EMBO/SourceData huggingface dataset:
Figure 6(A) Cell extracts from normoxic or hypoxic S2 cells were ultracentrifuged as described in fig.1B, protein extracts from pooled fractions corresponding to the same ribosomal content were analysed by western blot with anti-eIF4EHP and RpS23 antibodies.(B) Adult Canton S flies were kept at 21% or 1% O2 for 24h. Total protein extracts were prepared as described in the method section and fractionated by ultracentrifugation on sucrose gradients (15-50%). OD at 260 nm was measured in line during fractionation (upper panels). EIF4EHP was detected by western blot in representative fractions of the gradients.(C) Cross-Linking and Immunoprecipitation experiment (CLIP) was performed on cell extracts from normoxic and hypoxic (1% O2, 24h) UV irradiated WT or eIF4EHP KO S2 cells. eIF4EHP was immunoprecipitated with eIF4EHP antibody-coated protein G sepharose beads before Ldh and Rpl32 mRNA detection by RT-qPCR in total extract (input) or immunoprecipitation eluate. mRNA enrichment levels in eluate as compared to input from 3 independent experiments were calculated as described Data represent mean +/- SD. Statistical significance was tested by two-way ANOVA followed by Sidak's multiple comparisons test, * P<0.05.
Figure 7(A) Female or male flies with chromosomal insertions of an UAS_eIF4EHP (BDSC 36876) RNAi construct were crossed with male or female Tub>Gal4/Tm3 (BDSC 5138) driver flies. Two hours after mating, embryos were transferred to 6% O2 (lowest tolerated O2 concentration) or kept in normoxia until eclosion. Tub-Gal4/UAS-eIF4EHP_RNAi and Tm3/UAS-eIF4EHP_RNAi F1 individuals were counted based on the presence (control) or absence (eIF4EHP KD) of the Tm3 SB marker and results were expressed as % of total eclosions for each condition. Data represent mean +/-SD fro 2 independent biological replicates. Statistical significance was tested by two-way ANOVA followed by Sidak's multiple comparisons test. **** P<0.0001; *** P<0.001; ** P<0.01; * P<0.05.(B) Mobility of age-matched Tub-Gal4/UAS-eIF4EHP_RNAi (eIF4EHPRNAi), TM3/UAS-eIF4EHP_RNAi (control) (upper panels) or Tub-Gal4/UAS-mCherry_RNAi (m-cherryRNAi) (lower panel) females was assessed by negative geotaxis assay in normoxic conditions (21% O2) or 40 min. and 48h after an initial exposure to 1% O2 for 24h. Flies climbing past threshold within 120s were counted and expressed as % of total flies. Each assay was performed in triplicate with 20 flies. Data are mean +/- SD.
Figure 1A. Gentamicin survival assay showing bacteria captured by DC or B cells. Data information: Column bars in the figure represent the mean of at least three independent experiments. Error bars indicate the SD. Significant differences, analyzed by T test (two samples) or one way ANOVA (more than two samples), are represented by asterisk; *P<0.05, ***P<0.0005, non-significant differences are marked as (ns).B-C Confocal images of DCs decorated with HEL (not coloured) infected with L.moncytogenes (in red), coincubated during 30 min (B) or 120 min (C) with noninfected B cells (in blue) isolated from MD4 mice. Actin is shown in cyan. In C, the orthogonal views show intracellular bacteria in B cell. Scale bars represent 5 μm. White arrow in B point to a bacterium at the B cell-DC contact area surrounded by actin. Arrowheads in C indicate the XZ and YZ axes.G. Bacteria-infected and HEL-loaded DCs were co-incubated with B cells from MD4 mouse. After conjugate formation, BacB cells were re-isolated, and intracellular bacteria were detected by gentamicin survival assays at different times. Data were normalized compared to time 0. As control of the bacterial fitness, in parallel, HeLa cells were directly infected.H. WT B cells were allowed to form conjugates with Listeria-infected DC, after 30 min samples were treated with the indicated inhibitors; Leupeptin + NH4Cl, pepstatin-A + E64D, lactacystin, or PBS for controls. One hour later gentamicin was added to kill extracellular bacteria and two hours later B cells were reisolated by negative selection, lysed and plated in agar-containing plates. CFUs were counted; each colony represents one live intracellular bacterium inside B cells. Data information: Column bars in the figure represent the mean of at least three independent experiments. Error bars indicate the SD. Significant differences, analyzed by T test (two samples) or one way ANOVA (more than two samples), are represented by asterisk; *P<0.05, ***P<0.0005, non-significant differences are marked as (ns).
Figure 2CD80 (A-B) expression in B cells measured by flow cytometry on day 0 (non-instructed; naïve) and day 2 after L. monocytogenes capture (BacB). A show representative experiments, B show the mean of at least three independent experiments. Data information: Column bars in the figure represent the mean. Error bars indicate the SD. Significant differences, analyzed by T test are represented by asterisk; *P<0.05, ***P<0.0005, non-significant differences are marked as (ns).E-F. H-2Kb expression in B cells measured by flow cytometry on day 0 (naïve, pale grey) and day 1 after L. monocytogenes capture (BacB; dark grey). E shows a representative experiment, F shows the mean of four independent experiments Data information: Column bars in the figure represent the mean. Error bars indicate the SD. Significant differences, analyzed by T test are represented by asterisk; *P<0.05, ***P<0.0005, non-significant differences are marked as (ns).G-H. Expression of OVAp-I in the context of H-2Kb was detected by flow cytometry with anti-OVAp-I/H-2Kb antibody, comparing BacB capturing Listeria-WT (pale grey) and Listeria-OVA (dark grey). G shows a representative experiment, H shows the mean of two independent experiments. Data information: Column bars in the figure represent the mean. Error bars indicate the SD. Significant differences, analyzed by T test are represented by asterisk; *P<0.05, ***P<0.0005, non-significant differences are marked as (ns).I. Confocal images of Listeria-WT (left panels) or Listeria-OVA (right panels) BacB cells incubated with naïve OTI CD8+ T cells previously stained with CellTraceViolet (CV, red). Actin and CD3 fluorescence are shown on a "fire" scale and in cyan and white respectively in the merged images. Scale bars = 10 μm. * indicate CD8+ T cells. Arrow points to the IS. J. Quantification of actin accumulation at the IS on CD8+ T cells conjugated with Listeria-WT or Listeria-OVA BacB cells, analyzed using Synapse measures software from confocal images. Each dot corresponds to one IS. Three independent experiments were performed. Data information: Column bars in the figure represent the mean. Error bars indicate the SD. Significant differences, analyzed by T test are represented by asterisk; *P<0.05, ***P<0.0005, non-significant differences are marked as (ns).
Figure 3B. Mean of at least three experiments showing the % of naïve OTI CD8+ T cell proliferated after activation (days 1-3) with BacB capturing Listeria-WT (WT) or Listeria OVA (OVA). Non-activated, non-proliferative CD8+ T cells (NP) are shown as control. Data information: Column bars in the figure represent the mean of at least three independent experiments. Error bars indicate the SD. Statistical significant differences analyzed by one-way ANOVA is represented by *.*P<0.05, ***P<0.0005, non-significant differences are marked as (ns).C-D. CD25 expression in OTI CD8+ T cells on day 1 (light red), day 2 (red) and day 3 (dark red) after activation with Listeria-WT (left panel) or Listeria-OVA (right panel) BacB cells. FMO control is shown in grey and dash line. C, representative experient. D, Mean of at least three independent experiments. Data information: Column bars in the figure represent the mean of at least three independent experiments. Error bars indicate the SD. Statistical significant differences analyzed by one-way ANOVA is represented by *.*P<0.05, ***P<0.0005, non-significant differences are marked as (ns).G-H. Proliferation of OTI CD8+ T cells activated with Listeria-OVA BacB cells (H-2Kb) that captured bacteria from infected H-2Kb or H-2Kk iBM-DCs. Non-activated naïve CD8+ T cells are shown (thin grey line). B cells were isoated from WT mice. G shows a representative experiment, H shows the mean of the proliferation index from at least three independent experiments (relative to H-2Kk iBM-DCs donor cells). Data information: Column bars in the figure represent the mean of at least three independent experiments. Error bars indicate the SD. Statistical significant differences analyzed by one-way ANOVA is represented by *.*P<0.05, ***P<0.0005, non-significant differences are marked as (ns).J-L. Intracellular expression, measured by flow cytometry, of the indicated proteins in non-activated, naïve, CD8+ T cells or in CD8+ T cells activated with either antiCD3 + antiCD28 antibodies (αCD3/CD28), OVA-decorated splenocytes (OVA-pI), or Listeria-OVA BacB cells (BacB). The mean fluorescence intensity and the sd corresponding to three independent experiments is shown. Data information: Column bars in the figure represent the mean of at least three independent experiments. Error bars indicate the SD. Statistical significant differences analyzed by one-way ANOVA is represented by *.*P<0.05, ***P<0.0005, non-significant differences are marked as (ns).
Figure 4A-D Surface expression of H-2Kb (A-B) and CD80 (C-D) in L. monocytogenes (orange) or E. coli (purple) BacB cells analyzed by flow cytometry. A and C panels show representative experiments, B and D panels show the mean of at least three independent experiments.K. Representative data showing OTI CD8+ T cells proliferation 3 days after activation with Listeria-OVA (left panel, orange) or Listeria ∆hly -OVA (right panel, blue) BacB cells. Non-activated naïve CD8+ T cells (grey) are shown as a negative proliferation controls.M. Representative data showing OTI CD8+ T cells proliferation 3 days after activation with Listeria-OVA (orange), Listeria ∆hly-OVA (blue) or Listeria ∆hly::hly-OVA BacB cells (Purple).N. Proliferation index mean of at least three experiments as in H.
Figure 5B-D. Differentation of different CD8+ T cell populations (B, early effector cells, EECs (CD127lo KLRG1lo); C, memory precursor effector cells (MPECs (CD127hi KLRG1lo); D, short-lived effector cells, SLECs (CD127lo KLRG1hi)), gated on CD3+CD8+ T cells in the presence of the indicated B cells, 2 days after a second bacterial rechallenge (as indicated in A). Data information: Bars in the figure represent the mean. Error bars indicate the SD. Statistical significant differences, analyzed by ANOVA are represented by asterisk; *P<0.05, ***P<0.0005, non-significant differences are marked as (ns). …F. Kaplan Meier survival curve of Rag1-/- mice after adoptive transfer and bacteria challenge. Data information: Bars in the figure represent the mean. Error bars indicate the SD. Statistical significant differences, analyzed by ANOVA are represented by asterisk; *P<0.05, ***P<0.0005, non-significant differences are marked as (ns). …H. BacB cells reduce melanoma growth. B16 F10 melanoma cells (4 × 105) were injected s.c. in the mid-right flank of C57BL/6J (WT) host mice (4 mice/group; treated with BacB or untreated). Once tumors were formed and visible (6 days after imlantation) mice were treated with Listeria TRP2 BacB cells or with vehicle (PBS) (three consecutive injections in three days). Tumor growth were monitored every 1-3 days. It is shown the mean and the SD of the tumor volume in each group at the indicated time points. Mice with tumors ≥ 300 mm3 were sacrified. Data information: Bars in the figure represent the mean. Error bars indicate the SD. Statistical significant differences, analyzed by ANOVA are represented by asterisk; *P<0.05, ***P<0.0005, non-significant differences are marked as (ns). …
Figure 1(A) Serum starvation induces CP110 degradation. RPE-1 cells were either mock-treated or serum starved for 0.5, 1, 2, 4, 6 and 24 h and then lysed and subjected to immunoblotting with anti-CP110 antibodies (top panel) and anti-actin antibodies (loading control; bottom panel). (B) Graph shows CP110 levels normalized to actin. Normal distribution was determined by the Shapiro-Wilk normality test. (Inhibition of proteasomal degradation leads to CP110 stabilization on the m-centriole and impaired ciliogenesis. RPE-1 cells on cover-slips were either grown in media with serum, serum starved for 4 h in the absence of MG132, or serum starved for 4 h in the presence of MG132 prior to fixation and immunostaining with antibodies for acetylated tubulin (to detect cilia) and anti-CP110 antibodies. (E) Representative micrographs showing MG132 treatment impedes removal of CP110 from the m-centriole and impairs ciliogenesis. Bar, 10 μm. (F) Graph quantifying percent of cells displaying primary cilia. Normal distribution was determined by the D'Agostino and Pearson normality test.(H-I) EHD1 depletion prevents CP110 degradation upon serum starvation. (H) Mock-transfected RPE-1 cells or EHD1 siRNA-transfected RPE-1 cells were grown in media containing serum, or serum starved for 4 h in the absence or presence of MG132, and then lysed, separated by SDS-PAGE and immunoblotted with anti-CP110 antibodies (upper panel) or anti-actin antibodies (loading control; lower panel). Validation of EHD1 depletion is shown in the right panel. (I) Graph displaying densitometric analysis of the intensity of the CP110 bands shown in H normalized to actin (means and p-values are representative of 3 independent experiments). Normal distribution was determined by the Shapiro-Wilk normality test.
Figure 2(A-B) PCM1-depletion impedes CP110 ubiquitination upon serum starvation. (A) Mock-transfected RPE-1 cells were maintained in media with serum, and both mock-transfected and PCM1 siRNA-transfected cells were serum starved for 4 h in the presence of MG132 prior to lysis and immunoblotting with anti-CP110 antibodies (second panel from top), or first immunoprecipitated with anti-CP110 antibodies and then immunoblotted with antibodies to detect ubiquitinated CP110 (top panel). Bottom panels depict the efficacy of PCM1 depletion upon PCM1 siRNA-transfection, with actin as a loading control. (B) Graph displaying densitometric analysis of ubiquitinated CP110 levels (from the upper panel of A) normalized to total CP110 immunoprecipitated. Normal distribution was determined by the Shapiro-Wilk normality test.(D-G) Serum starved mock-transfected RPE-1 cells on cover-slips were fixed and immunostained with antibodies directed against acetylated tubulin and CP110 and display primary cilia generation (marked by acetylated tubulin; D, F and inset in G) and removal of CP110 from the m-centriole (E-G; see arrow marking CP110 only on the daughter centriole). (H-K) Serum starved PCM siRNA-transfected RPE-1 cells on cover-slips were fixed and immunostained with antibodies directed against acetylated tubulin and CP110 and display limited ciliogenesis (H, J and inset in K) and failure to remove CP110 from the m-centriole (I-K; see arrows identifying CP110 on both centrioles).EHD1 regulates the distribution of centriolar satellites to the centrosomal region. Mock-transfected RPE-1 cells on cover-slips were serum starved and immunostained with antibodies against acetylated tubulin and PCM1 and EHD1 siRNA-transfected cells on cover-slips were serum starved and immunostained with antibodies against acetylated tubulin and PCM1 The p-values are calculated for all non-ciliated and ciliated mock-transfected cells compared to all EHD1 siRNA non-ciliated and ciliated cells. Note that as expected, among the EHD1-depleted cells, there are minimal cells that display ciliation.
Figure 3(A) Validation of siRNA depletion. RPE-1 cells were mock-transfected or transfected with siRNA oligonucleotides to impair translation of Cullin3, HERC2, or MIB1. 72 h later, the cells were lysed, subjected to SDS-PAGE and immunoblotted. Immunoblotting with anti-actin antibodies was done as a loading control.(B) HERC2 or MIB1 depletion impairs CP110 ubiquitination. RPE-1 cells were grown in serum (first lane from left), or with MG132 in the absence of serum for 4 h (second lane from left), transfected with Cullin3 siRNA-oligonucleotides and treated with MG132 in the absence of serum for 4 h (third lane from left), transfected with HERC2 siRNA-oligonucleotides and treated with MG132 in the absence of serum for 4 h (fourth lane from left), or transfected with MIB1 siRNA-oligonucleotides and treated with MG132 in the absence of serum for 4 h (fifth lane from left). Cells were lysed and immunoprecipitated with anti-CP110 antibodies, and the resultant immunoprecipitates were immunoblotted with anti-ubiquitin to identify ubiquitinated CP110 (upper panel). The blot was then stripped and re-immunoblotted with anti-CP110 to reveal the amount of CP110 pulled-down from the lysate (lower panel).(D) CP110 interacts with HERC2 and MIB1, but not Cullin3. RPE-1 cells were lysed and immunoprecipitated with beads only or anti-CP110, and then subjected to SDS-PAGE and immunoblotting with CP110 to validate the immunoprecipitation (lower panel of each gel) and either Cullin3 (left gel, upper panel), HERC2 (middle gel, upper panel) or MIB1 (right gel, upper panel). 7% inputs are shown on the left of each gel.
Figure 4(B-M) (B-D) Mock-transfected cells, (E-G) MIB1 siRNA-transfected cells, (H-J) HERC2 siRNA-transfected cells, or (K-M) MIB1 and HERC2 siRNA-transfected cells on cover-slips were fixed and immunostained with antibodies against acetylated tubulin (insets; C, F, I, L: merged field; B, E, H, K) and CP110 (insets; D, G, J, M: merged field; B, E, H, K). Bars (B, E, H, K), 10 mm. Bars (C, D, F, G, I, J, L, M), 2 μm.
Figure 5(A) EHD1 is required for the interaction between HERC2 and CP110. RPE-1 cells were mock-transfected or transfected with EHD1 siRNA oligonucleotides for 72 h. Cells were lysed and immunoprecipitated with anti-CP110 or beads only (control) and separated by SDS-PAGE along with 7% of the lysate (input). After transfer to nitrocellulose, antibodies were used to detect CP110 (lower panel) and HERC2 (upper panel).(C-D) HERC2 partially localizes to PCM1-containing centriolar satellites. RPE-1 cells were serum starved for 4 h, fixed and immunostained with antibodies against PCM1 (red) and HERC2 (green). The micrographs show a single representative cell (C) and an inset (D). Bar (C), 10 μm. Bar (D), 1 μm.(G-H) Representative micrographs of RPE-1 cells on cover-slips that were mock-transfected (G) or EHD1 siRNA-transfected (H) and immunostained with antibodies to acetylated tubulin (red) or HERC2 (green). Dashed yellow regions of interest highlight the centrosomal area. Arrows denote the HERC2 signal observed in each micrograph. Bars, 1 μm.
Figure 6(A-D) RPE-1 cells on coverslips were mock-transfected (A), transfected with CP110 siRNA-oligonucleotides (B) transfected with HERC2 siRNA-oligonucleotides (C), or transfected with both CP110 and HERC2 siRNA-oligonucleotides (D). Cells were fixed and immunostained with acetylated tubulin to detect primary cilia (yellow arrows). Bars, 10 μm. : Graphs show standard deviation and p-values from 3 independent experiments. The micrographs (A-D) are representative ones from the 3 individual biological experiments. More than 50 cells were quantified in each experiment, although fewer ciliated cells were observed and measured under the HERC2 knock-down condition.(E) Immunoblot demonstrating the efficacy of depletion for CP110 (middle panel), HERC2 (top panel), and simultaneous CP110 and HERC2 depletion (middle and top panels). Actin was used as a loading control (bottom panel). : Graphs show standard deviation and p-values from 3 independent experiments. immunoblots (E) are representative ones from the 3 individual biological experiments.
Figure 1(C-F) C. Total fresh weight, D. Total dry weight, E. Primary root length and F. Lateral root density of 21-day-old, non-colonized (Mock) and SA190-colonized plants upon growth for 16 days on ½ MS or ½ MS + 25% PEG. Boxplot shows data from three independent biological replicates (technical replicates n = 18-24) where whiskers represents minimum and maximum values, "+" indicates mean and horizontal line represents median. Asterisks indicate the statistical differences based on the Student's t-test (*** p < 0.001).(G, H) G. Growth and H. Survival percentage of mock- and SA190-colonized plants in jiffy pots grown upon watering for 2 weeks, no watering for 3 weeks and after one week of re-watering. All plots represent the mean of 3 biological replicates (technical replicates n = 36). Whiskers represents minimum and maximum values, "+" indicates mean and horizontal line represents median. Asterisks indicate the statistical differences based on the Student's t-test (*** p < 0.001).J. Confocal microscopy showing the colonization of SA190:GFP on five days-old seedlings grown on ½ MS and 25% PEG. Green signal corresponds to GFP-tagged SA190 while in magenta are plant cell wall stained with propidium iodide (PI). Bar indicates 100 nm. K. SA190 colonization levels in Arabidopsis under normal (½ MS) and drought proxy (½ MS + 25% PEG) conditions as quantified by colony forming units of bacteria per mg of plant fresh weight. Boxplot shows data from four independent biological replicates where whiskers represents minimum and maximum values, "+" indicates mean and horizontal line represents median. Asterisks indicate the statistical differences based on the Student's t-test (* p < 0.05, *** p < 0.001).
Figure 2F-G. Primary root length and Lateral root density of 21-day-old, mock- and SA190-colonized Col-0, aba2-1 and qpyr/pyl plants upon growth for 16 days on ½ MS + 25% PEG. Data information: Boxplot shows data from three independent biological replicates (technical replicates n = 36) where whiskers represents minimum and maximum values, "+" indicates mean and horizontal line represents median. Asterisks indicate the statistical differences based on the Student's t-test (* p < 0.05, *** p < 0.001).
Figure 3B. Relative gene expression of aquaporin PIP and TIP genes by qRT-PCR analysis normalized to tubulin levels in 21-days old mock- and SA190-colonized roots of Arabidopsis grown for 16 days on ½ MS with or without 25% PEG. Data information: Values represent the means of three biological experiments. Error bars indicate SE. All plots represent the mean of 3 biological replicates (biological replicates n= 3). Asterisks indicate a statistical difference based on the Student's t-test (* p < 0.05; ** p < 0.01; *** p < 0.001).C. Relative gene expression of aquaporin PIP and TIP genes by qRT-PCR analysis in Col-0, aba2-1 and qpyr/pyl mutants normalized to tubulin levels in 21-days old mock- and SA190-colonized roots of Arabidopsis grown for 16 days on ½ MS with or without 25% PEG. Data information: Values represent the means of three biological experiments. Error bars indicate SE. All plots represent the mean of 3 biological replicates (biological replicates n= 3). Asterisks indicate a statistical difference based on the Student's t-test (* p < 0.05; ** p < 0.01; *** p < 0.001).
Figure 4Relative enrichment of the H3K4me3 mark at the indicated gene loci of selected PIPs and TIPs of non-colonized (Mock) and SA190-colonized (SA190) plants in Col-0 and aba2-1 mutant as determined by chromatin immunoprecipitation-qPCR (ChIP-qPCR). The regions targeted for amplification are labelled as R1, R2 and R3. R1 encompasses 5'-UTR, R2 is close to the transcription start site (TSS) while R3 is outside the gene body. Amplification values were normalized to input and H3 and region 3 (R3) of non-colonized (Mock) Col-0 plants. The plots represent the means of 2 biological replicates. Error bars represent SE. (ns) non-significant Asterisks indicate a statistical difference based on 2-way ANOVA (*p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001 for p value differences between the conditions when compared to Col-0 Mock using Dunnett's multiple comparison test).