EMBO/soda-nel huggingface dataset:
mTORC2 sustains thermogenesis via Aktinduced glucose uptake and glycolysis in BAT (2015)
Figure 6

Figure 6. Restoration of glucose uptake or Akt signaling suppresses the thermogenic defect in AdRiKO mice. (A) HKII mRNA expression level in BAT of AdRiKO and control mice infected with either AAV9-HKII or AAV9-empty (n=8/group). (B) Cold-induced 2-Deoxyglucose-6-phosphate (2DG6P) accumulation in BAT of AdRiKO and control mice infected with either AAV9-HKII or AAV9-empty housed at 4°C for 4h (n=8/group). (C) Body temperature of AdRiKO and control mice infected with either AAV9-HKII or AAV9-empty housed at 22°C (n=8/group). (D) Body temperature upon cold exposure of AdRiKO and control mice infected with either AAV9-HKII or AAV9-empty. The left panel represents body temperature after each hour of cold exposure while the right panel represents body temperature as a bar graph for the 3h cold exposure time point (n=8/group). a=significant difference between AdRiKO and control mice infected with AAV9-empty, b= significant difference between AdRiKO and control mice infected with AAV9-HKII, d= significant difference between AdRiKO mice infected with AAV9-empty and AAV9-HKII. (E) Immunoblot analysis of BAT from AdRiKO and control mice infected with either AAV8-Akt2S474D or AAV8-empty (n=6/group, each lane represents a mix of 3 mice). (F) Body temperature of AdRiKO and control mice infected with either AAV8-Akt2S474D or AAV8-empty housed at 22°C (n=11/group). (G) Body temperature upon cold exposure of AdRiKO and control mice infected with either AAV8-Akt2S474D or AAV8-empty. The left panel represents body temperature after each hour of cold exposure while the right panel represents body temperature as a bar graph for the 3h cold exposure time point (n=11/group). a=significant difference between AdRiKO and control mice infected with AAV8-empty, b= significant difference between AdRiKO and control mice infected with AAV8-Akt2S474D, d= significant difference between AdRiKO mice infected with AAV8-empty and AAV8-Akt2S474D. (H) Cold-induced 2-Deoxyglucose-6-phosphate (2DG6P) accumulation in BAT of AdRiKO and control mice infected with either AAV8-Akt2S474D or AAV8-empty housed at 4°C for 4h (n=7 (control AAV8-null), n=6 (AdRiKO AAV8-null), n=6 (control AAV8-AktS474D), n=6 (AdRiKO AAV8-AktS474D)). Data represent mean ± SEM. Statistically significant differences between AdRiKO and control mice were determined with unpaired Student’s t-test and are indicated with asterisks (*=p<0.05; **=p<0.01, ***=p<0.001). Statistically significant differences between viruses were determined with unpaired Student’s t-test and are indicated with a number sign (#=p<0.05; ##=p<0.01; ###=p<0.001). The exact p-value for each significant difference can be found in Appendix Table S2.
Glucose uptake to guard cells via STP transporters provides carbon sources for stomatal opening and plant growth (2020)
Figure 1

Figure 1.

Stomatal function is impaired in stp1stp4 plants.

Representative false colour images of leaf surface temperature captured by a thermal camera from WT, stp1-1, stp4-1, stp1stp4, stp13, stp1stp13 and stp4stp13 plants.

Normalized leaf surface temperature over the phenotyping period. Data shown are means ± SEM; n = 10 per genotype and time point.

Whole-plant recordings of changes in stomatal conductance (gs) from WT, stp1-1, stp4-1 and stp1stp4 plants. Data shown are means ± SEM; n ≥ 3 per genotype.

Whole-plant recordings of changes in stomatal conductance (gs) from self-grafted donor lines (WT/WT, stp1stp4/stp1stp4) and reciprocal grafting of shoot/root (WT/stp1stp4, stp1stp4/WT) plants. Data shown are means ± SEM; n = 3 per genotype.

Data information: (C, D) Plants have been illuminated with 150 µmol m-2 s-1 white light after the end of the night (EoN) under ambient-air CO2 concentrations. (B) Asterisk (*) indicates significant statistical difference between WT and stp plants for P < 0.05 determined by one-way ANOVA with post hoc Tukey's test. (C, D) Different letters indicate significant statistical differences amongst genotypes for the given time point for P < 0.05 determined by one-way ANOVA with post hoc Tukey's test.
Glucose uptake to guard cells via STP transporters provides carbon sources for stomatal opening and plant growth (2020)
Figure 2

Figure 2.

stp1stp4 plants have low levels of hexoses and are devoid of starch in guard cells.

Content of soluble sugars in guard cell-enriched epidermal peels of WT and stp1stp4 plants at the end of the night (EoN) and after 40 min of illumination with white light at 150 µmol m-2 s-1 following the EoN. Data for two independent experiments are shown; means ± SEM; n ≥ 7 per genotype and time point.

Representative confocal laser microscopy images of propidium iodide-stained guard cell starch granules of intact leaves of WT, stp1-1, stp4-1, stp1stp4, suc1 and suc3 plants. Scale bar, 10 µm.

Starch dynamics in guard cells of intact leaves of WT, stp1-1, stp4-1, stp1stp4, suc1 and suc3 plants at the end of the night (EoN) and after one and three hours of illumination with 150 µmol m-2 s-1 of white light. Data for three independent experiments are shown; means ± SEM; n = 120 individual guard cells per genotype and time point.

Data information: (A) Different letters indicate significant statistical differences amongst time points for the given genotype. Asterisk (*) indicates significant statistical difference between genotypes for the given time point for P< 0.05 determined by one-way ANOVA with post hoc Tukey's test. (C) Different letters indicate significant statistical differences amongst genotypes for the given time point. Asterisk (*) indicates significant statistical difference amongst time points for the given genotypes for P < 0.05 determined by one-way ANOVA with post hoc Tukey's test.
Glucose uptake to guard cells via STP transporters provides carbon sources for stomatal opening and plant growth (2020)
Figure 3

Figure 3.

stp1stp4 plants have reduced photosynthetic growth.

Representative false colour images of photosystem II (PSII) operating efficiency (ΦPSII) captured by chlorophyll fluorescence imaging from WT, stp1-1, stp4-1 and stp1stp4 plants. ΦPSII was measured at a photosynthetically active radiation (PAR) of 440 µmol m-2 s-1.

ΦPSII quantified over the phenotyping period. Data shown are means ± SEM; n = 10 per genotype and time point.

Whole-plant recordings of changes in CO2 assimilation (A) from WT, stp1-1, stp4-1 and stp1stp4 plants. Data shown are means ± SEM; n ≥ 3 per genotype.

Whole-plant recordings of changes in CO2 assimilation (A) from self-grafted donor lines (WT/WT, stp1stp4/stp1stp4) and reciprocal grafting of shoot/root (WT/stp1stp4, stp1stp4/WT) plants. Data shown are means ± SEM; n = 3 per genotype.

Quantification of leaf glucose (Glc). Data shown are means ± SEM; n = 8 per genotype and time point.

Quantification of leaf fructose (Fru). Data shown are means ± SEM; n = 8 per genotype and time point.

Quantification of leaf sucrose (Suc). Data shown are means ± SEM; n = 8 per genotype and time point.

Quantification of leaf starch. Data shown are means ± SEM; n = 8 per genotype and time point.

Representative Red Green Blue (RGB) images of 3-(day 0) and 4-week-old (day 7) WT, stp1-1, stp4-1 and stp1stp4 plants.

Projected rosette area over the phenotyping period. Data shown are means ± SEM; n = 10 per genotype and time point.

Data information: (B and J) Asterisk (*) indicates significant statistical difference between WT and stp plants for P < 0.05 determined by one-way ANOVA with post hoc Tukey's test. (C and D) Plants have been illuminated with 150 μmol m-2 s-1 white light after the end of the night (EoN) under ambient-air CO2 concentrations. Different letters indicate significant statistical differences amongst genotypes for the given time point for P < 0.05 determined by one-way ANOVA with post hoc Tukey's test. (E - H) Metabolites are from entire rosettes of WT, stp1-1, stp4-1 and stp1stp4 plants at the end of the night (EoN) and end of the day (EoD) in a 12-h-light/12-h-dark cycle. FW, fresh weight. Asterisk (*) indicates significant statistical difference between time points for the given genotype. Different letters indicate significant statistical differences amongst genotypes for the given time point for P < 0.05 determined by one-way ANOVA with post hoc Tukey's test.
Glucose uptake to guard cells via STP transporters provides carbon sources for stomatal opening and plant growth (2020)
Figure 4

Figure 4.

Elevated levels of CO2 rescue the stp1stp4 photosynthetic growth phenotype.

Photosynthetic CO2 assimilation (A) in dark-adapted WT and stp1stp4 plants in response to a step increase in CO2 concentrations from 0 to 1000 ppm. Data shown are means ± SEM; n=3 per genotype.

Quantification of leaf glucose (Glc). Data for two independent experiments are shown; means ± SEM; n ≥ 17 per genotype and time point.

Quantification of leaf fructose (Fru). Data for two independent experiments are shown; means ± SEM; n ≥ 17 per genotype and time point.

Quantification of leaf sucrose (Suc). Data for two independent experiments are shown; means ± SEM; n ≥ 17 per genotype and time point.

Quantification of leaf starch. Data for two independent experiments are shown; means ± SEM; n ≥ 17 per genotype and time point.

Representative Red Green Blue (RGB) images of 3-(day 0) and 4-week-old (day 6) WT and stp1stp4 plants grown under 600 ppm CO2.

Projected rosette area over the phenotyping period. Data shown are from two independent experiments; means ± SEM; n ≥ 24 per genotype and time point.

Representative confocal laser microscopy images of propidium iodide-stained guard cell starch granules of intact leaves of WT and stp1stp4 plants grown under 600 ppm CO2. Scale bar, 10 µm.

Starch dynamics in guard cells of intact leaves of WT and stp1stp4 plants grown under 600 ppm CO2 at the end of the night (EoN) and after one and three hours of illumination with 150 µmol m-2 s-1 of white light. Data for two independent experiments are shown; means ± SEM; n = 80 individual guard cells per genotype and time point.

Data information: (B - E) Metabolites are from entire rosettes of WT and stp1stp4 plants at the end of the night (EoN) and end of the day (EoD) in a 12-h-light/12-h-dark cycle. Plants were grown under 600 ppm CO2. FW, fresh weight. Asterisk (*) indicates significant statistical difference between time points for the given genotype. Different letters indicate significant statistical differences amongst genotypes for the given time point for P < 0.05 determined by one-way ANOVA with post hoc Tukey's test. (G) Asterisk (*) indicates significant statistical difference between genotypes for the given time point for P < 0.05 determined by one-way ANOVA with post hoc Tukey's test. (I) Different letters indicate significant statistical differences amongst time points for the given genotype for P < 0.05 determined by one-way ANOVA with post hoc Tukey's test.

Expanded View Figure Legends
Elp3-mediated codon-dependent translation promotes mTORC2 activation and regulates macrophage polarization (2022)
Figure 1

Figure 1: Elp3 negatively regulates M1 macrophage polarization.

A Generation of myeloid cells genetically inactivated for Elp3. Elp3 mRNA and protein levels were assessed by Real-Time PCR with extracts isolated from peritoneal macrophages of Elp3Control and Elp3∆Mye mice. mRNA levels in Elp3Control mice were set to 1 and levels in other experimental conditions were relative to that after normalization with Gapdh mRNA levels (n = 3 mice; mean +/- SD, Student T test, ***= p< 0.001). An anti-Elp3 western blot (WB) is also illustrated. The anti-Gapdh blot is shown for normalization purposes.

B Myeloid cells lacking Elp3 show decreased levels of mcm5S2 U34 tRNA modifications. Multiple tRNA modifications in BMDMs from both Elp3Control and Elp3∆Mye mice were quantified by Mass Spectrometry (see methods for details).

C and D Defective mTORC2 signaling upon Elp3 deficiency in myeloid cells. Peritoneal macrophages from Elp3Control and Elp3∆Mye mice were isolated and cultured ex-vivo. They were untreated or stimulated with a combination of LPS (100 ng/ml) and IFNγ (50 ng/ml) for the indicated periods of time and the resulting cell extracts were subjected to WB analyses.

E Enhanced mRNA levels of pro-inflammatory cytokines upon Elp3 inactivation in myeloid cells subjected to LPS or to both LPS and IFNγ stimulations. Peritoneal macrophage from Elp3Control or Elp3∆Mye mice were unstimulated (« Control ») or treated with IFNγ (50 ng/ml), LPS (100 ng/ml) or with both IFNγ and LPS for 24 hours and the resulting mRNAs of the listed pro-inflammatory cytokines were assessed by Real Time PCR. mRNA levels in Elp3Control mice were set to 1 and levels in other experimental conditions were relative to that after normalization with Gapdh mRNA levels (n = 6 mice; mean +/- SD, Student T test, **= p<0.01, *= p< 0.05).

F mTORC2 activation by M1 polarization signals relies on Ctu2. Control and Ctu2-depleted bone marrow-derived macrophages (BMDMs) (SiRNA-mediated depletions) were treated or not with IFNγ (50 ng/ml) and LPS (100 ng/ml) and the resulting cell extracts were subjected to WB analyses.

G Ctu2 deficiency potentiates M1 macrophage polarization. Control and Ctu2-depleted BMDMs were treated or not with IFNγ (50 ng/ml) and LPS (100 ng/ml) for 24 hours and the resulting mRNAs of the listed pro-inflammatory cytokines were assessed by Real Time PCR. mRNA levels in control BMDMs were set to 1 and levels in other experimental conditions were relative to that after normalization with Gapdh mRNA levels. Experiments were conducted in triplicates (mean +/- SD, Student T test, *= p< 0.05, **= p< 0.01, ***= p< 0.001).
Elp3-mediated codon-dependent translation promotes mTORC2 activation and regulates macrophage polarization (2022)
Figure 2

Figure 2: Elp3 expression in myeloid cells protects from intestinal damage in an experimental model of colitis.

A Decreased survival of mice lacking Elp3 expression in myeloid cells upon DSS administration (n = 10 mice per genotype; mean +/- SD, Student T test, *= p< 0.05).

B and C Elp3 deficiency in myeloid cells exacerbates the weight loss and the decrease of colon length upon DSS administration (n = 7 mice; mean +/- SD, **= p< 0.01).

D Elp3 deficiency in myeloid cells potentiates the production of cytokines and chemokines upon DSS administration. mRNA levels of the indicated candidates in Elp3Control mice were set to 1 and levels in other experimental conditions were relative to that after normalization with Gapdh mRNA levels (n = 5 mice per genotype; mean +/- SD, Student T test, *= p< 0.05, **= p< 0.01, ***= p< 0.001).

E More severe histological score in mice lacking Elp3 in myeloid cells upon DSS administration. Representative intestinal crypts from the indicated genotypes are illustrated. The histological score was established as described in methods (n = 5 mice per genotype; mean +/- SD, Student T test, *= p< 0.05).

F Increased infiltration of iNOS+/CD68+ cells upon DSS administration in Elp3∆Mye mice. The percentage of iNOS+ in CD68+ cells was established in both genotypes treated with DSS (n = 7 mice per genotype; mean +/- SD, Student T test, **= p< 0.01). Anti-iNOS and -CD68 immunofluorescence analyses carried out in intestinal crypts from both genotypes are illustrated.

G Patients suffering from ulcerative colitis (UC) show reduced levels of both Elp1 and Elp3. The central band represents the mean value of relative expression in the investigated cohort. Boxes represent 75th and 25th percentile. Whiskers represent maximum and minimum values before the upper and lower fence, respectively (**= p< 0.01, ***= p<0.001) (GSE9452-GEO DataSets, Definition of an ulcerative colitis pre-inflammatory state).
Elp3-mediated codon-dependent translation promotes mTORC2 activation and regulates macrophage polarization (2022)
Figure 4

Figure 4: The transcriptomic reprogramming during M2 macrophage polarization relies on Elp3.

A Elp3 deficiency potentiates the expression of genes induced by LPS-dependent pathways and impairs a Pparγ-dependent transcriptional signature. On the top, RNA-Seq combined with GSEA were carried out with total RNAs of BMDMs from Elp3Control and Elp3∆Mye mice. At the bottom, the figure shows some GSEA analyses carried out with total RNAs of BMDMs from Elp3Control and Elp3∆Mye mice treated with IL-4 ex-vivo.

B Defective induction of Pparγ by IL-4/IL-13 upon Elp3 deficiency. Cell extracts from peritoneal macrophages of Elp3Control and Elp3∆Mye mice treated or not with IL-4/IL-13 were subjected to WB analyses.

C Elp3 deficiency impairs the IL-4-dependent transcriptional signature. Cell extracts from BMDMs of Elp3Control and Elp3∆Mye mice treated or not with IL-4 for 2 hours were subjected to RNA-Seq experiments. A heatmap is illustrated to show the defective induction of 50 genes by IL-4 upon Elp3 deficiency.

D Defective Irf4 expression upon IL-4 stimulation in peritoneal macrophages lacking Elp3 expression. Irf4 mRNA levels in peritoneal macrophages from untreated Elp3Control mice were set to 1 and levels in other experimental conditions were relative to that after normalization with Gapdh mRNA levels (n = 3 mice; mean +/- SD, Student T test, **= p< 0.01).

E The IL-4-dependent transcriptomic signature requires Elp3. A volcano plot illustrating changes in mRNA levels in IL-4-stimulated BMDMs (2 hours) from Elp3Control and Elp3∆Mye mice is shown. F Identification of candidates whose protein but not mRNA levels are decreased in IL-4-treated BMDMs lacking Elp3. A volcano plot of correlation between changes in protein and mRNA levels is illustrated. Protein were extracted from BMDMs of Elp3Control and Elp3∆Mye mice treated for 4 hours with IL-4. Red dots indicate candidates whose protein but not mRNA levels were downregulated in BMDMs from Elp3Mye mice compared to BMDMs from Elp3Control mice.

G Identification of candidates enriched in LysAAA, GlnCAA, and GluGAA codons whose expression decreases in BMDMs lacking Elp3. A heatmap of the top 40 genes enriched in these codons is illustrated.
Elp3-mediated codon-dependent translation promotes mTORC2 activation and regulates macrophage polarization (2022)
Figure 7

Figure 7: Elp3 promotes Ric8b expression.

A The induction of Ric8b expression by IL-4 relies on Elp3. BMDMs from the indicated genotypes were treated or not with IL-4 (10 ng/ml) up to 24 hours and the resulting cell extracts were subjected to WB analyses.

B Elp3 promotes Ric8B mRNA translation. BMDMs from Elp3Control and Elp3∆Mye mice were treated by IL-4 for 24 hours, followed by a treatment with 10 μg/ml puromycin for 5 minutes. To detect newly synthesized Ric8b proteins, a Puro-PLA assay was conducted. Representative images detecting Ric8b (Red) and α-Tubulin+ microtubules (green) in BMDMs are illustrated. On the right, the graph shows a quantification of signals for Ric8b in α-Tubulin+ areas. A random of six different areas were counted (6 technical replicates, mean +/- SD, Student's t-test, *= p< 0.05).

C Ric8b is a direct target of Elp3. BMDMs from Elp3Control and Elp3∆Mye mice were infected with a lentiviral GFP construct (negative control), Flag-Ric8b WT or with Flag-Ricb MUT, as indicated and the resulting protein extracts were subjected to WB analyses to assess Ric8b and Elp3 protein levels. Gapdh was used as a loading control.

D and E Ric8b promotes IL-4-dependent mTORC2 activation. Control or Ric8b-depleted BMDMs were treated or not with IL-4 (10 ng/ml) for the indicated periods of time and the resulting cell extracts were subjected to WB analyses.

F and G Ric8b deficiency impairs the production of M2 markers upon IL-4/IL-13 stimulation. Control or Ric8b-depleted BMDMs were stimulated or not with IL-4/IL-13 (10 ng/ml) for 24 hours and the resulting RNAs were subjected to Real-Time PCR analyses. mRNA levels of the indicated candidates in control and unstimulated BMDMs were set to 1 and levels in other experimental conditions were relative to that after normalization with Gapdh mRNA levels (n = 3 mice per genotype; mean +/- SD, Student T test, ***= p< 0.001, **= p< 0.01).

H Ric8b controls glucose consumption in BMDMs. The glucose consumption rate was measured in BMDMs transfected with a siRNA CTRL or Ric8b. Data from three biological replicates (3 mice per group) are shown (mean +/- SD, Student T test, **= p< 0.01).
Elp3-mediated codon-dependent translation promotes mTORC2 activation and regulates macrophage polarization (2022)
Figure 8

Figure 8: Elp3 controls the expression of some mitochondrial proteins.

A Enrichment of candidates involved in mitochondrial translation in the proteomic signature of 206 candidates (Reactome pathway enrichment analysis).

B Enrichment of Mrpl proteins in the proteomic signature of 206 candidates. Proteins were classified according to the combined frequency of AAA, GAA and CAA codons in their open reading frame.

C Elp3 promotes mitochondrial translation. BMDMs from the indicated genotypes were treated or not with IL-4/IL-13 (10 ng/ml) for the indicated hours and the resulting cell extracts were subjected to WB analyses.

D Impaired expression of M2 markers upon Mrpl13 deficiency in IL-4-stimulated macrophages. BMDMs were transfected with a control siRNA or with a siRNA targeting Mrpl13 and the resulting cells were untreated or stimulated with IL-4 (10 ng/ml) for 24 hours. mRNA levels of the indicated candidates were quantified by Real-Time PCR experiments. mRNA levels in unstimulated BMDMs transfected with the control siRNA were set to 1 and levels in other experimental conditions were related to that. Experiments were conducted in triplicates (mean +/- SD, Student T test, ***= p< 0.001, **= p< 0.01, *= p< 0.05).
TLR3 downregulates expression of schizophrenia gene Disc1 via MYD88 to control neuronal morphology (2016)
Figure 1

Figure 1. TLR3 activation negatively regulates neuronal morphogenesis in vivo and in vitro.

(A, B) Poly(I:C) treatment inhibits axonal growth at 3 DIV (A) and dendritic growth at 6 DIV (B) in WT neurons. Neurons were transfected with GFP at 1 or 4 DIV. One day later, neurons were treated with poly(I:C) for 24 h before harvest. (C) Axon and dendrite morphology of Tlr3–/– neurons after poly(I:C) treatment. Data in A,B,C were analyzed by unpaired t-test. Mean values SEM of representatives of three independent experiments are shown. The numbers of analyzed neurons in the representative experiments are indicated in each column. ** P < 0.001, *** P < 0.0001. Scale bar, 20μm in A and B. (D, E) Neuronal morphology in saline- and poly(I:C)-treated Tlr3+/+;Thy1-Yfp (D) and Tlr3–/–;Thy1-Yfp (E) mouse brains. Total dendrite length, dendritic tip number and Sholl analysis of dendritic processes were used to examine the dendrite phenotype. Data of dendrite length and tip number were analyzed by unpaired t-test. The Sholl data were analyzed by two-way ANOVA with Bonferroni’s multiple comparison test. The numbers of analyzed neurons and mice are presented in each column. Mean values SEM are shown. * P < 0.05, ** P < 0.001, *** P < 0.0001. Scale bar, 50 μm in D and E.