EMBO/SourceData huggingface dataset:
Figure 5(A) HeLa cells were transfected with vector or ORF6-Flag for 12 h and treated with 200 μM OA for another 12 h, the medium was removed and replaced with fresh complete culture containing 1 μM DGAT1 inhibitor and 2 μM DGAT2 inhibitor for 24 h. Meanwhile, 50 μM ATGL inhibitor or 100 μM Chloroquine (CQ) was added and allowed to incubate for 24 h or 4 h to block lipolysis or lipophagy, respectively. Cells were fixed and stained with anti-Flag (red). LDs were labeled with BODIPY-493/503 (green). Cells were imaged by confocal microscopy. Scale bar represents 10 μm. White ROIs indicate cells expressing ORF6 and yellow ROIs indicate the cells without ORF6 expression.(C) HEK293T cells were transfected with ORF6-Flag. Protein interactions were detected by immunoprecipitation with anti-Flag beads and immunoblotting analysis with ATGL, HSL, and CGI58 antibodies.(D) Purified GST-ORF6 or indicated mutants were incubated with purified His tagged ATGL, and analyzed the interactions by GST pull-down.(H and I) The effect of ORF6-Flag or the ΔNTD mutant on the interactions of Flag-ATGL with GFP-Plin2 or GFP-UBXD8 was analyzed by immunoprecipitation.(K and L) Vero-E6 cells were transfected with Flag-ATGL and GFP-Plin2 or GFP-UBXD8 for 24 h and infected or non-infected with SARS-CoV-2 for 24 h. Protein interactions were analyzed by immunoprecipitation with anti-GFP beads and immunoblotting analysis.(M) SARS-CoV-2-infected Vero-E6 cells were treated with 50 μM ATGL inhibitor for 24 h. Cell viability was analyzed. Cell lysates were analyzed via WB. Supernatants were determined by plaque assays. Virus titer values represent mean ± SD for three independent replicates. Two-tailed Unpaired Student's t-test, *p < 0.05, ns means no significance. Error bars represent as the mean ± SD.
Figure 6(C) Representative transmission electron micrograph of ORF6-Flag stable expressing or vector HeLa cells upon OA treatment. Red arrows mark the contact sites between LDs and mitochondria. M, mitochondria. LD, lipid droplets. Scale bar represents 1 μm. (D) The number of mitochondria-LD contacts per cell was counted from 20 cells. The number of mitochondrial engaged in mitochondrial-LD contact per LD in (C) was counted from 20 LDs. Two independent experiments. Two-tailed Unpaired Student's t-test, ****p < 0.0001. Error bars represent the mean ± SD.(E) Representative transmission electron micrograph of non-infected or SARS-CoV-2 infected Vero-E6 cells. Red arrows mark the mitochondria-LD contact. LD, lipid droplets. M, mitochondria. Scale bar represents 1 μm. Quantification of number of mitochondria engaged in mitochondria-LD contact per LD. 20 LDs of two independent experiments were counted. Two-tailed Unpaired Student's t-test, ****p < 0.0001. Error bars represent the mean ± SD.(F and G) SARS-CoV-2-infected Vero-E6 cells were fixed and stained with anti-Tom20 (cyan). Tom20 represents mitochondria marker. LDs were labeled with LipidTOX Deep Red (red). Cells were imaged by confocal microscopy. Scale bar represents 10 μm. Colocalization of LDs and mitochondria (Pearson's Coefficient), n=20 cells, two independent experiments. Two-tailed Unpaired Student's t-test, **p < 0.01. Error bars represent the mean ± SD.(H) Cos7 cells expressing ORF6-Flag and vector were treated with 200 μM OA for 12 h, and then were fixed and stained with anti-Flag (white) and anti-Tom20 (red). Tom20 represents mitochondria marker. LDs were labeled with BODIPY-493/503 (green). Cells were imaged by confocal microscopy. Scale bar represents 10 μm.(L) Seahorse FAO assays to examine the oxidation of endogenous FAs in vector, or ORF6-Flag or ORF64Q-Flag over-expressed HeLa cells. Three biological replicates were performed with similar results. Error bars represent the mean ± SD.(M) The concentration of ATP in cells was analyzed according to manufacturer's instructions. Error bars, mean ± SD of four independent experiments. Two-tailed Unpaired Student's t-test, ***p < 0.001, ns means no significance.(N) Vero-E6 cells stable expressing GFP or GFP-Plin5 or GFP-Plin5Δ443-463 were infected with SARS-CoV-2 for 24 h. Cell viability was analyzed. Cell lysates were analyzed via WB. Supernatants were determined by plaque assays. Virus titer values represent mean ± SD for three independent replicates. Two-tailed Unpaired Student's t-test, **p < 0.01, ns means no significance. Error bars represent the mean ± SD.
Figure 7(C) HeLa cells were transfected with negative, or si-MTX1, or si-MTX2, or si-SAMM50 for 24 h and further transfected with ORF6-GFP 24 h. Subcellular fractions were isolated from cells. VDAC1 and ATP5A1 represent mitochondria markers, Tubulin represents cytosolic marker.(D) HeLa cells were transfected with negative or si-MTX1 and si-MTX2 for 24 h and further transfected with ORF6-GFP 24 h. Subcellular fractions were isolated from cells. VDAC1 and ATP5A1 represent mitochondria markers, Tubulin represents cytosolic marker.(F) Cos7 cells expressing ORF6-Flag were transfected with negative or si-MTX1/si-MTX2/si-SAMM50 (Triple KD) for 36 h and treated with 200 μM OA for another 12 h. Cells were fixed and stained with anti-Flag (white) and anti-Tom20 (red). Tom20 represents mitochondria marker. LDs were labeled with BODIPY-493/503 (green). Cells were imaged by confocal microscopy. Scale bar represents 10 μm. (G) Quantification of average number of LD-mitochondria contacts per LD from (F). 25 cells (Negative), 50 cells (Triple KD) from three independent experiments were calculated. Two-tailed Unpaired Student's t-test, ****p < 0.0001. Error bars represent the mean ± SD.(H) Vero-E6 cells were transfected with indicated siRNAs for 24 h and infected with SARS-CoV-2 for 24 h. Cell viability was analyzed. Cell lysates were analyzed via WB. Supernatants were determined by plaque assays. Virus titer values represent mean ± SD for three independent replicates. Two-tailed Unpaired Student's t-test, *p < 0.05, ***p < 0.001, ns means no significance. Error bars represent the mean ± SD.
Figure 2(B& Northern blot validation of new sRNAs. (B) Samples were taken in early exponential (EE), mid-exponential (ME), early late exponential (eLE), late exponential (LE), early stationary (eST) and stationary (ST) phase of growth in TY medium. The error bars represent the mean ± SD of four biological replicates(D) Coverage plots of several sRNA-IGR chimeras, where the IGR is located within poly-cistronic operons. Chimeric reads are highlighted in red; the RBS is depicted as a yellow box. The number of chimeras covering each interaction is provided and the respective interacting sRNA is highlighted in bold.
Figure 3(C) Northern blot validation of SpoY, SpoX and nc037 expression in early exponential (EE), mid-exponential (ME), early late exponential (eLE), late exponential (LE), early stationary (eST) and stationary (ST) phase of growth in TY medium. 5S rRNA served as a loading ctl. A representative image of three independent experiments is shown.
Figure 4(A) Quantification of EMSAs (error bars represent the mean ± SD of n=3 biological replicates performed with either 32P-labeled SpoY or SpoX (short isoform) with increasing concentrations of the spo0A target region, respectively. Purified Hfq was added to facilitate SpoY-spo0A complex formation. Mutating the respective sRNA seed region (Appendix Figure S4A, SpoY*/SpoX*) abolished the interaction, while introducing compensatory mutations into the spo0A target region (spo0A*C) slightly rescued the complex formation.(B) In-line probing of 0.2 pmol of 32P-labeled SpoY and SpoX (short isoform) in the absence (lane 4) or presence of increasing concentrations (lane 5-7) of the spo0A long 5′UTR (starting from pTSS) and first 69 nt of the CDS. RNase T1 and alkali-digested (OH) SpoY and SpoX serve as ladders respectively. Secondary structure and predicted seed region are highlighted. A representative image of three independent experiments is shown.(C) mCherry fluorescence of translational fusion constructs (error bars represent the mean ± SD of n=5 biological replicates, expressed in the respective sRNA knock-out background. Fluorescence intensity was normalized to that of the respective p[spo0A] ctl. Mutating the sRNA seed regions (Appendix Figure S4A, SpoY*/SpoX*) abolished the impact on spo0A translation, while introducing compensatory mutations into the spo0A target region (spo0A*C) partially rescued the effect. The long SpoX isoform used in reporter assays. Ordinary one-way ANOVA with Dunnett′s multiple comparison test was used to calculate statistical significance. Not significant (ns) P > 0.05; (∗) P ≤ 0.05; (∗∗∗∗) P ≤ 0.0001.
Figure 5(A) Western blot analysis comparing Spo0A protein levels in a WT strain (p[ctl]), sRNA knock-out mutants (ΔSpoY/ΔSpoX-p[ctl]) and strains constitutively expressing the respective sRNA (ΔSpoY/ΔSpoX-p[ΔSpoY/ΔSpoX]). Equal optical density (OD) units of total cell lysates (error bars represent the mean ± SD of n=3 biological replicates) were loaded from strains gown till mid-exponential (ME) or stationary (ST) phase of growth in TY. Band intensities were measured and Log2 fold changes were calculated relative to the respective WT. Western blot membranes were incubated with anti-Spo0A antibody. To calculate statistical significance, an ordinary one-way ANOVA with Dunnett′s multiple comparison test was applied in. Not significant (ns) P > 0.05; (∗) P ≤ 0.05; (∗∗∗) P ≤ 0.001. For western blot analysis Ponceau S staining of the blotting membrane served as loading control(B) Northern blot analysis validating sRNA expression in the respective growth conditions.
Figure 6Transcript levels of genes encoding sporulation-specific sigma factors and their respective regulon in sRNA knock-out mutants (ΔSpoY/ΔSpoX-p[ctl]) and strains constitutively expressing the respective sRNA (ΔSpoY/ΔSpoX-p[ΔSpoY/ΔSpoX]) relative to C. difficile 630 WT (p[ctl]). A schematic representation of the first four stages of sporulation in C. difficile is given on the left. RNA was extracted from samples (n = 3 biological replicates) taken at 9 h and 12 h post induction of sporulation on 70:30 sporulation plates. Log2 fold changes were calculated relative to the WT. Statistical significance was determined using two-way ANOVA with Dunnett′s multiple comparison test. P > 0.05; (∗) P ≤ 0.05; (∗∗) P ≤ 0.01; (∗∗∗) P ≤ 0.001; (∗∗∗∗) P ≤ 0.0001. Error bars represent the mean ± SD.
Figure 7(A) Representative phase-contrast images (n=4) of a WT strain (p[ctl]), sRNA knock-out mutants (ΔSpoY/ΔSpoX-p[ctl]) and strains constitutively expressing the respective sRNA (ΔSpoY/ΔSpoX-p[ΔSpoY/ΔSpoX]) at 6 h, 12 h, 24 h and 48 h post inoculation of 70:30 liquid sporulation medium. (B) Sporulation frequencies calculated based on phase-contrast microscopy. Error bars represent the mean ± SD of n=4 biological replicates (A). 2-way ANOVA with Dunnett′s multiple comparison test was used to calculate statistical significance. Not significant (ns) P > 0.05; (∗∗) P ≤ 0.01; (∗∗∗) P ≤ 0.001; (∗∗∗∗) P ≤ 0.0001.
Figure 8(B) Body weight loss over the course of 7 days (data points and error bars represent mean ± standard error of the mean; the dotted lines serve as a visual guideline) of mice infected with C. difficile 630 WT (n=15), ΔSpoY (n=15) or ΔSpoX (n=15)are indicated(C) final colon length at day 7 post infection of uninfected mice (n=10) and mice infected with C. difficile 630 WT (n=15), ΔSpoY (n=15) or ΔSpoX (n=15)are indicated (box-and-whisker plots show minimum, first quartile, median, third quartile, and maximum). Data information: Kruskal-Wallis was performed with Dunnett′s multiple comparison test to calculate statistical significance in (C) Not significant (ns) P > 0.05; (∗) P ≤ 0.05; (∗∗) P ≤ 0.01; (∗∗∗) P ≤ 0.001; (∗∗∗∗) P ≤ 0.0001.(D) Comparison of CFUs of C. difficile vegetative cells and spores in fecal pellets of mice at different time points during infection with C. difficile 630 WT (n=15), ΔSpoY (n=15) or ΔSpoX (n=15). Replicates with CFUs below the detection limit were set to 100. Box-and-whisker plots show minimum, first quartile, median, third quartile, and maximum. Data information: Kruskal-Wallis was performed with Dunnett′s multiple comparison test to calculate statistical significance in (D). Not significant (ns) P > 0.05; (∗) P ≤ 0.05; (∗∗) P ≤ 0.01; (∗∗∗) P ≤ 0.001; (∗∗∗∗) P ≤ 0.0001.
Figure 1(A) Relative mRNA expression of Foxc1 and Foxc2 in isolated blood endothelial cells (BECs) and lymphatic endothelial cells (LECs) from Foxc1fl/fl;Foxc2fl/fl mouse distal jejuna in sham and I/R-4h groups. Isolated epithelial cells (Epis) were used as a relative control, Data are box-and-whisker plots, Mann-Whitney U test, each symbol represents one mouse, N = 5~9, *P<0.05, **P<0.01, n.s. = not significant. The box-and-whisker plots in (A) display the median value (central band in the box), second and third quartiles (bottom and top ends of the box, respectively) as well as minimum/maximum values (whiskers blow/above the box) of the data sets.(B-C) Representative confocal images of the whole-mount intestine stained with CD31/LYVE1 (B), FOXC1/CD31/LYVE1 (C, left) and FOXC1/LYVE1 (C, right) in Foxc1fl/fl;Foxc2fl/fl mice. Images of maximum intensity projections (B) show the intestinal blood (green) and lymphatic (white) vasculatures from villous to submucosa. The regions in pink box 1~8 in Figure 1B are chosen for the specific images in Figure 1C. Scale bars = 50 μm. (C) Images of optical sections with high magnification chosen from specific regions shown in Figure 1B (B1~B8), with an additional channel of FOXC1 staining (red), show the up-regulation of FOXC1 in BECs (white arrows in blood vessels) and LECs (violet arrows in lymphatic vessels) at the level of villus and submucosa 4 h after I/R. Scale bars = 20 μm.(F) Relative mRNA expression of Foxc1 and Foxc2 in isolated CD45-CD31+ ECs from distal jejuna 12d after Tm treatment. Data are box-and-whisker plots, Mann-Whitney U test, each symbol represents one mouse, N = 4~5, *P<0.05. Data information: The box-and-whisker plots in (F) display the median value (central band in the box), second and third quartiles (bottom and top ends of the box, respectively) as well as minimum/maximum values (whiskers blow/above the box) of the data sets.
Figure 2(C-E) Quantification of Chiu Score for Control and EC-Foxc-DKO (C), Control and EC-Foxc1-KO (D), and Control and EC-Foxc2-KO groups (E) at I/R-24h. Data are box-and-whisker plots, Mann-Whitney U test, each symbol represents one mouse, N = 3 in C sham groups, N = 7~12 in C, D, E I/R-24h groups, *P<0.05, ****P<0.0001. The box-and-whisker plots in display the median value (central band in the box), second and third quartiles (bottom and top ends of the box, respectively) as well as minimum/maximum values (whiskers blow/above the box) of the data sets.(G) Representative immunostaining images of intestinal crypts labeled with BrdU (proliferative marker, injection performed 2h before tissue collection) and EpCAM (epithelial marker) show the proliferation of epithelial cells in crypts. Paraffin sections (4 µm), (H) Quantification of the number of BrdU+ epithelial cells per crypt based on Figure 2G. Data are box-and-whisker plots, Kruskal-Wallis One-way ANOVA test, each symbol represents one mouse, N = 3~6, *P<0.05, n.s. = not significant. The box-and-whisker plots in display the median value (central band in the box), second and third quartiles (bottom and top ends of the box, respectively) as well as minimum/maximum values (whiskers blow/above the box) of the data sets.(I) Representative images of intestinal crypts immunostained with β-catenin and the intestinal epithelial stem cell (ISC) marker OLFM4. At I/R-48h, the nuclear translocation of β-catenin in ISCs (dotted circles) was found in the control, whereas it was seldom found in EC-Foxc-DKO mice. Paraffin sections (4 µm), (J, K) (J) Quantification of relative fluorescent intensity (FI) of β-catenin immunostaining within ISC and (K) quantification of number of OLFM4+ ISCs per crypt were performed based on Figure 2I. Data are box-and-whisker plots, Mann-Whitney U test, each symbol represents one mouse, N = 4~5, *P<0.05, n.s. = not significant. The box-and-whisker plots in display the median value (central band in the box), second and third quartiles (bottom and top ends of the box, respectively) as well as minimum/maximum values (whiskers blow/above the box) of the data sets.(L) Representative images of intestinal mucosa labeled with Cyclin D1 (CCND1) in EC-Foxc-DKO mice compared with Control group in sham, 24h and 48h after I/R. Paraffin sections (4 µm), (M) Quantification of the number of CCND1+ epithelial cells per crypt based on Figure 2L. Data are box-and-whisker plots, Kruskal-Wallis One-way ANOVA test, each symbol represents one mouse, N = 4~6, **P<0.01, ****P<0.0001, n.s.=not significant. The box-and-whisker plots in display the median value (central band in the box), second and third quartiles (bottom and top ends of the box, respectively) as well as minimum/maximum values (whiskers blow/above the box) of the data sets.
Figure 3Chiu Score analysis from H&E stained distal jejunum 24h after I/R for Control (Foxc1f/f;Foxc2f/f) and LEC-Foxc-DKO (Vegfr3-CreERT2;Foxc1f/f;Foxc2f/f) mice (B), Control (Foxc1f/f) and LEC-Foxc1-KO (Vegfr3-CreERT2;Foxc1f/f) mice (C) Data are box-and-whisker plots, Mann-Whitney U test, each symbol represents one mouse, N=9~13, *P<0.05, **P<0.01. display the median value (central band in the box), second and third quartiles (bottom and top ends of the box, respectively) as well as minimum/maximum values (whiskers blow/above the box) of the data sets.(F) Representative images of crypts immunostained with OLFM4 and β-catenin in control and LEC-Foxc-DKO mice 24h after I/R. The accumulation of β-catenin in the nuclei of ISCs (dotted circles) was found in Control mice but inhibited in LEC-Foxc-DKO mice. Paraffin sections (4 µm), scale bars = 20 μm.
Figure 4(A) Representative images of intestinal whole-mount VEGFR2/CD31 (green/red) immunostaining show increased VEGFR2 (green) expression at the angiogenic front of villous blood capillaries in control mice at I/R-18.5h. In EC-Foxc-DKO mice, the increase of VEGFR2 is inhibited in villous blood vessels and the blood vasculatures are damaged (arrow). (B) Representative images of intestinal whole-mount VEGFR3/LYVE1 (red/green) immunostaining. 18.5h after I/R, VEGFR3 (red) is increased in lacteals especially at the lacteal tips in the control but is inhibited in the EC-Foxc-DKO lacteals (arrow).
Figure 5(A) Representative BrdU/CD31/LYVE1/DAPI immunostaining images of intestinal paraffin sections (15 µm) from mice injected with BrdU 18.5h before euthanasia for the analysis of proliferative BECs (B in 1, 3, 5, 7) and LECs (L in 2, 4, 6, 8) in intestines. Arrow heads show BrdU+ BECs or LECs. White/yellow bars = 100 or 10 µm, respectively. (B) The numbers of BrdU+ BECs and LECs per 0.1 mm2 blood vessel (CD31+LYVE1-) and lymphatic vessel (CD31+LYVE1+) area in intestinal mucosa were quantified respectively based on Figure 5A. Data are box-and-whisker plots, Kruskal-Wallis One-way ANOVA test, each symbol represents one mouse, N = 3~8, *P<0.05, **P<0.01, n.s. = not significant.(C) Representative TUNEL/CD31/LYVE1/DAPI immunostaining images of distal jejunums for the analysis of apoptotic BECs/LECs in intestinal paraffin sections (15 µm). High magnification images are from the dotted line boxes (1~4). Arrow heads show the apoptotic BECs (B in 1, 2) and LECs (L in 3, 4) at villus (1, 3) or submucosa (2, 4). White/yellow scale bars = 100 or 20 μm, respectively. (D) The numbers of apoptotic BECs and LECs per 0.1 mm2 blood vessel (CD31+LYVE1-) and lymphatic vessel (CD31+LYVE1+) area in intestinal mucosa were quantified respectively based on Figure 5C. Data are box-and-whisker plots, Mann Whitney U test, each symbol represents one mouse, N = 3~6, **P<0.01, n.s. = not significant.
Figure 6(D) Violin plots of the Rspo3 expression in trophocytes and LECs in intestine at I/R-18.5h. Mann-Whitney U test, each symbol represents one cell; N = 12 and 12 in control and EC-Foxc-DKO trophocytes, respectively; N=16 and 5 in control and EC-Foxc-DKO LECs.(E) Validation study by qPCR for the detection of relative mRNA expression of Rspo3 in the sorted intestinal LECs at I/R-18.5h. Data are box-and-whisker plots, Mann-Whitney U test, each symbol represents one mouse, N = 5~6, **P<0.01. The box-and-whisker plots display the median value (central band in the box), second and third quartiles (bottom and top ends of the box, respectively) as well as minimum/maximum values (whiskers blow/above the box) of the data sets.
Figure 7(D) Violin plots of the Cxcl12 expression in fibroblasts and telocytes/trophocytes. Mann-Whitney U test, each symbol represents one cell, N = 67 and 98 in control and EC-Foxc-DKO mice, respectively; n.s. = not significant.(F) Relative mRNA expression of Cxcl12 in Dynabeads-isolated ECs (CD45-CD31+) from distal jejunum. Data are box-and-whisker plots, Mann-Whitney U test, each symbol represents one mouse, N = 6~10, *P<0.05. The box-and-whisker plots display the median value (central band in the box), second and third quartiles (bottom and top ends of the box, respectively) as well as minimum/maximum values (whiskers blow/above the box) of the data sets.
Figure 8FOXC1 and FOXC2 co-occupy as well as the ECRs and promoters of CXCL12 in HUVECs (D). Quantitative-ChIP assay was performed using rabbit (R) anti-FOXC1 and mouse (M) anti-FOXC2 antibodies to analyze the recruitment of FOXC on promoters and/or ECRs in HUVECs respectively. Values were quantified against IgG controls. Data are mean ± SEM, paired t-test, each symbol represents data collected from one experiment, N = 4, * P<0.05, ** P<0.01, n.s.=not significant.