EMBO/sd-character-level-ner huggingface dataset:
mTOR S2159 phosphorylation promotes mTORC1 signaling, IRF3 nuclear translocation, and IFNβ production upon TLR3 activation in primary macrophages.Figure 8A. Reduced TLR3-induced mTORC1 signaling in mTOR S2159A knock-in primary macrophages (mTORA/A) relative to wild type (mTOR+/+): BMDMs were pre-treated with Torin 1 [100 nM] (30 min) and stimulated -/+ poly (I:C) [30 μg/ml] for 30 min. Whole cells lysate (WCL) was immunoblotted as indicated. The graph quantitates three independent experiments each with n=1 (n=3 total). The level of P-S6K1-T389 normalized to total protein in mTOR+/+ macrophages stimulated with poly (I:C) was set at 100%. *The confidence interval at 95% (52.7-59.1) indicates statistical significance between mTOR+/+ vs. mTORA/A BMDMs. B. Reduced TLR4-induced mTORC1 signaling in mTORA/A primary macrophages: BMDMs were treated as in A except stimulated -/+ LPS [100 ng/mL] (30 min). The graph quantitates three independent experiments each with n=1 (n=3 total). The level of P-S6K1-T389 normalized to total protein in mTOR+/+ macrophages stimulated with LPS was set at 100%. *The confidence interval at 95% (58.6-71.2%) indicates statistical significance between mTOR+/+ vs. mTORA/A BMDMs. C. Reduced TLR3-stimulated IFNβ protein production in mTORA/A primary macrophages: BMDMs were serum starved for 6 hr. and stimulated -/+ poly (I:C) [30 μg/ml] for 6 hr. The secretion of IFNβ was measured by ELISA. Results represent the mean +/- SEM from three independent experiments, n=8 samples total. *p=.0002 relative to mTOR+/+ + Poly(I:C) by unpaired t-test (equal variance; two-tailed). **p=.0001 relative to mTOR+/+ + Poly(I:C) by paired t-test (two-tailed). D. Reduced TLR3-stimulated IRF3 nuclear translocation in mTORA/A primary macrophages: BMDMs were treated -/+ poly (I:C) for 3 hr as in A, fixed, and processed for confocal immunofluorescence microscopy using an anti-IRF3-Alexa 488 antibody and Dapi stain. The graph represents the mean +/- SEM of at least 270 cells total from four independent experiments. *p=.001 relative to mTOR+/+ no Poly(I:C) by unpaired t-test (equal variance; two-tailed). **p=.02 relative to mTOR+/+ + Poly(I:C) by unpaired t-test (equal variance; two-tailed). Scale bar = 10 μM. E. Model: TLR3 and TLR4 signaling increase TBK1-mediated mTOR S2159 phosphorylation, resulting in increased mTORC1 catalytic activity, mTORC1 downstream signaling, IRF3 nuclear translocation, and IFNβ production.
Figure 1A. 3-OH-C10:0-induced ROS burst is suppressed by A23 and K252a. Two-week-old Col-0 seedlings were treated with 2μM K252a or 40 μM A23. One hour later, 1 μM 3-OH-C10:0 was used to trigger the ROS burst, and the photo count was recorded by a luminometer. Mock is solvent control. RLU, relative light unit. Values are means ± SD (n=3 biological replicates). B. A23 and K252a suppress 3-OH-C10:0-induced PTI marker gene expression. Treatments with 3-OH-C10:0 and inhibitors were same as in (A), and the plant leaves were collected for RT-qPCR 4 hrs later. Values are means ± SD (n=3 biological replicates). (ANOVA, P <0.01). C. MAPK cascade activation was inhibited by A23. Treatments with 3-OH-C10:0 and A23 were same as in (A). The plant leaves were collected at 0, 5, and 10 min for immunoblotting. Activated MAPKs were detected by immunoblotting with anti-phospho-p44/42 MAPK antibody. The corresponding bands are indicated for MPK3 and MPK6. Ponceau staining of Rubisco was used for estimating equal loading in each lane. The experiment was repeated three times with similar results. D. Tyrosine phosphorylation of LORE in vivo. Two-week-old 35S:LORE-FLAG complementation plants were subjected to immunoprecipitation with anti-FLAG agarose beads. A23 (40 μM) was used to inhibit the protein phosphorylation, and the treatments were same as in (A). Protein abundance of precipitated LORE-FLAG was detected by immunoblotting. The tyrosine phosphorylation was probed by anti-pTyrosine (α-pY) antibody. Similar results were observed in three biological replicates. E. LORE is an active tyrosine kinase. The α-pY antibody was used to probe tyrosine phosphorylation in recombinant protein MBP-tagged LORE-CD (cytosolic domain). The LORE activation loop conserved K516 was mutated to E, and D613 was mutated to V. The bottom gel showing protein abundance stained by Coomassie Brilliant Blue (CBB).
TBC1D15 is dispensable for mitochondria and peroxisome morphologies.Figure 1.(A) Total cell lysates prepared from the indicated HCT116 cell lines were analyzed by immunoblotting. For comparison, different amounts of proteins (1:3 ratio) were applied. An asterisk indicated non-specific crossreactive bands. (B) The indicated cell lines were analyzed by immunofluorescence microscopy using anti-Cytochrome c antibody for mitochondria and anti-PMP70 antibody for peroxisome staining. Images are displayed as z-stacks of 6 confocal slices. Magnified images are also shown for mitochondrial morphologies. Scale bars, 10 μm. (C) Quantification of mitochondrial morphologies in (B). Percentages of cells harboring fragmented, tubular, or elongated mitochondria are shown. Tubular and elongated denote normal tubular mitochondria seen in WT cells and highly connected mitochondrial network, respectively. The error bars represent ±SD from three independent replicates. Over 50 cells were counted in each of three replicate wells.
Figure 3A Overlap between differentially expressed (DEG) and spliced (DAS) genes in RNAi-ASCO and 35S:ASCO as compared to WT. B Scatter plot showing the respective gene expression fold change in RNAi-ASCO and 35S:ASCO lines as compared to WT. Genes showing significant changes in RNAi-ASCO and 35S:ASCO are highlighted as yellow and blue dots, respectively. C Scatter plots showing the respective Percent Spliced In difference (dPSI) in RNAi-ASCO and 35S:ASCO lines as compared to WT. Genes showing significant changes in RNAi-ASCO, 35S:ASCO or in both lines are highlighted as red, green or blue dot, respectively. Gray dots represent all AS events.
Fig.Figure 1The SnRK2 OST1 activates ABFs for expression of a luciferase reporter (LUC) in yeast. Luciferase activity is given on a Log10 scale as relative counts of light emission per second (rcps) normalized to the cell density. The OST1-mediated induction factor is indicated above the columns. In the absence of ABFs, the LUC activity was 3 x 103 rcps. Combining the ABA signaling components ABF2, OST1, the PP2C ABI1, and RCAR11 provides ABA-controlled LUC expression. The reporter is under the control of a minimal promoter that contains ABA-responsive cis elements (ABREs). Core ABA signaling involves the RCAR receptor that inhibits the ABA coreceptor (PP2C) in the presence of ABA and allows the activation of the protein kinase SnRK2. SnRK2 phosphorylates and activates the key transcription factor ABF, which targets promoters containing ABRE. Reporter activity was determined 16 h after exogenous ABA (0.1 mM) administration and referred to samples expressing ABF2 alone.
Figure 2A) Murine small (top) and large (bottom) intestines isolated from control (Cre negative) Lats1flox/flox Lats2flox/flox animals and Villin-CreERt Lats1flox/flox Lats2flox/flox animals treated with tamoxifen to induce homozygous deletion of Lats1/2 (dKO). Immunostaining for YAP and Ki67 shows a gradient of YAP expression along the crypt-villus axis in controls, with nuclear YAP and Ki67 positive cells restricted to the crypt base (representative images from n=5 mice). Tamoxifen treated (3 days i.p.) Villin-CreERt Lats1flox/flox Lats2flox/flox double homozygous mouse intestines show an enlarged crypt compartment after 7 days with strongly nuclear YAP immunostaining in all epithelial cells and an expanded proliferative zone marked by Ki67 positive cells. Note the gradient of YAP expression levels is maintained along the crypt-villus axis (representative images from n=5 mice for each genotype). (Right) Immunostaining for the Paneth cell marker Lyz reveals loss of this marker from the crypt base. (Bottom right) Q-PCR analysis of Wnt pathway target genes reveals that Lats1/2 dKO causes a mild reduction in Lgr5 expression, with complete loss of Olfm4. B) Axin2 mRNA expression was measured by RNAscope (red) and found to be increased in both intensity and uniformity of staining along the crypt-villus axis in Lats1/2 dKO after 3 days post i.p. injection (dpi) with tamoxifen to induce the homozygous deletion of Lats1 and Lats2. At 7dpi, Axin2 mRNA levels remain uniform along the crypt-villus axis in Lats1/2 dKO animals, although their total level has declined compared to 3dpi. C) Schematic diagram showing alteration of the Wnt signalling gradient upon Lats1/2 dKO in the intestine, with crypt expansion being accompanied by an elongation/flattening of the Wnt signalling gradient, whose elevated level gradually declines over time. D) Q-PCR analysis of YAP-TEAD and their target genes Ctgf and Cyr61 reveals that Lats1/2 dKO causes a strong induction of Ctgf and Cyr61 expression, as expected, as well as a strong induction of the common Wnt and YAP target gene CyclinD1.
Figure 4Figure 4. Sequencing analysis of the CRISPR/Cas9-induced disruption of the Tbr2 locus.(A-C) Next-generation DNA sequencing analysis of amplicons containing the Tbr2 locus and 4 off-target loci generated from PaprikaRFP-expressing cells isolated at E15.5 from neocortex of mouse embryos in utero electroporated at E13.5 with plasmids encoding Cas9_T2A_PaprikaRFP and either gTbr2 (black) or control gRNA (Con, white) as in Fig 3 A-D.(A) Quantification of the frequency of mismatches to the mouse genome sequence spanning 30 bp upstream and downstream of the Tbr2 target site and the off-target sites (OT1-4). Data are expressed as percentage of the total number of reads and are the mean of two independent experiments (8 and 5 embryos per condition, respectively).(B, C) Analysis of one of the experiments of (A) for deletions (B) and insertions (C) in the region spanning 80 bp upstream and downstream of the Tbr2 target site upon Cas9/gTbr2 plasmid electroporation.
Figure 5Sublingual infection of WT, Stim1fl/flVav-iCre and Stim1fl/flStim2fl/flVav-iCre mice with 2x107 CFU of C. albicans. (A) Body weight (BW) measured for 7 days and normalized to BW at day 0 of infection. Data are mean ± SEM measured from 4 repeat experiments and 13 WT, 7 Stim1fl/flVav-iCre, 8 Stim1fl/flStim2fl/flVav-iCre mice. Sublingual infection of WT, Stim1fl/flVav-iCre and Stim1fl/flStim2fl/flVav-iCre mice with 2x107 CFU of C. albicans. (B) Quantification of CFU of C. albicans per gram tongue tissue of the indicated mice at day 7 p.i. Data are mean ± SEM from 4 repeat experiments and 12 WT, 7 Stim1fl/flVav-iCre, 8 Stim1fl/flStim2fl/flVav-iCre mice. Sublingual infection of WT, Stim1fl/flVav-iCre and Stim1fl/flStim2fl/flVav-iCre mice with 2x107 CFU of C. albicans. (C) Macroscopic images of the C. albicans-infected tongues of mice at day 7 p.i. Arrows and dashes lines indicate C. albicans infiltrated areas. Sublingual infection of WT, Stim1fl/flVav-iCre and Stim1fl/flStim2fl/flVav-iCre mice with 2x107 CFU of C. albicans. (D) Histological images of H&E (left) and PAS (right) stained tongue tissue at day 7 p.i. Clusters of polymorphonuclear (PMN) cells (neutrophils, white arrows) and Candida (black arrows) are indicated. Sublingual infection of WT, Stim1fl/flVav-iCre and Stim1fl/flStim2fl/flVav-iCre mice with 2x107 CFU of C. albicans. (E) Frequencies of Cd11b+Gr1+ neutrophils in the tongues from the indicated mice at day 7 p.i. Bar graphs represent the mean ± SEM from 3 experiments and 9 WT, 7 Stim1fl/flVav-iCre, 8 Stim1fl/flStim2fl/flVav-iCre mice. (F,G) Cytokine production by CD4+ T cells isolated from submandibular lymph nodes of WT, Stim1fl/flVav-iCre, Stim1fl/flStim2fl/flVav-iCre and Stim1fl/flCd4Cre mice 7 days after sublingual infection with 2x107 CFU C. albicans. Representative contour plots of cytokine production (F) and percentages of CD4+ T cells producing TNF-α, IFN-γ, IL-17A, IL-17F and GM-CSF (G). Data are the mean ± SEM from two independent experiments and 9 WT, 7 Stim1fl/flVav-iCre, 8 Stim1fl/flStim2fl/flVav-iCre mice. Candicidal function of neutrophils depend on SOCE. (H) Coculture of Cd11b+Gr-1+ neutrophils from WT, Stim1fl/flMx1Cre and Stim1fl/flStim2fl/flMx1Cre mice with C.albicans in vitro for 0, 30, 60 and 90 min (MOI 0.5). Percentages of live C.albicans isolated from neutrophils that were lysed at the indicated time points. Data are the mean ± SEM from 6 mice per genotype and 3 independent repeat experiments. ROS production of neutrophils depend on SOCE. (I) ROS production by Cd11b+Gr-1+ neutrophils from WT, Stim1fl/flMx1Cre and Stim1fl/flStim2fl/flMx1Cre mice was measured after loading of cells with dihydrorhodamine 123 and stimulation with 10 nM PMA for 30 minutes. Representative histogram plots and bar graphs indicating the percentages of ROS+ neutrophils normalized to WT neutrophils stimulated with PMA.
RIPK3 complex and necroptosis (A) TNF-induced mRNA expression in indicated L929 cell lines measured via qRT-PCR (mean of three independent experiments ± standard deviation, two-tailed Student's t-test *P<0.Figure 2(A) TNF-induced mRNA expression in indicated L929 cell lines measured via qRT-PCR (mean of three independent experiments ± standard deviation, two-tailed Student's t-test *P<0.05, **P<0.01). (B) Immunoblot after co-immunoprecipitation (IP) of RIPK3. FT, flow through. (C) Relative quantification of A20 in RIPK3-IP fraction (means and statistical significance established for 0, 2, 4.5 and 8 hour time points from three independent experiments ± standard deviation; two-tailed Student's t-test *P<0.05, **P<0.01, n.s., P>0.05). (D) Histogram of volume normalized mRNA copy numbers measured by smFISH of NFκB target genes A20 and IκBα in L929 wildtype (wt) cells treated with TNF (representative data of three independent experiments; two additional independent experiments in Figure EV4H). (E) Fractions of "responder" cells (A20 count > 1 per cell) (mean of all three independent experiments ± standard deviation). (F) Fractional survival of wt cells after the early (<12 hours) and late phase of TNF time course obtained by microscopy (mean of three independent experiments ± standard deviation). (G) A20 mRNA expression via qRT-PCR in TNF-treated wt cells transfected with targeting (siA20) or non-targeting (siCon) siRNA (mean of three independent experiments ± standard deviation; two-tailed Student's t-test *P<0.05). (H) Immunoblot and relative quantification of basal A20 protein after siRNA-knockdown normalized to non-targeting siRNA control (mean of three independent experiments ± standard deviation; two-tailed Student's t-test revealed no statistically significant difference between targeting and non-targeting siRNA treatment, n.s., P>0.05). (I,J) (I) TNF-induced distributions of death times (representative data of three independent experiments), and (J) death rates obtained by live-cell microscopy (mean of three independent experiments ± standard deviation).
SOX9 is involved in liver fibrosis in vivo.Figure 2A Immunohistochemistry for SOX9 (brown) in wild-type (WT) mice following CCl4 or BDL induced fibrosis. SOX9 positive bile ducts (bd) and hepatocytes (arrowheads) indicated. B (Right panel) Immunofluorescence for SOX9 (green) and HNF4α (red) in WT fibrotic mice. Arrowheads (orange/yellow staining) indicate SOX9+/HNF4α+ hepatocytes and star (*; red) indicates SOX-/ HNF4α+ hepatocytes. (Left panel) Immunofluorescence for SOX9 (green nucleus) and CK19 (red cytoplasm) in WT fibrotic mice. Arrowheads (green) indicate SOX9+ hepatocytes and star (*) indicates SOX- hepatocytes identified by DAPI staining in Appendix Figure S1. C Co-localization by immunohistochemistry (top panel) and in situ hybridization (lower panel) for SOX9 (red) and αSMA (brown) in wild-type (WT) mice following CCl4 or BDL induced fibrosis. D Expression of SOX9 protein by immunoblotting of in vivo activated HSCs extracted from WT mice following CCl4 injections compared to olive oil control. E Immunohistochemistry for SOX9 (brown) in control (Sox9fl/fl;RosaCre-) or Sox9-null (Sox9fl/fl;RosaCre+) mice following fibrosis. Bile ducts (bd) and hepatocytes (arrowheads) indicated. SOX9 is absent, even in bile ducts in Sox9-null liver. Size bar, 25 μm (A, B and E), 10 μm (C).
Figure 3Heatmap of RNA-seq data for glycolysis and oxidative stress genes in diploid (2n) and tetraploid (4n) cells grown on YPD or PRE-SPO medium over 24 h. TTC assay results in diploid and tetraploid cells grown on YPD or PRE-SPO medium at 30°C or 37°C, respectively. Cells were grown for 24 h at which point a TTC overlay solution was applied and color allowed to develop for 15 min. Diploid and tetraploid strains were spot inoculated onto YPD or PRE-SPO medium supplemented with the pH indicator bromocresol purple and cultured for 24 h at 30°C or 37°C, respectively. Analysis of metabolic flux in diploid and tetraploid cells following growth for 24 h on YPD or PRE-SPO medium (30°C or 37°C, respectively), using a Seahorse instrument. OCR values represent rates of aerobic respiration while ECAR values represent rates of fermentation. Cell size and mean MitoTracker Green signal intensity in diploid and tetraploid cells grown on YPD medium for 24 h at 30°C (n=100 and n=5 for cell volume and MitoTracker Green measurements, respectively, error bars represent S.E.M.).
TSPO-PET imaging indicates rescue of microglial hyperactivation in Double-/- miceA.Figure 1A. Axial slices as indicated below (A1, A2, A3) show %-TSPO-PET differences between Grn-/-, Trem2-/- or Double-/- mice and WT at the group level. Images adjusted to an MRI template indicate increased microglial activity in the brain of Grn-/- mice (hot color scale), compensated microglial activity in the brain of Double-/- mice and decreased microglial activity in the brain of Trem2 -/- mice (cold color scale), each in contrast against age-matched WT mice. B. Scatter plot illustrates individual mouse TSPO-PET values derived from a whole brain volume of interest. 8-15 female mice per group at an average age of 11.1 ± 1.6 months (Grn-/- (n = 8), Trem2-/- (n = 9), Double-/- (n = 10), WT (n= 15)). Data represent mean ± SD. For statistical analysis one-way ANOVA with Tukey post hoc test was used. Statistical significance was set at *, p < 0.05; **, p < 0.01; ns, not significant.
Figure 2(A) Levels of KRAS, COUP-TFII, LDHA, and phosphorylated S6K (T389) in KRAS-silenced HCT116 and DLD-1 cells. (B) Levels of COUP-TFII, LDHA, and phosphorylated S6K (T389) in COUP-TFII-silenced HCT116 and DLD-1 cells. (C) Effect of lactate on phosphorylated S6K (T389), 4E-BP1 (T37/46), ULK1 (S757) in COUP-TFII-silenced HCT116 cells. (D) Effects of COUP-TFII and the inhibition of LDHA on phosphorylated S6K (T389) and Raptor (S792) in KRAS-silenced HCT116 cells. Asterisk indicates exogenous HA-COUP-TFII. Arrow indicates phosphorylated Raptor (S792). (E) Inhibition of COUP-TFII-silenced HCT116, DLD-1, and MIA PaCa-2 cell proliferation and its recovery by treatment with lactate. Data are expressed as the mean ± SEM of three independent experiments and normalized against values measured in control. *p<0.05, **p<0.01, and ***p<0.001. (F) Clonogenic assay of COUP-TFII-silenced HCT116, DLD-1, and MIA PaCa-2 cells in the presence or absence of lactate.
Figure 5A) Quantitative proteomics study of wild-type (WT) and OTUD1─/─ NCM460 cells. The expression of TFRC and IREB2 were highlighted with red color. Intensity indicates label-free quantification (LFQ) intensity of proteins in mass spectrometry. (B) Wild-type (WT) and OTUD1─/─ NCM460 cells were treated with indicated concentration of H2O2 for 24 hours and cell viability was measured by PI staining (n = 3 biological replicates, mean ± s.e.m., *P < 0.05, **P < 0.01, two-tailed unpaired Student's t-test). (C) CT26 cells stably expressing mock or OTUD1 were treated with 200 μM H2O2 together with various cell death inhibitors containing Fer-1 (10 μM), Z-VAD (25 μM) and nec-1 (20 μM). Cell viability was measured by PI staining n = 3 biological replicates, mean ± s.e.m., ns, not significant (P > 0.05), *P = 0.0119, **P = 0.0015, ***P < 0.001, two-tailed unpaired Student's t-test). (D) CT26 cells stably expressing mock, OTUD1 or OTUD1C320S were treated with Erastin (10 μM) and RSL3 (5 μM), and cell viability was measured by PI staining (n = 3 biological replicates, mean ± s.e.m., ns, not significant (P > 0.05), **P < 0.01, two-tailed unpaired Student's t-test). (E) Wild-type (WT) and OTUD1─/─ NCM460 cells were treated with Erastin (10 μM) and RSL3 (5 μM) for 24 hours and cell viability was measured by PI staining (n = 3 biological replicates, mean ± s.e.m., **P = 0.0082, ***P < 0.001, two-tailed unpaired Student's t-test). F) Intracellular ROS levels in mock, OTUD1 and OTUD1C320S overexpressing CT26 cells treated with Erastin (10 μM), AFC (50 μM) or H2O2 (100 μM) were detected by DCFDA staining. MFI, mean fluorescence intensity (n = 2 biological replicates). (G) Intracellular ROS levels in mock (n = 4 biological replicates), OTUD1 (n = 6 biological replicates) or OTUD1C320S (n = 5 biological replicates) expressing CT26 tumors (mean ± s.e.m., *P = 0.0189, **P = 0.0086, two-tailed unpaired Student's t-test) were detected by DCFDA staining. MFI, mean fluorescence intensity. (H) Schematic diagram of subcutaneous tumor treatment with nanoparticles-loading Vitamin E (NP-VE). BALB/c mice were transplanted with 1×106 mock or OTUD1 expressing CT26 cells. 50uL of 5M NP-VE was intratumorally injected every other day as shown. (I) Tumor volume (mock, n = 4 biological replicates; mock + NP-VE, n = 6 biological replicates; OTUD1, n = 4 biological replicates; OTUD1 + NP-VE, n = 6 biological replicates Flow cytometric analysis of DCFDA (n = 4 biological replicates, mean ± s.e.m., ***P < 0.001, two-tailed unpaired Student's t-test) of mock or OTUD1 expressing CT26 tumors treated with or without NP-VE. MFI, mean fluorescence intensity. Flow cytometric analysis of the percentage of tumor infiltrating CD8+ T cells (n = 4 biological replicates, mean ± s.e.m., *P = 0.0433, ***P = 0.0001, two-tailed unpaired Student's t-test) of mock or OTUD1 expressing CT26 tumors treated with or without NP-VE.