EMBO/SourceData huggingface dataset:
Figure 1(B) HCT116 cells in which eIF3k was homozygously modified with the mAID domain to trigger auxin-dependent degradation were exposed to 500 μM indole-3-acetic acid (IAA) for the periods shown, and cell lysate was subjected to immunoblotting with the indicated antibodies
Figure 2(A) Parental HCT116 and the five different conditional eIF3-mAID cell lines were treated with the vehicle DMSO or with IAA for the periods indicated. Metabolic activity (i.e. NAD(P)H) as a proxy of cell proliferation was determined by MTT assay. Graphs represent means ± SD, n = 5 - 6. Asterisks denote: * p < 0.005 , ** p < 0.00005, *** p < 0.000005(D) 1 x 106 eIF3-mAID cells were injected into nude mice. When tumors reached a diameter of ~ 4 mm, mice were treated with vehicle or 500 mg/kg IAA, and tumor growth was measured for 2 weeks. Graphs represent means ± SD, n = 5 to 7. Numbers indicate p values(E) Expression of the indicated eIF3 proteins in 97 colon adenocarcinomas versus 100 normal colonic mucosae and 110 clear cell renal cell carcinomas versus 84 normal kidney samples. Log2 spectral count data from CPTAC via UALCAN portal
Figure 3(A) The indicated conditional eIF3 cell lines were exposed to 500 μM IAA for 12 h to induce the degradation of the respective eIF3 subunits. Cell lysates were subjected to immunoblotting to quantify the expression of the indicated eIF3 subunits. Signals were normalized to the reference signal of actin and plotted relative to the vehicle DMSO. Bars represent means ± SD, n = 3. Numbers indicate p values (unpaired Student's t-test).(B) The indicated cell lines were exposed to IAA for 12 h, and cell lysates were separated by sucrose density gradient centrifugation. The monosomal (M) and polysomal (P) peaks were quantified, and P/M ratios relative to the vehicle DMSO were plotted. Bars represent means ± SD, n = 3. Numbers indicate p values (unpaired Student's t-test).(C) Scatter plots of changes in polysomal mRNA versus total mRNA upon depletion of the indicated eIF3 subunits.
Figure 4(A) eIF3k-mAID cells were maintained in media containing different concentrations of FBS and DMSO or IAA as indicated, and cell numbers were determined at various time points. Data represent means ± SD, n = 3 biological replicates. Numbers indicate p values(B) eIF3k-mAID cells were serum starved by maintaining in media containing 0% FBS for 24 h. During the last 12 h of starvation, DMSO or IAA was added as indicated. Cells were re-stimulated with media containing 10% FBS, and the expression of the indicated proteins was followed of a period of 120 minutes. The data from triplicate experiments were quantified and plotted as relative ratios of phosphorylated to unphosphorylated species. Bars represent means ± SD, n = 3 Numbers indicate p values (unpaired Student's t-test).(C) eIF3k-mAID cells were exposed to IAA or DMSO for 12 hours, followed by the addition of increasing concentrations of rapamycin for 1 h. The expression of the indicated proteins was determined by immunoblotting. The data from triplicate experiments were quantified and plotted as relative ratios of phosphorylated to unphosphorylated species. Bars represent means ± SD, n = 3 Numbers indicate p values (unpaired Student's t-test).
Figure 5(C) Total ribosome occupancy of the indicated mRNAs in eIF3k-mAID cells exposed to DMSO or IAA for 12 hours was determined by RT-qPCR of RNA across a sucrose density gradient Bars represent means ± SD, n = 3; numbers indicate p values (unpaired Student's t-test). Triplicate RT-qPCR data across the sucrose gradient are shown below the bar graphs.(D) Equal numbers of eIF3k-mAID cells stably expressing ectopic RPS15A (pCDH-RPS15A) or empty vector (pCDH) were plated and counted over a period of 6 days. Graphs represent means ± SD, n = 3. Numbers indicate p values(G) Equal numbers of eIF3k-mAID cells stably expressing ectopic RPS4X (pCDH-RPS4X) or empty vector (pCDH) were plated and counted over a period of 6 days. Graphs represent means ± SD, n = 3. Numbers indicate p values Total ribosome content of these cells was determined as described in (A).
Figure 6(B) Parental eIF3k-mAID cells or S15A-eIF3KO cells (clones #45 and # 361) were maintained in standard media, and cell numbers were determined at various time points. Data represent means ± SD, n = 3. Asterisks denote: * p < 0.005, ** p < 0.00005, *** p < 0.000005(D) Relative translational efficiencies (TEs) of mRNAs encoding RPS15A, RPS4X, and RPL7A before and after depletion of eIF3k were determined in the indicated cell lines. RT-qPCR was performed on total RNA and on RNA contained within polysomal fractions > 2 ribosomes, and TE was calculated according to the formula TE = polysomal mRNA / total mRNA. Bars represent means ± SD, n = 3; numbers indicate p values (unpaired Student's t-test).
Figure 7(A) Parental eIF3k-mAID cells or S15A-eIF3KO cells (clones #45 and # 361) were maintained in media with DMSO or IAA, and cell numbers were determined at various time points. Data represent means ± SD (too small to be visible), n = 3. All p values were > 0.45 except at the single time point where indicated otherwise.(D) Total ribosome occupancy of the indicated mRNAs in S15A-eIF3KO (clone #361) cells exposed to DMSO or IAA for 12 hours was determined by RT-qPCR of RNA across a sucrose density gradient Bars represent means ± SD, n = 3; numbers indicate p values (unpaired Student's t-test). Triplicate RT-qPCR data across the sucrose gradient are shown below the bar graphs.(E) RNA immunoprecipitation. eIF3k-mAID and S15A-eIF3KO (clone #361) cells were exposed to DMSO or IAA for 12 hours. Cell lysates were employed in immunoprecipitation with eIF3c antibodies and co-precipitated mRNAs were quantified by qPCR. Bars represent means ± SD, n = 4; numbers indicate p values (unpaired Student's t-test).
Figure 8(A) The indicated eIF3-mAID cell lines were treated with DMSO or IAA for 12 hours, followed by exposure to 2 μg/ml tunicamycin for up to 72 hours. Metabolic activity (i.e. NAD(P)H) as a proxy of cell viability was determined by CCK8 assay. Graphs represent the percentage of change in cell viability upon downregulating eIF3 subunits with IAA. Date are means ± SD, n = 3. (B) Same experiment as in (A) but cells were exposed to increasing concentrations of the oxidative stress inducer tert-butyl hydroperoxide (TBHP) for 2 hours.(C) The indicated eIF3-mAID cell lines were treated with DMSO or IAA for 12 hours, followed by exposure to 2 μg/ml tunicamycin for 24 hours. The expression of individual eIF3 subunits, ribosomal proteins, and the ER stress marker BIP were assessed by immunoblotting. Tubulin is show for reference. The data from triplicate experiments were quantified and to avoid overcrowding of the graph data obtained from IAA-treated cells were not plotted. Bars represent means ± SD, n = 3 Numbers indicate p values (unpaired Student's t-test).
Figure 1K-P Realtime qRT-PCR revealed the expression profiles of four zebrafish chs genes in different tissues (K), different gut sections (L), and different developmental stages (M-P). One representative example of three repeat experiments is shown. Data were the mean ± SEM of n=3 technical replicates. n.s. no significant, no label P < 0.05 by student's t-test.
Figure 2F-M Compared with wild-types (F-I), mutants (J-M) completely lost the CM (red) in the gut of 7dpf larva (F, J), as well as in the intestinal bulb (G, K), middle intestine (H, L) and posterior intestine (I, M) of adults. Red, SNAP-CBD stain; blue, DAPI stain; WT, wild-type; MU, chs1-/- mutant.
Figure 3A, B The survival rates of wild-types and mutants in the first eight days (A) and the first six months (B). Two independent batches (1 and 2) were conducted for each experiment. n, the initial numbers of zebrafish. * P < 0.05, ** P < 0.01, *** P < 0.001 by long-rank (Mantel-Cox) test. Please see Table EV1 for data on another three batches of independent experiments.
Figure 4E-J 6/7 dpf larva mutants were fed with 6-μm polystyrene beads. The beads jammed in the intestinal bulb and middle intestine (E, F, H, I). In some cases, the beads escaped the bead cord and stuck between villi (white triangles) (E, G, I, J). Blue rectangle indicates the intestinal bulb. Right panels were magnification of the red rectangle area in left panels.
Figure 5B Comparison of 3-month-old wild-types and mutants in the pairwise microbiota compositional dissimilarity (Jaccard distance) between the digesta (niche1) and the epithelia (niche2). This metric represents the radial gradient of the gut bacterial composition. The comparison was done on four taxonomy levels. Data of n=14 individuals were represented in box (interquartile range) and whisker (min to max) plots. Individual data points were plotted as black dots. * P < 0.05, ** P < 0.01 by the conservative, nonparametric Kruskal-Wallis test. Please see Appendix Fig S10 for another way of data presentation, and Dataset EV1 for the details of the sample information and raw statistics. Note that each 16S-rRNA library contains only one sample from one animal.Comparison of the bacterial community richness of the intestine samples (S3-S6; including both the digesta and the epithelia) between six 3-month-old wild-types and six 3-month-old mutants. Three alpha diversity metrics were used: Ace (abundance-based coverage estimator), Chao1 Data of n=6 individuals were represented in box (interquartile range) and whisker (min to max) plots. Individual data points were plotted as black dots. * P < 0.05 by Kruskal-Wallis test.
Figure 6E Immunohistochemical detection of BrdU-labeled cells in the intestine bulb (section S2) of two 3-month-old adult wild-types and two 3-month-old adult mutants to measure the rate of cell proliferation (new cells were stained bright green) and migration. Compared with wild-types (WT), the mutants had more green spots along the villi 2 hours after treatment, and mutants also have more green spots concentrated in the tip of villi 12 hours after treatments. The violin plots (interquartile range with mean line) show the relative numbers of green puncta from tip to base of villi, which were calculated from 3-5 villi from the raw version of these images. Please see Appendix Fig S19 for the images of more animals and time points.
Figure 7Comparison of the body length, weight and external appearance of five wild-types and five mutants after the alternative treatments of two sets of antibiotic mixtures. The treatments were started with 8-week-old fish and continued for six weeks. The measurements and photoshoots were conducted five weeks later. Data of n=5 are represented in box (min to max with mean line) plots. Individual data points for each animal were plotted as grey dots. n.s., no significant, ** P < 0.01 by student's t-test. The raw statistics was listed in Table EV3.
Figure 1(A) Western blotting of Mcrs1 at the GV (0 h), GVBD (4 h), MI (8 h), and MII (12 h) stages of mouse oocyte meiosis. The blots were probed with anti-Mcrs1 and anti-actin antibodies.(G) Percentages of control (n = 210), Mcrs1-KD (n = 182), and Mcrs1-rescue (n = 201) oocytes that underwent GVBD and PBE. The bars are representing the mean ± SEM. The P-values were calculated using Student's t-test. GVBD%: ***P = 0.0003; *P = 0.01, PB1%: *P = 0.03; *P = 0.02.
Figure 2(A) Representative images of the occurrence of GVBD in control, Mcrs1-KD, and Mcrs1-rescue (Mcrs1 siRNA + Mcrs1 mRNA) oocytes at 3 h following release from IBMX. Scale bar, 100 μm. (B) The incidence of GVBD at 1, 2, and 3 h post-IBMX release in control (n = 155), Mcrs1-KD (n = 157), and Mcrs1-rescue (n = 150) oocytes.(D) Expression levels of CDK1 in Mcrs1-KD and control oocytes. The bars represent the mean (± SEM) of 5 biological replicates. The P-values were calculated using Student's t-test. *P = 0.01. (E) Expression levels of p-CDK1(Y15) in Mcrs1-KD and control oocytes at 1 h following release from IBMX (200 oocytes per sample). **P = 0.006. (F) Expression levels of cyclin B1 in control and Mcrs1-KD oocytes at 1 h following release from IBMX (200 oocytes per sample). The bars represent the mean (± SEM) of 3 biological replicates. The P-values were calculated using Student's t-test. **P = 0.0042.(I) The incidence of GVBD at 1, 2, and 3 h post-IBMX release in control oocytes (n = 112), Mcrs1-KD oocytes (n = 98), and Mcrs1-KD oocytes treated with 5 mM WEE1-1N-4 (n = 102). (J) The incidence of GVBD at 1, 2, and 3 h post-IBMX release in control (n = 123), Mcrs1-KD (n = 134), and Mcrs1 siRNA + cyclin B1-GFP (n = 95) oocytes. (K) The incidence of GVBD at 1, 2, and 3 h post-IBMX release in control (n = 92), Mcrs1-KD (n = 97), and Mcrs1 siRNA + cyclin B1-GFP oocytes treated with 5 mM WEE1-1N-4 (n = 106).
Figure 3(B) Detection of newly synthesized RNAs by EU incorporation in control, Mcrs1-KD, and Mcrs1-rescue GV oocytes. The scattergram shows the relative fluorescence intensity of EU in control (n = 43), Mcrs1-KD (n = 52), and Mcrs1-rescue (n = 30) GV oocytes. Scale bar, 10 μm. ***P = 0.0001.(C) Immunofluorescence analysis of H3K4me2 in control and Mcrs1-KD oocytes. Scale bar, 10 μm. Relative fluorescence intensity of H3K4me2 signals in control (n = 26) and Mcrs1-KD (n = 30) GV oocytes. ***P = 0.0001.(E) Immunofluorescence analysis of H3K9me2 in control and Mcrs1-KD oocytes. Scale bar, 10 μm. Relative fluorescence intensity of H3K9me2 signals in control (n = 29) and Mcrs1-KD (n = 22) GV oocytes. **P = 0.0035. (F) Immunofluorescence analysis of H4K16ac in control and Mcrs1-KD oocytes. Scale bar, 10 μm. Relative fluorescence intensity of H4K16ac signals in control (n = 30) and Mcrs1-KD (n = 28) GV oocytes. *P = 0.022.(G) Immunofluorescence analysis of HDAC2 in control and Mcrs1-KD oocytes. Scale bar, 10 μm. Relative fluorescence intensity of HDAC2 signals in control (n = 27) and Mcrs1-KD (n = 24) GV oocytes. ***P = 0.0005.
Figure 4(A) Representative images of spindle assembly at 3 h post-release from IBMX in control and Mcrs1-KD oocytes. Scale bar, 20 μm.(G) Immunofluorescence analysis of H3S10ph and α-tubulin in control and Mcrs1-KD oocytes. Scale bar, 20 μm. The scattergram shows the relative fluorescence intensity of H3S10ph signals in control (n = 21) and Mcrs1-KD (n = 23) oocytes. *P = 0.017.(H) Immunofluorescence analysis of γ-tubulin and α-tubulin in control and Mcrs1-KD oocytes. Scale bar, 20 μm. The histogram shows the percentages of control (n = 55) and Mcrs1-KD (n = 63) oocytes with abnormal γ-tubulin. *P = 0.014. (I) Immunofluorescence analysis of acetylated tubulin and α-tubulin in control and Mcrs1-KD oocytes. Scale bar, 20 μm. The scattergram shows the relative fluorescence intensity of acetylated tubulin signals in control (n = 83) and Mcrs1-KD (n = 66) oocytes. **P = 0.0046.
Figure 5(C) Levels of p-Aurka and p-Aurkc in control and Mcrs1-KD MI oocytes. The blots were probed with anti-p-Aurora A/B/C and anti-actin antibodies. (D) Co-IP was performed with an anti-Mcrs1 antibody. The immunoblots of protein precipitates were probed with an anti-Aurka antibody.(G) Percentages of control (n = 92), Mcrs1-KD (n = 73), and Aurka-rescue (n = 67) oocytes that underwent PBE. *P = 0.033; *P = 0.018.(H) Percentages of control (n = 69), Mcrs1-KD (n = 76), and Aurka-rescue (n = 43) oocytes with abnormal spindles. *P = 0.018; *P = 0.024.(K) Immunofluorescence analysis of Kif2A in control and Mcrs1-KD oocytes. Scale bar, 20 μm. The histogram shows the percentages of control (n = 50) and Mcrs1-KD (n = 61) oocytes with abnormal Kif2A. **P = 0.003.(L) Immunofluorescence analysis of TPX2 and α-tubulin in control and Mcrs1-KD oocytes. Scale bar, 20 μm. The scattergram shows the relative fluorescence intensity of TPX2 signals in control (n = 48) and Mcrs1-KD (n = 52) oocytes.
Figure 1A Arabidopsis seedlings grown for 7 days in LD at 20°C or 28°C. B Root lengths of seedlings from day 2 to day 12 after sowing (grown as in A).C Seedlings of Brassica oleracea and Solanum lycopersicum (lycop.) grown for 7 days at 20°C or 28°C. D Root lengths of 7 days-old seedlings grown at 20°C or 28°C (n = 18-22).
Figure 2A Elongation responses of detached roots. Shoots were removed from 4 days-old Arabidopsis thaliana (n = 18-19), 4 days-old Brassica oleracea (n = 11) and 5 days-old Solanum lycopersicum (lycop., n = 10-12) seedlings grown at 20°C. Detached roots were grown for additional 4 days at 20°C or 28°C,B-D 9 days-old seedlings were hypocotyl-grafted, recovered for 7 days and then cultivated at 20°C or 28°C for additional 7 days (n = 16-26). Boxplots show medians, interquartile ranges and min-max values with individual data points superimposed as colored dots. Diﬀerent letters denote statistical diﬀerences at P < 0.05 as assessed by one-way two-way (A-D) ANOVA and Tukey's HSD posthoc test.
Figure 3Total length of root zones between days 2 and 7 after sowing of Arabidopsis seedlings grown in LD at 20°C or 28°C. A Meristem, B Elongation zone, C Differentiation zone. A-C Solid lines and points show mean root zone lengths with half-transparent ribbons denoting SEM (n = 5-7 individual roots).E EdU staining marks cells that were in S-phase during the 1h incubation time prior to microscopic imaging. Scale bar = 50 µm, n= 12. F DAPI staining was used to identify cells currently in mitosis (examples are highlighted by yellow asterisks).
Figure 4A Root growth assay of seedlings grown for 7 days in the presence of increasing concentrations of auxin biosynthesis inhibitors kynurenine and yucasin (n = 14-26, both inhibitors always in equal concentrations) at the indicated temperatures.D Temperature induced root growth is unaffected in 8 days-old seedlings if 0.5 mM NPA is applied to the root shoot junction on day 5 (n = 13-16) at the indicated temperatures.G (Co-)incubation of 5 days-old seedlings for 3h with 10 μM EdU only or in combination with either 100 nM NAA or 50 μM PEO-IAA (n = 11-24),
Figure 5B Imaging and quantification of DR5::GFP reporter activity in root tips of 5 days-old seedlings (scale bar = 50 µm, n= 9-14).C EdU staining of 5 days-old seedlings at indicated temperatures (n = 11-14).D qRT-PCR expression analyses of PIN1-4 from whole root and roo tip samples, respectively, Col-0 RNA was extracted with respective organs and first-strand cDNA was then synthesized (n = 3).
Figure 1Verification of screen hits by mtKeima-based FACS analysis of mitophagy. Upper: representative FACS analysis of mitophagy in HeLa cells expressing sgNTC and sgFBXL4. Bottom: quantitative analysis of mitophagy levels in HeLa cells expressing the indicated sgRNAs. The percentage of cells with high mitophagy level is indicated in red. Two independent sgRNAs were used for each gene. NTC: non-targeting control.Imaging analysis of mitophagy levels in the indicated HeLa cells. Left: sequencing result of WT and FBXL4-KO cells and representative mitophagy images, white arrows point to mitolysosomes (FBXL4-KO cells were not labeled because of the large numbers of mitolysosomes); middle: percentage of cells with mitophagy (mitolysosome-positive); right: quantification of mitolysosome/mitochondria area. n: number of cells analyzed (middle); number of imaging areas (20-30 cells/area) analyzed (right).FACS analysis of mitophagy levels in the indicated HeLa cells. Left: representative FACS results; right: quantitative analysis of mitophagy levels.
Figure 2Immunoblot analysis of the indicated HeLa cells. B: BNIP3; N: NIX. Two clones were shown for each double and triple knockout cells.Representative live cell imaging (E) of mitophagy levels in the indicated HeLa cells. n: number of cells analyzed (left); number of imaging areas analyzed (right).
Figure 3Determination of the submitochondrial localization of FBXL4 by Protease K and trypsin digestion. Mitochondria from HeLa FBXL4-FLAG stable line were purified and stored as intact mitochondria or treated with hypotonic swelling buffer or lysed with Triton X-100 buffer. Different mitochondrial preparations were then digested with Protease K or trypsin.Analysis of the association of FBXL4 with membrane by alkaline carbonate extraction. Purified mitochondria from HeLa FBXL4-FLAG stable line were treated with alkaline carbonate buffer at the indicated pH and then centrifuged to collect the supernatant and the pellet fractions. S: supernatant; P: pellet.Determination of the submitochondrial localization of FBXL4(1-23)-RFP by Protease K digestion. Experiments were performed similarly as (A).
Figure 4Immunoprecipitation analysis of the FBXL4-Skp1-Cullin1 (SCF-FBXL4) complex. HeLa cells were infected with lentiviruses expressing the indicated genes. ∆F: the F-box deletion mutant of FBXL4-FLAG; 4A: FBXL4-4A mutant as shown in (A).Immunoprecipitation analysis of the ubiquitination of BNIP3. FLAG-BNIP3 (knockin), FBXL4-KO, HA-ubiquitin (Ub) HeLa cells were rescued with WT or ∆F FBXL4, and treated with DMSO or MG132 (20 μM) for 8 hours.   Immunoprecipitation analysis of the ubiquitination of NIX. FLAG-NIX (knockin), FBXL4-KO, HA-Ub HeLa cells were treated the same as (F).  Immunoblot analysis of the indicated HeLa cells. FBXL4-KO HeLa cells were rescued with vector, FBXL4-FLAG, FBXL4(4A)-FLAG or FBXL4(∆F)-FLAG.
Figure 5Immunoblot analysis of the indicated HeLa cells. WT and FBXL4-KO HeLa cells were rescued with vector, WT or mutant FBXL4-FLAG.Immunoprecipitation analysis of FBXL4-substrate interaction. FBXL4-KO HeLa cells were rescued with vector, FBXL4(∆F)-FLAG or mutant FBXL4(∆F)-FLAG.Immunoprecipitation analysis of the integrity of the SCF-FBXL4 complex. Upper: FBXL4-KO HeLa cells expressing vector, WT or mutant FBXL4-FLAG were crosslinked with DSP and subject to anti-FLAG immunoprecipitation; lower: quantification of the ratio of Cullin1/FLAG band intensities.
Figure 6Viability of the indicated mice. n: number of mice analyzed.   Immunoblot analysis of organs from the indicated mice at P0 of age. Elevated BNIP3 and NIX levels are highlighted by red boxes; reduced mitochondrial proteins are highlighted by blue boxes. Three mice for each genotype were analyzed. Protein samples from HeLa cells treated with DMSO or Actinomycin D (ActD) were used as positive control for PARP cleavage.  Live cell confocal imaging of mitophagy level in the indicated mouse liver. Left: representative images. The dashed line indicates cell boundary. Right: quantitative analysis of mitophagy levels in hepatocytes. n: number of cells analyzed. Data are mean ± SD. Statistics: two-tailed unpaired Student's t-test; ***P < 0.001.
Figure 7Viability of the indicated mice. n: number of mice analyzed.Immunoblot analysis of liver samples from the indicated mice at P0 of age. Left: immunoblot analysis, three mice for each genotype were analyzed; right: quantification of the ratio of mitochondrial protein/actin band intensities. Data are mean + SD from three biological replicates. Statistics: two-tailed unpaired Student's t-test; *P < 0.05; **P < 0.01; ***P < 0.001; ns, not significant.