EMBO/SourceData huggingface dataset:
Figure 4A. A549 cells were transfected with control plasmid pcDNA 3.1 or plasmids encoding FLAG-tagged WT or mutant NS5 proteins. At 48 hrs post-transfection cells were treated with IFN-α (100 U/mL) for 2 hrs and then processed for indirect immunofluorescence. Images were acquired on a spinning disk confocal microscope with a 40X objective. NS5 positive cells are indicated by dashed white circles. Representative panels are shown. The experiment was repeated three times.B. Quantification of nuclearSTAT2 was performed using Volocity image analyses software. A minimum of 20 cells were counted for each sample. N.D.= not detected. Values are expressed as mean ± standard error. *P<0.05, **P<0.01 (Student's t-test); N=3C. A549 cells were transfected with plasmids encoding FLAG-tagged ZIKVNS5 or FLAG alone for 24 hrs and then treated with epoxomicin (400 nM) for 24 hrs. Cells were harvested and process for immunoprecipitation (IP) using a mouse anti-FLAG or anti-myc antibody followed by SDS-PAGE and Immunoblotting. Representative panels are shown. The experiment was repeated three times.D. A549 cells were transfected with plasmids encoding FLAG-tagged WT, mutant NS5 proteins or FLAG alone for 48 hrs. Cells were harvested and process for immunoprecipitation (IP) using rabbit anti-STAT2 followed by SDS-PAGE and immunoblotting. Arrows indicate co-immunoprecipitated WT NS5 and NS5 mutants that are in a complex with STAT2. Representative panels are shown. The experiment was repeated two times.
Figure 1 (A) Bcl-xfl/fl;RosaCreERT2+/Ki (n=12) or, as controls, Bcl-xfl/fl (n=6) and RosaCreERT2+/Ki (n=16) mice (age 9-12 weeks, males and females) were treated with tamoxifen (200 mg/kg/body weight administered in 3 daily doses by oral gavage) to induce CreERT2-mediated deletion of the floxed Bcl-x alleles. Mice were monitored for up to 200 days post-treatment with tamoxifen. Data are presented as % survival post-treatment with tamoxifen and statistical significance was assessed using the Mantel-Cox (Log-rank) test; ****p<0.0001.  (B) Bcl-xfl/fl;RosaCreERT2+/Ki (n=24) or, as controls, Bcl-xfl/fl (n=9) and RosaCreERT2+/Ki (n=6) mice (males and females, aged 8-14 weeks, numbers also indicated in the figure legend) were lethally γ-irradiated (2 x 5.5 Gy, 3 h apart) and reconstituted with bone marrow from UBC-GFP mice (referred to as GFP-Chimeras). After 8 weeks, reconstituted mice were treated with tamoxifen (200 mg/kg body weight administered in 3 daily doses oral gavage) and monitored for up to 200 days (termination of the experiment). Data are presented as % survival post-treatment with tamoxifen and statistical significance was assessed using the Mantel-Cox (Log-rank) test; ****p<0.0001.  (C-F) Total counts of (C) platelets, (D) red blood cells (RBC), (E) hemoglobin (HGB) content and (F) hematocrit (HCT) of tamoxifen-treated Bcl-xfl/fl;RosaCreERT2+/Ki (n=8) or, as controls, Bcl-xfl/fl (n=6) and RosaCreERT2+/Ki (n=12) and Bcl-xfl/fl;RosaCreERT2+/Ki;GFP-Chimera (n=21) or, as controls, Bcl-xfl/fl;GFP-Chimera (n=6) and RosaCreERT2+/Ki;GFP-Chimera (n=6) mice were determined by ADVIA. Data are presented as mean ±SEM. Each data point represents an individual mouse. Statistical significance was assessed using the Student's t-test; ****p<0.0001. (G) Spleen weights were measured in sick mice (at sacrifice) or, for the healthy control mice, at the termination of the experiment. Data are presented as mean ±SEM. Each data point represents an individual mouse and numbers are indicated. Statistical significance was assessed using the Student's t-test; ****p<0.0001.  (H) Representative image of enlarged spleens from two Bcl-xfl/fl;RosaCreERT2Ki/+ mice and age-matched control Bcl-xfl/fl and RosaCreERT2+/Ki mice (34 days after treatment).  (I) Histological analysis of H&E-stained sections of spleens of tamoxifen-treated mice of the indicated genotypes.
Figure 2Bcl-xfl/fl;RosaCreERT2+/Ki (n=8) or, as controls, Bcl-xfl/fl (n=6) and RosaCreERT2+/Ki (n=12) mice as well as Bcl-xfl/fl;RosaCreERT2+/Ki;GFP-Chimeras (n=21), or as controls, Bcl-xfl/fl;GFP-Chimeras (n=6) and RosaCreERT2+/Ki;GFP-Chimeras (n=6) (age 8-14 weeks, males and females) were treated with tamoxifen (200 mg/kg/body weight administered in 3 daily doses by oral gavage) to induce CreERT2-mediated deletion of the floxed Bcl-x alleles. (A) Total white blood cell counts (WBC) were analyzed by ADVIA in sick mice or at the termination of the experiment (healthy control mice). Data are presented as mean ±SEM. Each data point represents an individual mouse and n numbers are indicated above. Statistical significance was assessed using the Student's t-test. No statistically significant differences were observed.  (B) The neutrophil/lymphocyte ratio (NLR) is presented as mean ±SEM for sick Bcl-xfl/fl;RosaCreERT2+/Ki;GFP-Chimeras (n=21) or healthy control RosaCreERT2+/Ki;GFP-Chimeras (n=6) at the termination of the experiment. Data are presented as mean ±SEM. Each data point represents an individual mouse. Statistical significance was assessed using the Student's t-test; *p<0.05.  (C) Histological analysis of H&E-stained sections of the sternum of sick Bcl-xfl/fl;RosaCreERT2+/Ki;GFP-Chimeras or healthy wild-type and RosaCreERT2+/Ki;GFP-Chimera control mice at the indicated time points post-treatment with tamoxifen (dpt=days post-treatment).  (D) Histological analysis of H&E-stained sections of the livers of sick mice or age-matched healthy control wild-type mice or healthy RosaCreERT2+/Ki;GFP-Chimeras at the termination of the experiment (dpt=days post-treatment).  (E) ALT (left panel), AST (middle panel) and bilirubin levels (right panel) in the serum were determined in sick Bcl-xfl/fl;RosaCreERT2+/Ki;GFP-Chimeras (n=18) or in healthy RosaCreERT2+/Ki;GFP-Chimeras (n=4) at the termination of the experiment. Data are presented as mean ±SEM. Each data point represents one individual mouse. Statistical significance was assessed using Student's t-test; **p<0.01. nd=not detected.
Figure 3 Bcl-xfl/fl;RosaCreERT2+/Ki (n=7) or as controls, Bcl-xfl/fl (n=8) and RosaCreERT2+/Ki (n=16) mice as well as Bcl-xfl/fl;RosaCreERT2+/Ki;GFP-Chimeras (n=5), Bcl-xfl/fl;GFP-Chimeras (n=8) and RosaCreERT2+/Ki;GFP-Chimeras (n=6) (age 8-14 weeks, males and females) were treated with tamoxifen (200 mg/kg/body weight administered in 3 daily doses by oral gavage) to induce CreERT2-mediated deletion of the floxed Bcl-x alleles. (A) Kidney weights were measured in sick Bcl-xfl/fl;RosaCreERT2+/Ki;GFP-Chimeras (at sacrifice) or, for the healthy control mice, at the termination of the experiment. Data are presented as mean ±SEM. Each data point represents one individual mouse. Statistical significance was assessed using Student's t-test; *p<0.05, ***p<0.001.  (B) Picture showing both kidneys from age-matched healthy control wild-type mice (left) and sick Bcl-xfl/fl;RosaCreERT2+/Ki;GFP-Chimeras (right).  (C) Histological analysis of H&E-stained sections of the kidneys of age-matched healthy control mice or sick Bcl-xfl/fl;RosaCreERT2+/Ki;GFP-Chimeras as indicated, showing from left to right, shrunken scarred kidney, segmental chronic tubulo-interstitial disease, accumulation of cellular debris and amorphous material within the collecting ducts of the papilla. The higher magnification shows tubular epithelial degeneration, apoptosis and segmental secondary glomerular sclerosis. Pictures are representative of at least 5 mice for each genotype and treatment.  (D) Multiphoton analysis of fixed sections of the kidneys of age-matched healthy RosaCreERT2+/Ki;GFP-Chimeras (left) or sick Bcl-xfl/fl;RosaCreERT2+/Ki;GFP-Chimeras (right). Pictures are representative of at least 3 mice for each genotype and treatment. green=second harmony, blue=short wavelength autofluorescence.
Figure 4 (A) TUNEL staining of kidney sections from control RosaCreERT2+/Ki, Bcl-xfl/fl;RosaCreERT2+/Ki, RosaCreERT2+/Ki;GFP-Chimeras or Bcl-xfl/fl;RosaCreERT2+/Ki;GFP-Chimeras at the indicated time points post-tamoxifen treatment (200 mg/kg/body weight administered in 3 daily doses by oral gavage) or γ-irradiation (2 x 550 Rad), as indicated. Slides were counterstained with hematoxylin. Arrow heads indicate examples of TUNEL+ (apoptotic) cells. (B) TUNEL+ cells were quantified using a personalized script for the Image J software. Kidney sections were divided into ~40 microscopic images and the total numbers of blue and brown (TUNEL+) nuclei were determined. The percentages of TUNEL+ nuclei were calculated for each picture. The average value for each kidney section was then determined and plotted as a single data point. Error bars represent SEM from at least 3 independent samples for each genotype and treatment. Statistical significance was assessed using one-way ANOVA analysis with Tukey's multiple comparisons test (comparing control RosaCreERT2+/Ki (24 h post-tamoxifen n=2, 24 h post-γ-irradiation n=9) with Bcl-xfl/fl;RosaCreERT2+/Ki (24 h post-tamoxifen n=6, 24 h post-γ-irradiation n=5) and control RosaCreERT2+/Ki;GFP-Chimeras (24 h post-tamoxifen n=7, End point n=8) with Bcl-xfl/fl;RosaCreERT2+/Ki;GFP-Chimeras (24 h post-tamoxifen n=8, End point n=6) (***p<0.001).
Figure 5 (A, B) Data are presented as % survival and statistical significance was assessed using the Mantel-Cox (Log-rank) test comparing tamoxifen-treated Bcl-xfl/fl;RosaCreERT2+/Ki;GFP-Chimeras (n=25) with Bcl-xfl/fl;RosaCreERT2+/Ki;Bim+/-;GFP-Chimeras (n=25), Bcl-xfl/fl;RosaCreERT2+/Ki;Bim-/-;GFP-Chimeras (n=18), Bcl-xfl/fl;RosaCreERT2+/Ki;Puma+/-;GFP-Chimeras (n=21), Bcl-xfl/fl;RosaCreERT2+/Ki;Puma-/-;GFP-Chimeras (n=15) and Bcl-xfl/fl;RosaCreERT2+/Ki; Bim+/-;Puma+/-;GFP-Chimeras (n=22). *p<0.05, ****p<0.0001. The survival curves for the Bcl-xfl/fl;RosaCreERT2+/Ki;GFP-Chimeras  (C) Histological analysis of H&E-stained sections of the kidneys of sick mice of the indicated genotypes at the indicated time points (dpt = days post-tamoxifen treatment). Pictures are representative of at least 3 mice for each genotype and treatment.  (D-F) (D) Total red blood cell (RBC) count, (E) hematocrit (HCT) and (F) hemoglobin (HGB) content were determined by ADVIA in the blood of sick mice of the indicated genotypes or at the termination of the experiment for healthy controls. Data are presented as mean ±SEM. Each data point represents one individual mouse. Statistical significance was assessed using Student's t-test comparing tamoxifen-treated RosaCreERT2+/Ki;GFP-Chimeras (n=6) with Bcl-xfl/fl;RosaCreERT2+/Ki;GFP-Chimeras (n=21), Bcl-xfl/fl;RosaCreERT2+/Ki;Bim+/-;GFP-Chimeras (n=20), Bcl-xfl/fl;RosaCreERT2+/Ki;Bim-/-;GFP-Chimeras (n=12), Bcl-xfl/fl;RosaCreERT2+/Ki;Puma+/-;GFP-Chimeras (n=15), Bcl-xfl/fl;RosaCreERT2+/Ki;Puma-/-;GFP-Chimeras (n=10) and Bcl-xfl/fl;RosaCreERT2+/Ki; Bim+/-;Puma+/-;GFP-Chimeras (n=17) ; ***p<0.001; ****p<0.0001. Data for the Bcl-xfl/fl;RosaCreERT2+/Ki;GFP-Chimeras and RosaCreERT2+/Ki;GFP-Chimeras
Figure 6 (A) GFP-Chimeras were treated with the BCL-XL inhibitor A1331852 (n=8, 5 doses by oral gavage, 25 mg/kg body weight each dose). Control groups (n=8 each group) include untreated GFP-Chimeras and un-irradiated C57BL/6-Ly5.1 (wild-type) mice treated with the BCL-XL inhibitor A1331852 or left untreated. Mice were monitored for up to 200 days post-treatment (dpt). Data are presented as % survival.  (B) C57BL/6-Ly5.1 (wild-type) mice (females, aged 10 weeks) were treated with Cyclophosphamide (n=7, 150 mg/kg body weight, 1 dose i.v.) or 5-Fluorouracil (5-FU, n=7, 100 mg/kg body weight, 1 dose i.v.) and after 5 days additionally treated with the BCL-XL inhibitor A1331852 (5 doses by oral gavage, 100 mg/kg body weight each dose). Control groups (n=7 each group) include C57BL/6-Ly5.1 (wild-type) mice (females, aged 10 weeks) treated with A1331852 alone, Cyclophosphamide alone, 5-FU alone or left untreated. Mice were monitored for up to 150 days post-treatment (dpt). Data are presented as % survival.  (C) C57BL/6-Ly5.1 (wild-type) mice (females, aged 10 weeks) were treated with the BCL-XL inhibitor A1331852 (n=7, 5 doses by oral gavage, 25 or 100 mg/kg body weight each dose). Total platelet counts were determined by ADVIA at the indicated time points. Data are presented as mean ±SEM. Each data point represents one individual mouse. Statistical significance was assessed using one-way ANOVA analysis with Tukey's multiple comparisons test comparing untreated and drug-treated mice within each group (**p<0.01, ***p<0.001, ****p>0.0001).  Total red blood cell count (RBC), platelet count, hemoglobin (HGB) content and hematocrit (HCT) were determined by ADVIA as indicated in the blood of drug-treated GFP-Chimeras or control mice (n=8 each group) at the termination of the experiment Data are presented as mean ±SEM. Each data point represents one individual mouse. Statistical significance was assessed using one-way ANOVA analysis with Tukey's multiple comparisons test comparing untreated and drug-treated mice within each group (*p<0.05, **p<0.01, ***p<0.001, ****p>0.0001).  Total red blood cell count (RBC), platelet count, hemoglobin (HGB) content and hematocrit (HCT) were determined by ADVIA as indicated in the blood of in drug-treated C57BL/6-Ly5.1 (wild-type) mice or control mice at the indicated time points (n=7 each group). Data are presented as mean ±SEM. Each data point represents one individual mouse. Statistical significance was assessed using one-way ANOVA analysis with Tukey's multiple comparisons test comparing untreated and drug-treated mice within each group (*p<0.05, **p<0.01, ***p<0.001, ****p>0.0001).
Figure 7 (A) GFP-Chimeras were treated with the BCL-XL inhibitor A1331852 (n=8, 5 doses by oral gavage, 25 mg/kg body weight each dose). Control groups (n=8 each group) include untreated GFP-Chimera and un-irradiated C57BL/6-Ly5.1 (wild-type) mice treated with the BCL-XL inhibitor A1331852 or left untreated. Kidney (left panel) and spleen weights (right panel) were measured in drug-treated GFP-Chimeras or control mice at the termination of the experiment (n=8 each group). Data are presented as mean ±SEM. Each data point represents one individual mouse. Statistical significance was assessed using one-way ANOVA analysis with Tukey's multiple comparisons test. No statistically significant differences were observed.  (B) C57BL/6-Ly5.1 (wild-type) mice (females, aged 10 weeks) were treated with Cyclophosphamide (n=7, 150 mg/kg body weight, 1 dose i.v.) or 5-Fluorouracil (5-FU, n=7, 100 mg/kg body weight, 1 dose i.v.) and after 5 days additionally treated with the BCL-XL inhibitor A1331852 (5 doses by oral gavage, 100 mg/kg body weight each dose). Control groups (n=7 each group) include C57BL/6-Ly5.1 (wild-type) mice (females, aged 10 weeks) treated with the BCL-XL inhibitor A1331852 alone, Cyclophosphamide alone, 5-FU alone or left untreated. Kidney (left panel) and spleen weights (right panel) were measured in drug-treated mice or control mice at the termination of the experiment (n=7 each group). Data are presented as mean ±SEM. Each data point represents one individual mouse. Statistical significance was assessed using one-way ANOVA analysis with Tukey's multiple comparisons test. No statistically significant differences were observed.  (C) Histological analysis of H&E-stained sections of the kidneys of drug-treated GFP-Chimeras or control mice at the termination of the experiment. Pictures are representative of at least 3 mice for each treatment group.  (D) Histological analysis of H&E-stained sections of the kidneys of drug-treated mice or control untreated mice at the termination of the experiment. Pictures are representative of at least 3 mice for each treatment group.
Figure 2The location of causal candidate LN1 is indicated as is the position of the linked TAS rs2303565 associated with ALS. The structures of transcripts indicate the exons (blue) and introns (gray with >). The color key indicates the chromatin states as defined by ChromHMM analysis of epigenomic data from the NIH Roadmap Epigenomics Consortium. Chromatin states are shown for brain tissues, neural progenitor cells (NPCs), and a variety of other tissues. Next to the color key, NCBI dbVar ID for TE, SNP ID for TAS, genic location of TAS, TE name, and r2 value are shown. * r2 value verified by genotyping PCR. ChIP-seq peaks of transcription factors from ENCODE are shown below ChromHMM chromatin states.
Figure 3The location of causal candidate LN2 is indicated as is the position of the linked TAS rs11759769 associated with migraine disorder. The structures of transcripts indicate the exons (blue) and introns (gray with >). L1MA5 indicates the position of an L1 element fixed in the genome. The color key indicates the chromatin states as defined by ChromHMM analysis of epigenomic data from the NIH Roadmap Epigenomics Consortium. Chromatin states are shown for brain tissues, NPCs, and a variety of other tissues. Next to the color key, NCBI dbVar ID for TE, SNP ID for TAS, genic location of TAS, TE name, and r2 value are shown. * r2 value verified by genotyping PCR. ChIP-seq peaks of transcription factors from ENCODE are shown below ChromHMM chromatin states and peaks for two transcription factors known for heterochromatin formation located around the site of LN2 insertion are labeled.
Figure 4The location of causal candidate LN16 is indicated as is the position of the linked TAS rs12185268 associated with PD. The structures of transcripts indicate the exons (blue) and introns (gray with >). The color key indicates the chromatin states as defined by ChromHMM analysis of epigenomic data from the NIH Roadmap Epigenomics Consortium. Chromatin states are shown for brain tissues, NPCs, and a variety of other tissues. Next to the color key, NCBI dbVar ID for TE, SNP ID for TAS, genic location of TAS, TE name, and r2 value are shown. * r2 value verified by genotyping PCR. ChIP-seq peaks of transcription factors from ENCODE are shown below ChromHMM chromatin states. The position of the LN16 TE is enlarged in an inset.
Figure 5Approximately 500 bp of genomic sequence, with and without Alu inserts was assayed for transcription activity when inserted upstream of a minimal promoter in a luciferase vector transfected into NCRM1 cells. The orientation (white triangle) of the Alu and its associated genomic sequence (yellow box) is diagramed at top. Forward orientation (Fwd) indicates plus strand sequence of the reference genome. The minimal promoter is symbolized by a black rectangle with an arrow indicating the direction of transcription. The causal TE candidates tested, their linked TASs, and their ChromHMM status are indicated at right. Luciferase values are normalized to transfection efficiency (renilla) for each of four independent transfections and subsequently to luciferase expression from the minimal promoter alone. Mean values (± SD) were determined, and statistical significance was analyzed by Student's two-sample unequal variance t-Test with a one-tailed distribution. Each locus was examined in four independent experiments and a representative dataset is shown here. ☨: p < 0.05 in two experiments and p > 0.05 in two experiments. ns: P > 0.05.
Figure 1B. TBK1/IKKε phosphorylate mTOR S2159 directly in vitro: In vitro kinase (IVK) assays with recombinant (re) active TBK1 or IKKε [50 ng] (Invitrogen) and recombinant GST-mTOR substrate [200 ng] for 30 min at 30oC. Reactions were pre-incubated on ice 30 min with amlexanox [500, 250 or 50 μM], BX-795 [10 μM] or MRT-67307 [10 μM] and immunoblotted (IB) as indicated.C. TBK1/IKKε phosphorylate full-length mTOR on S2159: Myc-mTOR wild type (WT) and S2159A were immunoprecipitated (IP) from transfected HEK293 cells and incubated with re-TBK1 or re-IKKε. IVK assays were performed as above and immunoblotted (IB) as indicated.J. LPS increases mTOR P-S2159 in vivo: Mice (C57BL6, 6 wks old) were injected intraperitoneally with PBS or LPS [1 mg/kg-BW] (2 hr.). mTOR was immunoprecipitated from spleen tissue, and IPs and WCL were immunoblotted as indicated. The graph depicts levels of mTOR P-S2159 relative to total mTOR in spleen tissue -/+ LPS. n=8 from 3 independent experiments +/- SD. *p=.004 relative to PBS treated control mice by paired t-test (two-tailed).E. TBK1 and IKKε overexpression increases mTOR P-S2159 within mTORC1: HEK293-TLR3 cells were co-transfected with Flag-TBK1 or Flag-IKKε, Myc-mTOR, and HA-raptor. HA-raptor immunoprecipitates and whole cell lysates (WCL) were immunoblotted (IB) as indicated.F. Flag-TBK1 and Flag-IKKε co-immunoprecipitate with endogenous mTORC1: HEK293T cells were transfected with Flag-TBK1 or Flag-IKKε wild type (+) or kinase dead (KD). Endogenous raptor immunoprecipitates and WCL were immunoblotted (IB) as indicated.G. mTOR is phosphorylated on S2159 in wild type but not TBK1 null MEFs: TBK1+/+ and TBK1-/- MEFs were serum starved (20 hr.) and stimulated -/+ EGF [25 ng/mL]. WCL was immunoblotted (IB) as indicated.H. The TBK1- and IKKε-activating agonists poly(I:C) and LPS increase mTOR P-S2159 in a BX-795 sensitive manner in cultured macrophages: RAW264.7 macrophages were pre-treated with BX-795 [10 μM] (2 hr.) and stimulated -/+ poly (I:C) [30 μg/ml] or LPS [100 ng/ml] (60 min).I. Poly(I:C) and LPS increase mTOR P-S2159 in primary bone marrow-derived macrophages (BMDMs): BMDMs were stimulated -/+ poly (I:C) [30 μg/ml] or LPS [100 ng/ml] (60 min).J. LPS increases mTOR P-S2159 in vivo: Mice (C57BL6, 6 wks old) were injected intraperitoneally with PBS or LPS [1 mg/kg-BW] (2 hr.). mTOR was immunoprecipitated from spleen tissue, and IPs and WCL were immunoblotted as indicated. The graph depicts levels of mTOR P-S2159 relative to total mTOR in spleen tissue -/+ LPS. n=8 from 3 independent experiments +/- SD. *p=.004 relative to PBS treated control mice by paired t-test (two-tailed).
Figure 2A. EGF increases mTORC1 signaling in a TBK1-dependent manner: TBK1+/+ and TBK1-/- MEFs were serum starved (20 hr.), pre-treated with Ku-0063794 [1 μM] (30 min), and stimulated -/+ EGF [25 ng/mL] (0-60 min). Whole cell lysate (WCL) were immunoblotted (IB) as indicated.B. Ectopic expression of TBK1 rescues mTORC1 signaling in TBK1-/- MEFs: TBK1-/- MEFs were transiently transfected with vector control (V), wild type (WT), or kinase dead (KD) Flag-TBK1, serum-starved (20 hr.), and analyzed as above.C. EGF-receptor signaling remains intact in TBK1-/- MEFs: TBK1+/+ and TBK1-/- MEFs were serum-starved (20 hr.) and stimulated -/+EGF [25 ng/mL] (0, 5, or 15 min).D. TBK1 is required for EGF-stimulated mTORC1 and mTORC2 signaling: TBK1+/+ and TBK1-/- MEFs were stimulated with EGF as in A.E. TBK1 is not required for insulin-stimulated mTORC1 signaling: TBK1+/+ and TBK1-/- MEFs were stimulated with insulin as in C.F. Side-by-side comparison of EGF- vs. insulin-stimulated mTORC1 and mTORC2 signaling in TBK1+/+ vs. TBK1-/- MEFs: MEFs were treated as in C.G. Pharmacologic TBK1 inhibition reduces EGF-induced mTORC1 signaling: HEK293 cells were serum starved (20 hr.), pre-treated with Ku-0063794 [1 μM] (30 min) or amlexanox [50 μM] (2 hr.), and stimulated -/+EGF [25 ng/mL] (30 min) or insulin (INS) [100 nM] (30 min).H. EGF but not insulin increases mTOR P-S2159: TBK1+/+ vs. TBK1-/- MEFs: MEFs were treated as in C.
Figure 3A. mTORC1 signaling remains intact in MEFs lacking mTORC2 function: Rictor-/- MEFs stably expressing vector control (V) or rescued stably with HA-rictor were serum starved (20 hr.), pre-treated with Ku-0063794 [1 μM] (30 min), and stimulated -/+epidermal growth factor (EGF) [25 ng/mL] (30 min). Whole cell lysate (WCL) were immunoblotted as indicated.B. TBK1 is required for mTORC1 signaling in MEFs with reduced Akt S473 phosphorylation: TBK1 expression was reduced using lentivirally-delivered TBK1 shRNA in Rictor-/- MEFs stably expressing vector control (V). Cells were EGF stimulated as above, except Torin1 [100 nM] was used to inhibit mTOR.