT-F This paper qPCR primers GCCGGAGAGGT CGTTTGCCC Sequencebased reagent NR4A2-qRT-R This paper qPCR primers AGGGTTCGCCT GGAACCTGGAA Sequencebased reagent INSL4-qRT-F This paper qPCR primers GATGTGGTCCC CGATTTGGA Sequencebased reagent INSL4-qRT-R This paper qPCR primers AGGTTGACACCA TTTCTTTGGG Sequencebased reagent CPS1-qRT-F This paper qPCR primers CTGATGCTGCC CACACAAAC Sequencebased reagent CPS1-qRT-R This paper qPCR primers AGGGGAAGGA TCGAGAAGCT Sequencebased reagent PDK4-qRT-F This paper qPCR primers ACAGACAGGAA ACCCAAGCC Continued on next page Zhou et al. eLife 2021;10:e66095.

Techniques: Targeted Gene Expression, Western Blot, Stable Transfection, Transduction, Knock-Out, Clone Assay, Control, Quantitative RT-PCR, Cell Culture, Trypan Blue Exclusion Assay, Staining, Flow Cytometry, Microscopy

Journal: eLife

Article Title: Dependency of human and murine LKB1-inactivated lung cancer on aberrant CRTC-CREB activation

doi: 10.7554/elife.66095

Figure Lengend Snippet: Figure 5. dnCRTC expression suppressed the growth of LKB1-null but not LKB1-positive lung cancer cells. (A) Two LKB1-null (A549 and H157) and two LKB1-positive (H322 and H522) NSCLC cells were transduced with dnCRTC or control GFP retroviruses. The MOI was optimized to obtain an infection rate of 40–60%, and then the percentage of GFP-positive cells was determined by FACS analysis every 3 days for a total of 24 days starting at day 3 post-infection. The percentage of GFP-positive cells at day three post-infection was considered as 100%, and the remaining data were normalized (n = 3). (B, C) The GFP-positive cells for dnCRTC- and GFP-transduced cells were sorted and confirmed for dnCRTC and GFP expression by western blotting (B). Sorted cells were also cultured at 2 105 (for H322 and H522) or 3 105 (for A549 and H157) cells/well in the six-well plates for 96 hr and viable cells were counted using trypan blue exclusion test (C) (n = 3). (D) Transduced cells were cultured at 400 cells/well in six-well plates for 14 days and colonies were stained by crystal violet and photographed. The number of colonies in each well was counted using ImageJ. Assays were performed Figure 5 continued on next page

Article Snippet: DOI: https://doi.org/10.7554/eLife.66095 18 of 30 Continued Reagent type (species) or resource Designation Source or reference Identifiers Additional information Recombinant DNA reagent pcDNA FLAG TORC1 (plasmid) Addgene Plasmid #25718 Recombinant DNA reagent pcDNA FLAG TORC2 (plasmid) Addgene Plasmid #22975 Recombinant DNA reagent pcDNA FLAG TORC3 (plasmid) Addgene Plasmid #22976 Recombinant DNA reagent lentiCas9Blast (plasmid) Addgene Plasmid #52962 Recombinant DNA reagent non-targeting control gRNA (plasmid) Addgene Plasmid #80180 Recombinant DNA reagent STK11 gRNA1 (plasmid) Addgene Plasmid #75912 Recombinant DNA reagent STK11 gRNA-2 (plasmid) Addgene Plasmid #75913 Recombinant DNA reagent pMD2.G (plasmid) Addgene Plasmid #12259 Lentiviral Envelope Recombinant DNA reagent psPAX2 (plasmid) Addgene Plasmid #12260 Lentiviral Packaging Sequencebased reagent CRTC1-qRT-F This paper qPCR primers TGTCTCTCTGACC CCCTTCCAATCC Sequencebased reagent CRTC1-qRT-R This paper qPCR primers GTCCGCGGGTGGT GAGAGGTA Sequencebased reagent CRTC2-qRT-F This paper qPCR primers AGCCCCCTGA GTTTGCTCGC Sequencebased reagent CRTC2-qRT-R This paper qPCR primers TGGGGGTAACCGC TGGTCAGT Sequencebased reagent CRTC3-qRT-F This paper qPCR primers TGACCAGCAGTC CATGAGGCCA Sequencebased reagent CRTC3-qRT-R This paper qPCR primers GGTCTTTGAACAG GCTGGTGCTGG Sequencebased reagent LINC00473-qRT-F This paper qPCR primers AAACGCGAACG TGAGCCCCG Sequencebased reagent LINC00473-qRT-R This paper qPCR primers CGCCATGCTCT GGCGCAGTT Sequencebased reagent FOS-qRT-F This paper qPCR primers CACTCCAAGC GGAGACAG Sequencebased reagent FOS-qRT-R This paper qPCR primers AGGTCATCAGG GATCTTGCAG Sequencebased reagent NR4A2-qRT-F This paper qPCR primers GCCGGAGAGGT CGTTTGCCC Sequencebased reagent NR4A2-qRT-R This paper qPCR primers AGGGTTCGCCT GGAACCTGGAA Sequencebased reagent INSL4-qRT-F This paper qPCR primers GATGTGGTCCC CGATTTGGA Sequencebased reagent INSL4-qRT-R This paper qPCR primers AGGTTGACACCA TTTCTTTGGG Sequencebased reagent CPS1-qRT-F This paper qPCR primers CTGATGCTGCC CACACAAAC Sequencebased reagent CPS1-qRT-R This paper qPCR primers AGGGGAAGGA TCGAGAAGCT Sequencebased reagent PDK4-qRT-F This paper qPCR primers ACAGACAGGAA ACCCAAGCC Continued on next page Zhou et al. eLife 2021;10:e66095.

Techniques: Expressing, Transduction, Control, Infection, Western Blot, Cell Culture, Staining

Journal: eLife

Article Title: Dependency of human and murine LKB1-inactivated lung cancer on aberrant CRTC-CREB activation

doi: 10.7554/elife.66095

Figure Lengend Snippet: Figure 6. Expression of dnCRTC significantly inhibited the growth of LKB1-null NSCLC xenograft tumors. (A–E) A549-luc were transduced with GFP control or dnCRTC for 72 hr and the transduced cells (1 106 per mouse) were injected subcutaneously to the right flanks of NOD/SCID mice. Tumor volumes of two cohorts (n = 5 each) were measured every two days starting from day 14 until day 48 (A). The bioluminescent images of mice (B), excised tumors (C) and tumor weights (D) as well as Ki-67 immunohistochemical staining of xenograft tumor sections (E) were shown. (F–J) H157-luc Figure 6 continued on next page

Article Snippet: DOI: https://doi.org/10.7554/eLife.66095 18 of 30 Continued Reagent type (species) or resource Designation Source or reference Identifiers Additional information Recombinant DNA reagent pcDNA FLAG TORC1 (plasmid) Addgene Plasmid #25718 Recombinant DNA reagent pcDNA FLAG TORC2 (plasmid) Addgene Plasmid #22975 Recombinant DNA reagent pcDNA FLAG TORC3 (plasmid) Addgene Plasmid #22976 Recombinant DNA reagent lentiCas9Blast (plasmid) Addgene Plasmid #52962 Recombinant DNA reagent non-targeting control gRNA (plasmid) Addgene Plasmid #80180 Recombinant DNA reagent STK11 gRNA1 (plasmid) Addgene Plasmid #75912 Recombinant DNA reagent STK11 gRNA-2 (plasmid) Addgene Plasmid #75913 Recombinant DNA reagent pMD2.G (plasmid) Addgene Plasmid #12259 Lentiviral Envelope Recombinant DNA reagent psPAX2 (plasmid) Addgene Plasmid #12260 Lentiviral Packaging Sequencebased reagent CRTC1-qRT-F This paper qPCR primers TGTCTCTCTGACC CCCTTCCAATCC Sequencebased reagent CRTC1-qRT-R This paper qPCR primers GTCCGCGGGTGGT GAGAGGTA Sequencebased reagent CRTC2-qRT-F This paper qPCR primers AGCCCCCTGA GTTTGCTCGC Sequencebased reagent CRTC2-qRT-R This paper qPCR primers TGGGGGTAACCGC TGGTCAGT Sequencebased reagent CRTC3-qRT-F This paper qPCR primers TGACCAGCAGTC CATGAGGCCA Sequencebased reagent CRTC3-qRT-R This paper qPCR primers GGTCTTTGAACAG GCTGGTGCTGG Sequencebased reagent LINC00473-qRT-F This paper qPCR primers AAACGCGAACG TGAGCCCCG Sequencebased reagent LINC00473-qRT-R This paper qPCR primers CGCCATGCTCT GGCGCAGTT Sequencebased reagent FOS-qRT-F This paper qPCR primers CACTCCAAGC GGAGACAG Sequencebased reagent FOS-qRT-R This paper qPCR primers AGGTCATCAGG GATCTTGCAG Sequencebased reagent NR4A2-qRT-F This paper qPCR primers GCCGGAGAGGT CGTTTGCCC Sequencebased reagent NR4A2-qRT-R This paper qPCR primers AGGGTTCGCCT GGAACCTGGAA Sequencebased reagent INSL4-qRT-F This paper qPCR primers GATGTGGTCCC CGATTTGGA Sequencebased reagent INSL4-qRT-R This paper qPCR primers AGGTTGACACCA TTTCTTTGGG Sequencebased reagent CPS1-qRT-F This paper qPCR primers CTGATGCTGCC CACACAAAC Sequencebased reagent CPS1-qRT-R This paper qPCR primers AGGGGAAGGA TCGAGAAGCT Sequencebased reagent PDK4-qRT-F This paper qPCR primers ACAGACAGGAA ACCCAAGCC Continued on next page Zhou et al. eLife 2021;10:e66095.

Techniques: Expressing, Transduction, Control, Injection, Immunohistochemical staining, Staining

Journal: eLife

Article Title: Dependency of human and murine LKB1-inactivated lung cancer on aberrant CRTC-CREB activation

doi: 10.7554/elife.66095

Figure Lengend Snippet: Figure 7. Expression of dnCRTC reduced lung colonization of LKB1-null lung cancer cells. (A–C) Luciferase-expressing LKB1-null A549 cells (A549-luc) were transduced with retroviruses expressing GFP control or dnCRTC for 72 hr, and transduced cells (2 106 cells per mouse) were intravenously injected to NOD/SCID mice (n = 6 each). Eight weeks after injection, lung colonization was assessed by bioluminescent imaging (A). Lungs were dissected and bright field and GFP fluorescence images were shown (B). The number of surface tumor nodules with visible GFP signal per lung of each mouse was quantified and presented (right panel). Representative H and E staining of lung sections were shown (C). Tumor area was calculated from multiple H and E-stained lung sections from each mouse and presented as a percentage of tumor area to total lung area (right panel). (D–F) Luciferase- expressing LKB1-null H157 lung cancer cells (H157-luc) were transduced with retrovirus expressing GFP control or dnCRTC for 72 hr, and transduced cells (2 106 cells per mouse) were intravenously injected to NOD/SCID mice (n = 6 each). Four weeks after injection, lung colonization was assessed by bioluminescent imaging (D). Lungs were dissected and bright field and GFP fluorescence images were shown (E). The number of tumor nodules with visible GFP signal per lung of each mouse was quantified (right panel). Representative H and E staining images of lung sections were shown (F). Tumor area was calculated from multiple H and E-stained lung sections from each mouse and presented as a percentage of tumor area to total lung area (right panel). The p values were calculated by two-tailed student’s t-test (**p<0.01, ***p<0.001, ****p<0.0001). Figure 7 continued on next page

Article Snippet: DOI: https://doi.org/10.7554/eLife.66095 18 of 30 Continued Reagent type (species) or resource Designation Source or reference Identifiers Additional information Recombinant DNA reagent pcDNA FLAG TORC1 (plasmid) Addgene Plasmid #25718 Recombinant DNA reagent pcDNA FLAG TORC2 (plasmid) Addgene Plasmid #22975 Recombinant DNA reagent pcDNA FLAG TORC3 (plasmid) Addgene Plasmid #22976 Recombinant DNA reagent lentiCas9Blast (plasmid) Addgene Plasmid #52962 Recombinant DNA reagent non-targeting control gRNA (plasmid) Addgene Plasmid #80180 Recombinant DNA reagent STK11 gRNA1 (plasmid) Addgene Plasmid #75912 Recombinant DNA reagent STK11 gRNA-2 (plasmid) Addgene Plasmid #75913 Recombinant DNA reagent pMD2.G (plasmid) Addgene Plasmid #12259 Lentiviral Envelope Recombinant DNA reagent psPAX2 (plasmid) Addgene Plasmid #12260 Lentiviral Packaging Sequencebased reagent CRTC1-qRT-F This paper qPCR primers TGTCTCTCTGACC CCCTTCCAATCC Sequencebased reagent CRTC1-qRT-R This paper qPCR primers GTCCGCGGGTGGT GAGAGGTA Sequencebased reagent CRTC2-qRT-F This paper qPCR primers AGCCCCCTGA GTTTGCTCGC Sequencebased reagent CRTC2-qRT-R This paper qPCR primers TGGGGGTAACCGC TGGTCAGT Sequencebased reagent CRTC3-qRT-F This paper qPCR primers TGACCAGCAGTC CATGAGGCCA Sequencebased reagent CRTC3-qRT-R This paper qPCR primers GGTCTTTGAACAG GCTGGTGCTGG Sequencebased reagent LINC00473-qRT-F This paper qPCR primers AAACGCGAACG TGAGCCCCG Sequencebased reagent LINC00473-qRT-R This paper qPCR primers CGCCATGCTCT GGCGCAGTT Sequencebased reagent FOS-qRT-F This paper qPCR primers CACTCCAAGC GGAGACAG Sequencebased reagent FOS-qRT-R This paper qPCR primers AGGTCATCAGG GATCTTGCAG Sequencebased reagent NR4A2-qRT-F This paper qPCR primers GCCGGAGAGGT CGTTTGCCC Sequencebased reagent NR4A2-qRT-R This paper qPCR primers AGGGTTCGCCT GGAACCTGGAA Sequencebased reagent INSL4-qRT-F This paper qPCR primers GATGTGGTCCC CGATTTGGA Sequencebased reagent INSL4-qRT-R This paper qPCR primers AGGTTGACACCA TTTCTTTGGG Sequencebased reagent CPS1-qRT-F This paper qPCR primers CTGATGCTGCC CACACAAAC Sequencebased reagent CPS1-qRT-R This paper qPCR primers AGGGGAAGGA TCGAGAAGCT Sequencebased reagent PDK4-qRT-F This paper qPCR primers ACAGACAGGAA ACCCAAGCC Continued on next page Zhou et al. eLife 2021;10:e66095.

Techniques: Expressing, Luciferase, Transduction, Control, Injection, Imaging, Fluorescence, Staining, Two Tailed Test