EMBO/SourceData huggingface dataset:
Figure 5Growth of indicated Y. pestis strains in zinc-limited medium (cPMH2) (n=3), One-way ANOVA with Dunnett test compared to znuBC mutant, **p≤0.01, ****p≤0.0001. Data represents the mean ±SD of three biological replicates. For some points, error bars are too small to visualize on the graph.
Figure 1A. Representative confocal micrographs of HeLa cells infected or not with B. abortus 544 for the indicated times, then fixed and immunostained for TOMM20 (Alexa Fluor 488 - Green) and B. abortus LPS (Alexa Fluor 568 - Magenta). DNA was stained with Hoechst 33258 (Blue). Scale bars: 20 µm.
Figure 2A. Representative confocal micrographs of HeLa cells infected or not with B. abortus 544 GFP for 48 h, then fixed and immunostained for the β-subunit of the ATP synthase (Alexa Fluor 633 - Magenta) and LC3 (Alexa Fluor 568 - Green). DNA was stained with Hoechst 33258 (Blue). Arrows indicate LC3-ATP synthase β-positive punctae. Scale bars: 20 µm. B. Quantification of the number of LC3-β-subunit of the ATP synthase-positive punctae per HeLa cell infected or not (NI) with B. abortus 544 GFP for 24, 48 and 72 h from micrographs shown in (A) Data are presented as means ± SD from n=3 (biological replicates independent experiments (the numbers indicated in the columns represent the number of cells analysed per condition). Statistical analyses were performed using a two-way ANOVA followed by a Šidàk's multiple comparisons test; asterisks indicate significant differences compared to the control (NI); ns: not significant; ***: p <0.001; ****: p <0,0001; hashtags indicate significant differences between infected conditions throughout time; #: p <0.05; ##: p <0.01.C. Representative confocal micrographs of HeLa cells transfected with a FIS1-GFP(Green)-mCherry(Magenta) expression construct, infected or not with B. abortus 544 for 48 h, then fixed and immunostained for B. abortus LPS (Alexa Fluor 633 - Red). DNA was stained with Hoechst 33258 (Blue). Arrows indicate FIS1-mCherry-positive-GFP-negative punctae. Scale bars: 20 µm. D. Quantification of the number of FIS1-mCherry-positive-GFP-negative punctae per HeLa cell infected or not (NI) with B. abortus 544 for 48 h from micrographs shown in (C). Data are presented as means ± SD from n=3 (biological replicates) independent experiments (the numbers indicated in the columns represent the number of cells analysed per condition). Statistical analyses were performed using an unpaired two-tailed Student's t-test; ***: p <0.001 (p = 0.0008).
Figure 3A. Representative confocal micrographs of HeLa cells infected or not with B. abortus 544 GFP (Red) for 24 h, 48 h, and 72 h then fixed and immunostained for HIF-1α (Alexa 568 - Green). DNA was stained with Hoechst 33258 (Blue). B. Quantification of the percentages of cells positive for a nuclear localisation of HIF-1α from HeLa cells infected or not (NI) with B. abortus 544 GFP for 24, 48 and 72 h from micrographs shown in (A.B.C). Data are presented as means ± SD from n=3 (biological replicates) independent experiments (the numbers indicated in the columns represent the number of cells analysed per condition). Statistical analyses were performed using a two-way ANOVA followed by a Šidàk's multiple comparisons test; asterisks indicate significant differences compared to the control (NI); ****: p <0.0001.C. Representative confocal micrographs of HeLa cells infected or not with B. abortus 544 GFP (Red) for 48 h, then fixed and immunostained for BNIP3L (Alexa 568 - Green) and TOMM20 (Alexa 633 - Magenta). DNA was stained with Hoechst 33258 (Blue). D. Relative median fluorescence intensity (MFI) of BNIP3L immunostaining from HeLa cells infected or not (NI) with B. abortus 544 GFP for 48 h as measured by flow cytometry. Data are presented as means ± SD from n=4 (biological replicates) independent experiments (8,296 cells analysed in total per condition). Statistical analyses were performed using a one sample t-test; *: p <0.05 (p = 0.0351).
Figure 4D. Representative confocal micrographs of HeLa cells infected or not (NI) with B. abortus 544 GFP (Red) treated or not (ctrl) with 500 µM FeCl2 for 48 h, then fixed and immunostained for BNIP3L (Alexa 568 - Green). DNA was stained with Hoechst 33258 (Blue). E. Relative median fluorescence intensity (MFI) of BNIP3L immunostaining from HeLa cells infected or not (NI) with B. abortus 544 GFP treated or not (ctrl) with 500 µM FeCl2 for 48 h as measured by flow cytometry. Data are presented as means ± SD from n=3 (biological replicates) independent experiments (10,199 cells analysed in total per condition). Statistical analyses were performed using a two-way ANOVA followed by a Šidàk's multiple comparisons test; asterisks indicate significant differences compared to the control (NI); ns: not significant; *: p <0,05; hashtags indicate significant differences compared to the infected condition without FeCl2; #: p <0.05.F. Representative confocal micrographs of iBMDM infected or not (NI) with B. abortus 544 GFP (Red) treated or not (ctrl) with 500 µM FeCl2 for 48 h, then fixed and immunostained for BNIP3L (Alexa 568 - Green). DNA was stained with Hoechst 33258 (Blue). G. Relative median fluorescence intensity (MFI) of BNIP3L immunostaining from iBMDM infected or not (NI) with B. abortus 544 GFP treated or not (ctrl) with 500 µM FeCl2 for 48 h as measured by flow cytometry. Data are presented as means ± SD from n=3 (biological replicates) independent experiments (14,152 cells analysed in total per condition). Statistical analyses were performed using a two-way ANOVA followed by a Šidàk's multiple comparisons test; asterisks indicate significant differences compared to the control (NI); ns: not significant; *: p <0,05; hashtags indicate significant differences compared to the infected condition without FeCl2; ###: p <0.001.
Figure 5A. Representative confocal micrographs of HeLa cells transfected with a non-targeting siRNA pool (siNT - 40 nM) or a BNIP3L siRNA SMARTpool (siBNIP3L - 40 nM), infected or not (NI) with B. abortus 544 GFP (Red) for 48 h, then fixed and immunostained for BNIP3L (Alexa Fluor 568 - Magenta) and TOMM20 (Alexa Fluor 633 - Green). DNA was stained with Hoechst 33258 (Blue). B.,C. Quantification of the mitochondrial population morphology by assessing the AR (B) and EBR (C) of the mitochondria of HeLa cells from micrographs shown in (A). Data are presented as means ± SD from n=3 (biological replicates) independent experiments (the numbers indicated in the columns represent the number of cells analysed per condition). Statistical analyses were performed using a multiple Mann-Whitney test followed by a Holm-Šidàk's multiple comparisons test; asterisks indicate significant differences compared to the control (NI); *: p <0.05; **: p <0.01; hashtags indicate significant differences compared to the infected condition transfected with a siNT; #: p <0.05.D. Representative confocal micrographs of HeLa cells transfected with a FIS1-GFP(Green)-mCherry(Magenta) expression construct, infected or not (NI) with B. abortus 544 for 48 h while being transfected with a non-targeting siRNA pool (siNT - 40 nM) or a BNIP3L siRNA SMARTpool (siBNIP3L - 40 nM), then fixed and immunostained for B. abortus LPS (Alexa Fluor 633 - Red). DNA was stained with Hoechst 33258 (Blue). Arrows indicate FIS1-mCherry-positive-GFP-negative punctae. Scale bars: 20 µm. E. Quantification of the number of FIS1-mCherry-positive-GFP-negative punctae per cell of HeLa cells from micrographs t shown in (D). Data are presented as means ± SD from n=3 (biological replicates) independent experiments (the numbers indicated in the columns represent the number of cells analysed per condition). Statistical analyses were performed using a two-way ANOVA followed by a Šidàk's multiple comparisons test; asterisks indicate significant differences compared to the control (NI); ns: not significant; **: p <0.01; hashtags indicate significant differences compared to the infected condition transfected with a siNT; #: p <0.05.
Figure 6A. Representative confocal micrographs of HeLa cells infected with B. abortus 544 GFP (Green) and transfected with a non-targeting siRNA pool (siNT - 40 nM) or a BNIP3L siRNA SMARTpool (siBNIP3L - 40 nM) for 48 h, then incubated under reinfection-permissive conditions for 24 h before analysis at 72 h pi. Cells were fixed and DNA was stained with Hoechst 33258 (Blue). Arrows indicate reinfected cells. B. Quantification of the percentages of reinfection foci per infected cell at 72 h pi of HeLa cells from micrographs shown in (A). Data are presented as means ± SD from n=3 (biological replicates) independent experiments (the numbers indicated in the columns represent the number of cells analysed per condition). Statistical analyses were performed using an unpaired two-tailed Student's t-test; **: p <0.01 (p = 0.0032). C. CFU assay expressing Log (CFU/ml) from the supernatant of HeLa cells infected with B. abortus 544 GFP (Green) and transfected with a non-targeting siRNA pool (siNT - 40 nM) or a BNIP3L siRNA SMARTpool (siBNIP3L - 40 nM) for 48 h, then incubated under reinfection-permissive conditions for 24 h before collecting the supernatant at 72 h pi for analysis. Data are presented as means ± SD from n=5 (biological replicates) independent experiments; Statistical analyses were performed using an unpaired two-tailed Student's t-test; (p = 0.0042).D. Representative confocal micrographs of HeLa cells infected with B. abortus 544 GFP (Red) and transfected with a non-targeting siRNA pool (siNT - 40 nM) or a BNIP3L siRNA SMARTpool (siBNIP3L - 40 nM) for 72 h pi, then fixed and immunostained for LAMP-1 (Alexa Fluor 568 - Green). DNA was stained with Hoechst 33258 (Blue). Arrows indicate LAMP-1-positive BCVs (aBCVs). Scale bars: 20 µm. E. Quantification of the number of LAMP-1-positive BCVs (aBCVs) per infected HeLa cells from micrographs shown in (E). Data are presented as means ± SD from n=3 (biological replicates) independent experiments (the numbers indicated in the columns represent the number of cells analysed per condition). Statistical analyses were performed using an unpaired two-tailed Student's t-test; *: p <0.05 (p = 0.0117).
Figure 7A. Representative confocal micrographs of HeLa cells infected with B. abortus 544 GFP (Green) treated or not (ctrl) with FeCl2 (500 µM) for 48 h, then incubated under reinfection-permissive conditions (with or without FeCl2) for 24 h before analysis at 72 h pi. Cells were fixed and DNA was stained with Hoechst 33258 (Blue). Arrows indicate reinfected cells. B. Quantification of the percentages of reinfection foci per infected cell at 72 h pi of HeLa cells from micrographs shown in (A). Data are presented as means ± SD from n=3 (biological replicates) independent experiments (the numbers indicated in the columns represent the number of cells analysed per condition). Statistical analyses were performed using an unpaired two-tailed Student's t-test; ***: p <0.001 (p = 0.0008).C. Representative confocal micrographs of HeLa cells infected or not with B. abortus 544 GFP (Magenta) treated or not (ctrl) with FeCl2 (500 µM) for 72 h, then fixed and immunostained for LAMP-1 (Alexa Fluor 568 - Green). DNA was stained with Hoechst 33258 (Blue). Arrows indicate LAMP-1-positive BCVs (aBCVs). D. Quantification of the number of LAMP-1-positive BCVs (aBCVs) per infected HeLa cells from micrographs shown in (C). Data are presented as means ± SD from n=3 (biological replicates) independent experiments (the numbers indicated in the columns represent the number of cells analysed per condition). Statistical analyses were performed using an unpaired two-tailed Student's t-test; *: p <0.05 (p = 0.0368).E. Representative confocal micrographs of iBMDM infected with B. abortus 544 GFP (Green) treated or not (ctrl) with FeCl2 (500 µM) for 48 h, then incubated under reinfection-permissive conditions (with or without FeCl2) for 24 h before analysis at 72 h pi. Cells were fixed and DNA was stained with Hoechst 33258 (Blue). Arrows indicate reinfected cells. F. Quantification of the percentages of reinfection foci per infected cell at 72 h pi of iBMDM from micrographs shown in (E). Data are presented as means ± SD from n=3 (biological replicates) independent experiments (the numbers indicated in the columns represent the number of cells analysed per condition). Statistical analyses were performed using an unpaired two-tailed Student's t-test; **: p <0.01 (p = 0.0043).
Figure 8A. Representative confocal micrographs of HeLa cells infected with B. abortus 544 GFP (Magenta) for 48 and 72 h, then fixed and immunostained for TOMM20 (Alexa Fluor 647 - Green). DNA was stained with Hoechst 33258 (Blue). Arrows indicate when B. abortus was found inside a mitochondrion (mBCVs). Scale bars: 20 µ B. Quantification of the percentage of infected HeLa cells displaying TOMM20-positive BCVs (mBCVs) at the indicated times, from micrographs shown in (A). Data are presented as means ± SD from n=3 (biological replicates) independent experiments (the numbers indicated in the columns represent the number of cells analysed per condition). Statistical analyses were performed using an unpaired two-tailed Student's t-test; ***: p <0.001 (p = 0.0001). C. Quantification of the number of TOMM20-positive BCVs (mBCVs) per infected HeLa cells, at the indicated times, from micrographs shown in (E). Data are presented as means ± SD from n=3 (biological replicates) independent experiments (the numbers indicated in the columns represent the number of cells analysed per condition). Statistical analyses were performed using an unpaired two-tailed Student's t-test; *: p = 0.05.D. Representative confocal micrographs of iBMDM infected with B. abortus 544 GFP (Magenta) for 48 and 72 h, then fixed and immunostained for TOMM20 (Alexa Fluor 647 - Green). DNA was stained with Hoechst 33258 (Blue). Arrows indicate when B. abortus was found inside a mitochondrion (mBCVs). Scale bars: 20 µm. E. Quantification of the percentage of infected iBMDM displaying TOMM20-positive BCVs (mBCVs) at the indicated times, from micrographs shown in (D). Data are presented as means ± SD from n=3 (biological replicates) independent experiments (the numbers indicated in the columns represent the number of cells analysed per condition). Statistical analyses were performed using an unpaired two-tailed Student's t-test; ***: p <0.001 (p = 0.0009). F. Quantification of the number of TOMM20-positive BCVs (mBCVs) per infected HeLa cells, at the indicated times, from micrographs shown in (E). Data are presented as means ± SD from n=3 (biological replicates) independent experiments (the numbers indicated in the columns represent the number of cells analysed per condition). Statistical analyses were performed using an unpaired two-tailed Student's t-test; ns: not significant (p = 0.3739).
Figure 1(B) Detection of S-acylation of BSKs in plant cells. 12 BSK proteins were fused with GFP at their C terminus and expressed in Arabidopsis protoplasts for 24 hours and the cells were collected for Biotin-switch assay. Cell lysates are shown as input, and the S-acylated proteins enriched on the resin which is dependent on hydroxylamine (NH2OH) are indicated as pulldown samples. All images of anti-GFP immunoblots are representative of three biological independent experiments.(D) Effect of 2-BP on subcellular localization of BSK1 in root cells of intact transgenic plants. Six-day-old seedlings of 35S:BSK1-GFP were treated with 80 μM 2-BP for 2 h; DMSO was supplemented in the control samples. Representative GFP (green) and merged (with bright field) signals from three biologically independent experiments are shown. Bars, 10 µm.(E) Effect of 2-BP on the localization of BSK1-GFP in transgenic plants using a cell fractionation assay. Six-day-old seedlings were treated with 80 μM 2-BP (DMSO was used in the control sample) for 3 h before protein extraction. Total proteins (T) were divided into soluble (S) and pellet (P) fractions using ultra-centrifugation. The representative immunoblot using an anti-GFP antibody is shown.
Figure 2(A, B) Identification of S-acylation sites on BSK1 and BSK3. The wild-type (WT) and indicated mutant versions of BSK1-GFP (A) and BSK3-GFP (B) were expressed in protoplasts for biotin-switch assay. Images of anti-GFP immunoblots are representative of three biological independent experiments.(E, F) Effect of S-acylation site mutation on the localization of BSK1 and BSK3 in intact root cells. Six-day-old seedlings of the WT or S-acylation site mutant versions of 35S:BSK1-GFP (E) and 35S:BSK3-GFP (F) were subjected to localization analysis using confocal microscopy. Representative GFP (green) and merged (with bright field) signals in root cells from three biologically independent experiments are shown. Bars, 10 µm.(G, H) The effect of S-acylation site mutations on the localization of BSK1 and BSK3 in a cell fractionation assay. The WT and mutant versions of BSK1-GFP (G) and BSK3-GFP (H) were expressed in protoplasts and the total proteins (T) were divided into soluble (S) and pellet (P) fractions using ultra-centrifugation. Representative immunoblotting data using an anti-GFP antibody are shown.
Figure 3(B) Co-IP result for measuring the function of BSK1 S-acylation in its interaction with BRI1. Free GFP (negative control) or the WT/C3S version of BSK1-GFP was co-expressed with BRI1-MYC in plant cells for co-IP using an anti-GFP resin. The proteins in the input and IP samples were detected via immunoblots with anti-MYC or anti-GFP antibodies.(E, F) The effect of S-acylation on the function of BSK3 in BR responses. The WT or 3M (C5, 10, 11S) version of BSK3 driven by a native promoter was introduced into the bsk3-1 mutant to generate complementation lines. The phenotype of 8-day-old seedlings with and without 100 nM of BL treatment is shown in (E). Bars, 1 cm. Quantitative data of hypocotyl and root length under the control or BL treatment condition are shown in (F). Data are means ± SD from 20 seedlings in four independent experiments. Significance analysis using one-way ANOVA followed by Tukey's multiple comparison test is shown in different lower-case letters (P < 0.05).
Figure 4(C) Detection of the regulation of BSK1 S-acylation mediated by SA. BSK1-GFP was expressed in protoplasts with 0-, 20-, 40-, or 60-min treatment with 100 µM of SA. Cells were collected for S-acylation detection in a biotin-switch assay. Representative immunoblots are shown in the top graph. Immunoblot signals were quantified by ImageJ and the S-acylation levels were calculated from relative signals ([pulldown+/input+] - [pulldown-/ input-]). The relative S-acylation level of the 0 min sample was set to 1. The data in the bottom graph are means ± SD from three biologically independent experiments. Significance analysis using one-way ANOVA followed by Tukey's multiple comparison test is shown in different lower-case letters (P < 0.05).(D, E) Effect of SA on the physiological function of BSK1. The indicated seeds were germinated on the MS medium with or without 40 µM of SA. The photographs were taken 8 days after germination. Representative images are shown in (D). Bars, 1 cm. Quantitative data of fresh weight (per 10 seedlings) of different genotypes are shown in (E). Data are means ± SD from three biologically independent experiments. Significance analysis using one-way ANOVA followed by Tukey's multiple comparison test is shown in different lower-case letters (P < 0.05).
Figure 5(A) Identification of the de-S-acylation enzyme of BSK1. BSK1-RFP was co-overexpressed with 11 GFP-fused ABAPT members in protoplasts respectively (free GFP was used as a control). Cells with both RFP and GFP signals were used for analyzing the localization of BSK1-RFP. The predominant localization patterns of BSK1-RFP are shown. The RFP (magenta), GFP (green), and the merged (with bright field in gray) signals are included in the top graph. Bars, 5 µm. The quantitative data of the percentages of cells with mislocalization of BSK1-GFP (100 cells for each sample) are shown in the bottom graph. Data are means ± SD from three biologically independent experiments. Significance analysis using one-way ANOVA followed by Tukey's multiple comparison test is shown in different lower-case letters (P < 0.05).(B) Detection of the effect of ABAPT11 on the localization of BSK members. 12 BSK-GFP fusion proteins were co-overexpressed with RFP (control) or ABAPT11-RFP in protoplasts. BSK-GFP localization in cells with both RFP and GFP signals was recorded. Representative localization patterns of BSK-GFP with RFP or ABAPT11-RFP are shown in the top graph. Bars, 5 µm. Quantitative data of the percentages of cells with localization changes of BSK-GFP (100 cells for each sample) are shown in the bottom graph. Data are means ± SD from three biologically independent experiments. ****P < 0.0001, ***P < 0.001, *P < 0.05, n.s. (no significance) P > 0.05, Student's t-test (unpaired, two-tailed).
Figure 6(B) Effect of ABAPT11 overexpression on the BSK1 membrane association in a cell fractionation assay. BSK1-GFP was expressed in protoplasts with or without ABAPT11 overexpression. Total proteins (T) were separated into soluble (S) and pellet (P) fractions using ultra-centrifugation. A representative immunoblot using an anti-GFP antibody is shown. The percentages of BSK1-GFP in the soluble fraction were calculated from relative immunoblotting signals (S/[S+P]). Data are means ± SD from three biologically independent experiments. **P < 0.01, Student's t-test (unpaired, two-tailed).(C, D) Effect of ABAPT11 overexpression on S-acylation of BSK1 and BSK3. The S-acylation level of BSK1-GFP (C) and BSK3-GFP (D) with or without ABAPT11 overexpression in protoplasts was detected in a biotin-switch assay. Representative immunoblotting image is shown in the top graphs. Quantification of relative S-acylation levels was included in the bottom graphs. Immunoblotting signals were quantified by ImageJ and the S-acylation levels were calculated from relative signals ([pulldown+/input+] - [pulldown-/ input-]). The relative S-acylation level of the control sample was set to 1. Data are means ± SD from three biologically independent experiments. **P < 0.01, Student's t-test (unpaired, two-tailed).