EMBO/SourceData huggingface dataset:
Figure 4Detection of PUAR in the nuclear and non-nuclear fractions by RT-qPCR. U6 and tRNA were used as RNA markers for the nuclear and non-nuclear fractions, respectively. The seedlings were grown under white light for 6 d, and then maintained under white light, or transferred to shade for 4 h. WL, white light (R = 20 µE, B = 20 µE, FR = 5 µE). SH, shade (R = 10 µE, B = 5 µE, FR = 60 µE). Different letters indicate statistically significant differences (P-value < 0.05) by one-way ANOVA with Tukey's HSD test. The data shown are the mean ± SDs (n = 4, n refers to technical replicates).Remaining levels of PHYA and GAPDH at different time points post 1 mM cordycepin in Col-0 and PUAR-S1ox lines. 6 d-old seedlings were shade-treated for 4 h prior to cordycepin treatment. SH, shade (R = 10 µE, B = 5 µE, FR = 60 µE). Data are presented as means ± SD (n = 3, n refers to biological replicates).PIF7 specifically binds to PUAR in vitro. Purified TF-His-PIF7 from E. coli was incubated with the biotinylated PUAR at 4 °C for 1 h, and then non-biotinylated PUAR was added according to the dose as shown in the figure at 4 °C for 1 h. These reaction systems were further incubated with streptavidin-coupled agarose beads in the reaction mix to isolate RNA complex at 4 °C for 2 h.Interaction between PUAR and PIF7 was determined using an in vivo pull-down assay. RNA-protein extracts were from plants that overexpressed PIF7-Flash*Col-0 and PIF7-Flash*PUAR-S1ox, respectively. The seedlings were grown under white light for 6 d, and then maintained under white light or transferred to shade for 4 h. WL, white light (R = 20 µE, B = 20 µE, FR = 5 µE). SH, shade (R = 10 µE, B = 5 µE, FR = 60 µE). Streptavidin-coupled agarose beads were added in the reaction mix to isolate RNA complex for 2 h.
Figure 5Relative expression levels of PHYA in Col-0, pif7-1, and PIF7-Flash by RT-qPCR. The seedlings were grown under white light for 6 d, and then maintained under white light or transferred to shade for 1 h. WL, white light (R = 20 µE, B = 20 µE, FR = 5 µE). SH, shade (R = 10 µE, B = 5 µE, FR = 60 µE). Different letters indicate statistically significant differences (P-value < 0.05) by one-way ANOVA with Tukey's HSD test. The data shown are the mean ± SDs (n = 3, n refers to biological replicates).   Relative expression levels of PHYA in Col-0, phyB-9, and pif7-1*phyB-9 by RT-qPCR. The seedlings were grown under white light for 6 d. WL, white light (R = 20 µE, B = 20 µE, FR = 5 µE). Different letters indicate statistically significant differences (P-value < 0.05) by one-way ANOVA with Tukey's HSD test. The data shown are the mean ± SDs (n = 3, n refers to biological replicates).  PIF7 specifically binds to 5′ UTR of the PHYA in vitro. Purified His-PIF7 from E. coli was incubated with the biotinylated 5′ UTR of the PHYA fragment at 4 °C for 1 h, and then non-biotinylated 5′ UTR of the PHYA fragment was added according to the dose, as shown in the figure, at 4 °C for 1 h. These reaction systems were further incubated with streptavidin-coupled agarose beads in the reaction mix to isolate DNA complex for at 4 °C for 1 h.Hypocotyl phenotypes of Col-0, pif7-1, phyA-211, and pif7-1*phyA-211 under WL and SH. The seedlings were grown under white light for 4 d, and then maintained under white light or transferred to shade for 3 d. WL, white light (R = 20 µE, B = 20 µE, FR = 5 µE). SH, shade (R = 10 µE, B = 5 µE, FR = 60 µE). The scale bar represents 2 mm. Different letters indicate statistically significant differences (P-value < 0.05) by one-way ANOVA with Tukey's HSD test. The data shown are the mean ± SDs (n ≥ 16, n refers to biological replicates).
Figure 6Hypocotyl phenotypes of Col-0, pif7-1, PUARox, and PUARox/pif7-1. The seedlings were grown under white light for 4 d, and then maintained under white light or transferred to shade for 3 d. WL, white light (R = 20 µE, B = 20 µE, FR = 5 µE). SH, shade (R = 10 µE, B = 5 µE, FR = 60 µE). The scale bar represents 2 mm. Different letters indicate statistically significant differences (P-value < 0.05) by one-way ANOVA with Tukey's HSD test. The data shown are the mean ± SDs (n ≥ 16, n refers to biological replicates).Relative expression levels of PHYA in Col-0, pif7-1, PUARox, and PUARox/pif7-1 by RT-qPCR. The seedlings were grown under white light for 6 d, and then maintained under white light or transferred to shade for 4 h. WL, white light (R = 20 µE, B = 20 µE, FR = 5 µE). SH, shade (R = 10 µE, B = 5 µE, FR = 60 µE). Different letters indicate statistically significant differences (P-value < 0.05) by one-way ANOVA with Tukey's HSD test. The data shown are the mean ± SDs (n = 3, n refers to biological replicates).PUAR specifically blocks the binding of PIF7 to the 5′ UTR of PHYA in vitro. Purified TF-His-PIF7 from E. coli was incubated with the biotinylated 5′ UTR of the PHYA fragment or fragment of the PIL1 promoter at 4 °C for 1 h, and then PUAR or anti-PUAR was added according to the dose as shown in the figure, at 4 °C for 1 h. These reaction systems were further incubated with streptavidin-coupled agarose beads in the reaction mix to isolate DNA complex for at 4 °C for 1 h.I-J. Relative expression levels of PHYA and PIL1 in Col-0, PIF7-Flash, PUAR-S1ox, PIF7-Flash*Col-0, and PIF7-Flash*PUAR-S1ox by RT-qPCR. The seedlings were grown under white light for 6 d, and then maintained under white light or transferred to shade for 1 h. Different letters indicate statistically significant differences (P-value < 0.05) by one-way ANOVA with Tukey's HSD test. The data shown are the mean ± SDs (n = 3, n refers to biological replicates).
Figure 1B. Structured Illumination Microscopy (SIM) images of S. aureus cells labelled with the membrane dye Nile Red. White arrowheads point to tetrads of cells that result from deficient cell splitting. Scale bars, 1μm.D. SIM images of 8325-4∆sle1 and 8325-4∆ftsK S. aureus cells incubated with purified HisTag-Sle1-SP. External addition of the protein split the tetrads observed in sle1 and ftsK knock-out strains. Scale bars, 1μm.
Figure 2A. Absence of Sle1 in (from top to bottom) autolytic, cell wall or total cellular extracts and spent medium of 8325-4∆sle1 and 8325-4∆ftsK mutants, but not in the parental strain NCTC8325-4, was confirmed by western blot analysis using an anti-Sle1 antibody. All western blot experiments were performed with three biological replicates and representative images are shown.B. Quantification of Sle1 total cellular levels, detected by western blot (representative gel on the bottom) in S. aureus mutant 8325-4∆ftsK and ClpX inactivated mutants 8325-4 ClpXR95C and 8325-4∆ftsK ClpXR95C showing that ClpX inactivation restores the presence of Sle1 in the ftsK deletion mutant. Data information: Graphs show ratio between Sle1 amount in different samples versus the amount in parental strain NCTC8325-4 indicated by dashed line. Data represented in scatter dot plot column graphs, where column height represents mean, and error bars are the standard deviation. Data from at least four biological replicates.C. Quantification of Sle1 total cellular levels, detected by western blot (representative gel on the bottom) in FtsK C-terminal deletion (8325-4∆ftsKc) or inactivated (8325-4FtsKK971A) mutants showing that Sle1 levels increase in the absence of FtsK DNA translocase activity. Data information: Graphs show ratio between Sle1 amount in different samples versus the amount in parental strain NCTC8325-4 indicated by dashed line. Data represented in scatter dot plot column graphs, where column height represents mean, and error bars are the standard deviation. Data from at least four biological replicates.
Figure 3A. Total protein extracts of strains NCTC8325-4, 8325-4 TF-GFP, 8325-4 MurC-GFP and 8325-4 TF-GFP Sle1Tn (the latter lacking Sle1) were immunoprecipitated with GFP-Trap Magnetic Particles. Western blot analysis of the pulled-downed fractions using antibodies against Sle1 and GFP shows that Sle1 is pulled down with TF-GFP but not with negative control MurC-GFP, suggesting a specific interaction between TF and Sle1.B. Total protein extracts of strains 8325-4 TF-3XFLAG, 8325-4 GFP-FtsK TF-3XFLAG, 8325-4sfgfp-ftsK and 8325-4 TF-3XFLAG GFP-HU (Input extracts, bottom panel) were immunoprecipitated with DYKDDDDK Fab-Trap Agarose that binds FLAG-tagged proteins. Western blot analysis of the pulled-downed fractions (top panels) using antibodies against FLAG and GFP shows that GFP-FtsK, but not the negative control GFP-HU, is pulled down with TF-3XFLAG, suggesting an interaction between TF and FtsK. For each assay, a representative image of three independent biological replicates is shown.
Figure 4A. SIM images of cells expressing TF-GFP labeled with membrane dye FM5-95. TF-GFP forms a gradient towards the septum in the background of parental strain NCTC8325-4 (8325-4 TF-GFP, top panels). This localization pattern is lost in cells lacking FtsK (8325-4∆ftsK TF-GFP, middle panels), but not in 8325-4∆ftsKc TF-GFP mutant, expressing FtsK without its C-terminal domain (bottom panels). Scale bars, 1 µm.
Figure 5B. Deconvolved epifluorescence images of live cells of parental strain 8325-4 Sle1-3XFLAG ∆spa and TF deletion mutant 8325-4 Sle1-3XFLAG ∆spa ∆tig, producing Sle1-3XFLAG as the sole Sle1 copy in the cell. Normal localization pattern of Sle1 (enriched at midcell, top panel) is altered in the absence of TF (bottom panel) with the protein becoming more dispersed over the cell surface. See image S1 for more images of Sle1-3XFLAG surface localization in the parental strain and the negative control of the immunofluorescence assay. Cells were incubated with fluorescent D-amino acid HADA, which is incorporated into peptidoglycan, for cell wall visualization (colored blue). Cell surface Sle1-3XFLAG was detected with Anti-FLAG Monoclonal Antibody-Alexa Fluor 488 (colored green, projection of a 33 slices Z-stack shown). Scale bars, 1 µm.D. Frequency of cells in each cell cycle phase in the TF knockout mutant 8325-4Δtig and NCTC8325-4 parental strain, showing that the TF mutant is delayed in cell cycle phase 3, taking longer to split the division septum. Data from three biological replicates of NCTC8325-4 (Ntotal=728) and 8325-4Δtig (Ntotal=699). Data represented in column graphs where column height represents mean and error bars are the standard deviation.
Figure 6A. Quantification of Sle1 total cellular levels, detected by western blot (representative gel on the bottom), in S. aureus cultures of wild-type strain NCTC8325-4 grown in the presence (1/2 X MIC) or absence of DNA targeting antibiotics mitomycin C and nalidixic acid or protein synthesis inhibitor erythromycin. The results show a decrease in Sle1 cellular levels upon DNA replication defects or DNA damage. Graphs show ratio between Sle1 amount in different samples versus the amount in parental strain NCTC8325-4 indicated by dashed line. Data represented in scatter dot plot column graphs, where column height represents mean, and error bars are the standard deviation. Data from four biological replicates. Statistical significance based on a Mann-Whitney U test, where * p< 0.05.C. SIM image and heat map representation of TF-GFP localization in 8325-4 TF-GFP cells grown in the presence or absence of nalidixic acid, showing the loss of TF-GFP gradient towards the septum in most cells grown in the presence of the antibiotic. Scale bars, 1 µm. Heatmaps represent the average of TF-GFP localization in 50 cells in phase 2 of the cell cycle. The color scale depicts the average normalized TF-GFP fluorescence signal in each pixel, ranging between 0 and 1. The fraction of the area of each cell model where the normalized signal intensity is above 70% of the maximum signal is 0.47 and 0.61 in the absence or presence of nalidixic acid, respectively, confirming that TF-GFP becomes more dispersed over the cytoplasm in the presence of the antibiotic.
Figure 1Representative images of EGFP-Nix-FKBP (green) Rapalog-induced recruitment to mtKeima labeled mitochondria (Keima excitation 488, cyan) and subsequent mitophagy (Keima excitation 561, magenta). Scale bar: 10 µm. Data Information: Area shading indicates standard deviation between wells. Over one thousand cells quantified per condition.Confirmation of decreased mitochondrial proteins in WT but not in FIP200 ko cells expressing CID Nix either untreated or treated for 24 hours with Rapalog by immunoblotting. Quantification of MTCO2 relative to Actin over three independent experiments after treatment with Rapalog over 24 hours by immunoblotting. Cells in this figure express mtKeima-P2A-FRB-Fis1tTand EGFP-Nix1-188-FKBP. Data Information: Area shading indicates standard deviation between wells. Over one thousand cells quantified per condition. Statistics performed by two-way ANOVA; ** indicates a p-value<0.01, *** indicates a p-value<0.001. Experiments repeated at least 3 times.
Figure 2Mitophagy time course by mtKeima of different Nix truncation mutants recruited with CID, measured by percentage of Keima positive pixels above threshold. Data Information: Area shading indicates standard deviation between wells. Over one thousand cells quantified per condition. Experiments repeated at least 3 times.Mitophagy by mtKeima of Nix1-70 when treated with Torin, Rapalog, or both compared to control. Mitophagy by mtKeima of Nix1-87 when treated with Torin, Rapalog, or both compared to control. Data Information: Area shading indicates standard deviation between wells. Over one thousand cells quantified per condition. Experiments repeated at least 3 times.
Figure 3Volcano plot generated from one-tailed t-test of proteins that interact with Nix1-87 more so than Nix1-70 by IP-mass spectrometry. Red dots indicate proteins over 2-fold enrichment and p-value<0.05 Data Information: Statistics: one-tailed unpaired Student's ﻿t-test. Fold change calculated from spectral counts (sc) by (sc1-87+1)/(sc1-70+1) so that zero values could be included.IP western blot of FKBP-EGFP-Nix1-188, Nix1-87, or Nix1-70, comparing 1% input to GFP-pulldown and blotted against GFP or WIPI2 after 4 hours of Rapalog. Data Information: Statistics: one-tailed unpaired Student's ﻿t-test. Fold change calculated from spectral counts (sc) by (sc1-87+1)/(sc1-70+1) so that zero values could be included. IP Experiments repeated at least 3 times.HeLa cells expressing FKBP-EGFP- p62327-348 or FKBP-EGFP-WIPI2b were treated for 16 hours with phenanthroline to induce endogenous Nix and BNIP3 expression, then underwent a GFP-pulldown and compared to 1% input by immunoblot. Data Information IP Experiments repeated at least 3 times.IP western blot of FKBP-EGFP-Nix1-188 compared to Nix60-188, comparing 1% input to GFP-pulldown and blotted against GFP or WIPI2 after 4 hours of Rapalog. Data Information IP Experiments repeated at least 3 times.
Figure 4Representative images of WT or 6ko HeLa cells expressing EGFP-Nix1-188-FKBP (green), mtKeima, and FRB-Fis1T with or without 4 hours of Rapalog, immunostained for endogenous Tom20 (red) and WIPI2 (white). Scale bar: 50µm. Automated puncta counting of WIPI2 in WT or 6ko cells normalized to cell number in control conditions or after 4 hours with Rapalog. Data Information: Error bars indicate standard deviation. Statistics performed by two-way ANOVA; ** indicates a p-value<0.01, *** indicates a p-value<0.001. ns indicates not significant. Over one thousand cells quantified per condition. Experiments repeated at least 3 times.Representative images of WT or WIPI2 ko HeLa cells expressing EGFP-Nix1-188-FKBP (green), mtKeima, and FRB-Fis1T with or without 4 hours of Rapalog immunostained for endogenous Tom20 (red) and GABARAP (white). Scale bar: 50µm. Automated puncta counting of GABARAP in WT or WIPI2 ko cells normalized to cell number in control conditions or after 4 hours with Rapalog. Data Information: Error bars indicate standard deviation. Statistics performed by two-way ANOVA; ** indicates a p-value<0.01, *** indicates a p-value<0.001. ns indicates not significant. Over one thousand cells quantified per condition. Experiments repeated at least 3 times.GFP pulldown of EGFP-Nix1-188-FKBP in WT, WIPI2 ko, or FIP200 ko cells after 4 hours of Rapalog treatment. FIP200 cropped due to the appearance of non-specific bands after IP. Data Information: Error bars indicate standard deviation. Statistics performed by two-way ANOVA; ** indicates a p-value<0.01, *** indicates a p-value<0.001. ns indicates not significant. Over one thousand cells quantified per condition. Experiments repeated at least 3 times.
Figure 5Representative images of endogenous Halo -WIPI2 in U2OS cells and stained with JF646 Halo dye (magenta) expressing mtKeima (cyan), FRB-Fis1T, and FKBP-EGFP-Nix1-87­, FKBP-EGFP-Nix60-188, or FKBP-EGFP-Nix1-70 (green) after 16 hours of Rapalog treatment. Data Information: Error bars indicate standard deviation between wells. Over one thousand cells quantified per condition. Statistics performed by two-way ANOVA;* indicates a p-value<0.05, ** indicates a p-value<0.01, **** indicates a p-value<0.0001. ns indicates not significant. Experiments repeated at least 3 times.Automated puncta counting of endogenous Halo -WIPI2 in U2OS cells expressing either FKBP-EGFP-Nix1-87 (LIR and MER), Nix60-188 (MER), or Nix1-70 (LIR) after 16 hours of Rapalog treatment then fixed. Data Information: Error bars indicate standard deviation between wells. Over one thousand cells quantified per condition. Statistics performed by two-way ANOVA;* indicates a p-value<0.05, ** indicates a p-value<0.01, **** indicates a p-value<0.0001. ns indicates not significant. Experiments repeated at least 3 times.
Figure 7Percent cells above threshold line shown in panel A as indication of mitophagy measured by mtKeima after one week of stably expressing EGFP, or EGFP-Nix as WT or with W36A/L39A or L75A mutations. Data Information: Error bars indicate standard deviation between wells. Over one thousand cells quantified per condition. Statistics: Panel B - two-tailed unpaired student's t-test ** indicates a p-value<0.01, **** indicates a p-value<0.0001. ns indicates not significant. Experiments repeated at least 3 times.Automated puncta counting of endogenous Halo -WIPI2 in U2OS cells expressing FKBP-EGFP-Nix1-188 as WT or with W36A/L39 (LIR) or L75A (MER) mutations after 16 hours of Rapalog treatment then fixed. Data Information: Error bars indicate standard deviation between wells. Over one thousand cells quantified per condition. Statistics: panels C two-way ANOVA; ** indicates a p-value<0.01, **** indicates a p-value<0.0001. ns indicates not significant. Experiments repeated at least 3 times.GFP pulldown of EGFP alone, EGFP-Nix1-188-FKBP as WT, W36A/L39A, or L75A mutants after 4 hours of Rapalog treatment.GFP pulldown of FKBP-EGFP-WIPI2 as WT or with R108E/R125E mutations after 24 hours of Phenanthroline treatment.
Figure 1S2 cells were incubated at 21% or 1% O2 concentrations for 24h hours. A) Puromycin was added 20 min. before cell harvesting. Total puromycin incorporation and Actin level were revealed by western blot.S2 cells were incubated at 21% or 1% O2 concentrations for 24h hours. (C) LDH and RpL32 protein levels were detected by western blot in total cell extracts from S2 cells exposed to normoxic conditions or to 1% O2 environment for 24 and 48 hours.S2 cells were incubated at 21% or 1% O2 concentrations for 24h hours. (E) One representative experiment performed as in (D) is shown.
Figure 2S2 cells were transiently transfected with the indicated constructs and exposed to 1% O2 for 24h. (A, B) (A) Activities of Firefly (Fluc) and Renilla (Rluc) luciferase, produce from reporter genes under control of the Ldh gene promoter, were measured in total cell extracts by dual luciferase assay and the ratio of both measures was calculated for each transfection. (B) Relative FLuc and RLuc mRNA levels were measured by RT-qPCR.S2 cells were transiently transfected with the indicated constructs and exposed to 1% O2 for 24h. (D) Indicated stably transfected cell lines were exposed to 21% or 1% O2 for 24h before fractionation of cell extracts by ultracentrifugation on linear sucrose gradients (15%-50% The distribution of FLuc mRNA, produced from reporter genes under control of the pMT promoter was measured by northern blot in each cell types. Fractions numbers from top to bottom of the gradient are indicated.
Figure 3(A) Control (TM3>UAS-eIF4EHP_RNAi) or eIF4EHP KD (Tub-GAL4>UAS-eIF4EHP_RNAi) flies were maintained in either normoxia or hypoxia (1%O2, 24h). Protein extracts were prepared from whole flies and eIF4EHP, LDH and Actin were detected by western blot. (B) Quantification of LDH/Actin ratio in 3 independent experiments performed as in A. Values represent mean +/- SD, n=3 biological replicates, P-value of two-ways ANOVA.(C) S2 cells lines inactivated for Yellow or eIF4EHP genes were established by a CRISPR-Cas9 strategy as described in the methods section. After exposure to normoxic or hypoxic conditions, cells were lysed and Actin, LDH and eIF4EHP protein levels were detected by western blot. (D) Quantification of LDH/Actin ratio in 6 independent experiments performed as in C. Values represent mean +/- SD, P-values of two-way ANOVA.(E) Cell extracts from Yellow, eIF4EHP or eIF4E6 KO cells incubated at 21 or 1% O2 were separated by centrifugation on linear sucrose gradients and Ldh mRNA was detected by northern blot in the different fractions. Fractions numbers from top to bottom of the gradient are indicated.(G) Indicated S2 cell lines were cultivated at 1% O2 for 24h and labelled with puromycin (5µg/ml) for 20 min. before harvesting. Puromycin signal in total cell extracts was quantified by western blot and expressed relative to the level detected in wild-type cells (n=6 biological replicates, t-test, two-sided).
Figure 4B) Polysome profiles of FLuc-Ldh 3'UTR and Fluc-CA-rich 3'UTR reporter mRNA in control (Yellow KO) and eIF4EHP KO S2 cells in hypoxia.(D) WT and eIF4EHP KO cells were co-transfected with plasmids encoding V5-tagged D. persimilis eIF4EHP (Dp eIF4EHP) or GFP, along with FLuc-Ldh3'UTR and RLuc reporters. eIF4EHP proteins were detected by western blot using eIF4EHP antibody (cross-reacting to Dp eIF4EHP).(E, F) 24h after transfection, cells were cultivated in hypoxia (1%O2) for an additional 24h before measurement of luciferase activity by dual luciferase reporter assay (D) and luciferase mRNA levels by qPCR (E). Values represent mean ± SD, n=3 biological replicates, P-value of one-way ANOVA, *-P ≤ 0.05, ns- P>0.05.
Figure 5(C) Combined detection of eIF4EHP by immunofluorescence (green) and polyA mRNA by oligo-dT FISH (red) in normoxic and hypoxic WT S2 cells. nuclei were counterstained with DAPI (blue). Red and green signal intensities were quantified with the Zen software (Zeiss).(D) Combined detection of GFP-Rox8 or GFP-Dcp1 (green) and endogenous eIF4EHP (red) in transfected S2 cells cultivated in normoxia (21% O2), in hypoxia (1% O2 for 24h) or after oxidative stress (250μM arsenite for 1h). Representative images of confocal imaging (Z-stack maximum intensity projection) and zooms are shown. , nuclei were counterstained with DAPI (blue). Red and green signal intensities were quantified with the Zen software (Zeiss).