EMBO/SourceData huggingface dataset:
Figure 7Xenogeneic inoculation of 786-0 cells lacking GDH1 with restored expression of WT, ED or 3KR mutant GDH1 into the left groin of nude mice. The solid tumor formed by the indicated 786-0 cells is shown (B).Xenogeneic inoculation of 786-0 cells lacking GDH1 with restored expression of WT, ED or 3KR mutant GDH1 into the left groin of nude mice. The staining of Ki67 (G, upper panel) and TUNEL (G, lower panel) using tumors from (B) are shown and analyzed (G, right panel). We used 6 mice in each group. Scale bars: 50 μm. Data information: All statistical analyses were conducted using unpaired t-test (n = 6). (N.S.: not significant; *p < 0.05; **p < 0.01; ***p < 0.001).
Figure 2  The heat map shows the position (left to right), identity (top row) and frequency (color code) of variant nucleotide positions detected by SARS-CoV-2 full genome amplicon sequencing in (A) 20 samples of MPP-R workers tested positive on May 20 or 25, (B) 15 samples of MPP-R workers tested positive between June 5 and 17, and (C) two workers from MPP-D who may have transmitted the virus to cases B1 and/or B2. Individual collection dates are shown to the right of each sample. Variant sequences are given relative to the Wuhan reference strain NC_045512. The two silent mutations which define the prototype of the investigated outbreak are marked with an asterisk. Frequencies below 100% mean that only a fraction of viral genomes shows nucleotide variations, indicating the presence of viral intra-host sub-populations. White rectangles denote nucleotide positions which were not covered by amplicon-seq reads within the respective sample. For nucleotide positions in coding regions, the corresponding viral ORF(s) are shown above the variant position. Variants without such information are located in non-coding regions.
Figure 3 (A) Distance (in meters) of PCR-tested workers from the suspected index case B1 at the workplace. For workers without fixed position in the beef processing plant (marked with an asterisk) coordinates indicate estimated average location during the early shift. Squares and diamonds denote prototype or variant SARS-CoV-2 genotypes, respectively. Filled blue circles denote cases for which viral genomes were not sequenced (i.e., workers tested positive after 5/25/2020). (B) Top panel: Observed accumulated percentage of positive cases (red line) within the indicated distance from the suspected index case. The gray dashed line shows the average infection rate that would be expected for a random spatial distribution of positive cases. Bottom panel: -log10 p-value for the frequency of accumulated positive cases within the given distance being significantly higher than expected based on a random spatial distribution of positive cases Only employees with fixed work positions were included in the calculation.
Figure 1HEK293T cells were transfected for 48 hours with each mitoTev-TALE (11.5 and 8.5 RVDs) and total cell extracts were used for western-blots with anti-T2A antibody. Molecular weights of the two different mitoTev-TALEs are ~85KD and ~70KDa, respectivelyImmunocytochemistry using the same antibody along with Mitotracker red was performed in COS7 cells, 24 hours after transfection. Nuclei were counterstained with DAPI (blue) 11.5RVD and 8.5RVD mitoTev-TALEs both co-localized with mitochondria, as seen in the merge image. Scale bar: 10 µm
Figure 2MtDNA heteroplasmy analyzed by PCR/RFLP, 24 hours post mitoTev-TALE and mitoTALEN transfection. The RFLP analysis shows increased %WT mtDNA in sorted cells when compared to the the untransfected. mitoTALEN monomers positive for both eGFP and mCherry were isolated as "Yellow". The "Black" cells represent the mitoTALEN sorted population of cells negative for eGFP and mCherry and the "GFP-+" population represents mitoTev-TALE sorted cells with low levels of eGFP fluorescence. GFP++ were cells positive for GFP after mitoTev-TALEs transfectionsQuantification of the % of WT load in cybrid sorted populations after transfectionRepresentative RFLP analysis showing the change in heteroplasmy at 1 day and 15 days, after sortingQuantification of the heteroplasmic shift over timeDetermination of the levels of mtDNA (ND1/ACTB) by qPCR of mutant cybrids transfected with MERRF mitoTev-TALEmtDNA levels in wild-type sorted cells transfected with the MERRF mitoTev-TALE. Data is expressed as percentage of the Untransfected cells (%UNT)
Figure 3RFLP analyses of high mutant cybrids transfected with the MERRF mitoTev-TALE. The sorted cells were analyzed at different times of growth by PCR/RFLP as previously describedQuantification of WT loads of 6-8 independent experiments/sortings, analyzed at different time-pointsTotal mtDNA levels determined by qPCR in 6-7 separate sortings/experimentsFlow cytometry diagram of gated sorted cells. The GFP-+ represent the cells with low levels of fluorescence, GFP++ showed intermediate levels of fluorescence while GFP+++ had the highest levelsQuantification of mtDNA levels by PrimeTime qPCR probes. The mtDNA levels were analyzed in the different populations of sorted cells. Data are expressed as mean±SEM of 3-4 separate experiments mtDNA levels were also determined in the WT cybrids as described above. Data are expressed as mean±SEM of 4-7 separate experiments
Figure 4Oxygen consumption rate (OCR) upon sequential injection of oligomycin (Oligo), FCCP and rotenone (Rot) + antimycin (AA), in untransfected, sorted "GFP-+" and "GFP++". The error bars represent the mean±SEM of 5-7 separate experimentsQuantification of 4-6 independent experiments comparing the basal respiration, maximal respiration, spare respiratory capacity and ATP-linked respiration of the UNT versus "GFP-+", "GFP++" and WT cells. The cells were analyzed between 25-28 days after sortingCoupling efficiency, measured as the ratio of ATP-linked/Basal respiration x 100. Error bars represent the mean±SEM of 4-6 separate experimentsRepresentative membrane of a mitochondrial protein synthesis experiment. The results were normalized to the gel loadingWestern-blot showing the levels of NDUFB8 and COXI in untransfected and sorted cells. The SDHA and tubulin levels were also analyzed. The panel also shows the quantification of these markers normalized by the stain-free loading of 6 independent sortings/experiments
Figure 1(G) Sorted human monocytes were incubated with control medium or SARS-CoV-2 viruses at an MOI of 1 for 1 h, followed by washing away extracellular viruses and further culture for 1 h. Cells were fixed and stained with the indicated antibodies. Otherwise, monocytes were challenged with 1 μg/ml LPS for 3 h and 10 μM nigericin for 30 min, followed by fixing and staining with the indicated antibodies. Treatment with medium from uninfected Vero cells served as a control. Scale bar: 5 μm. Data information: results were repeated for three times with similar results.Supernatants from control or SARS-CoV-2 infected CD14+ monocytes with or without LPS and nigericin stimulation were collected and subjected to an ELISA assay for IL-1β levels (H). Student's t-test was used and data were shown as means±SD of three technical replicates. *, P<0.05; **, P<0.01; ***, P<0.001; NS, non-significant. Results were repeated for three times with similar results. Data information: , Student's t-test was used and data were shown as means±SD of three technical replicates. *, P<0.05; **, P<0.01; ***, P<0.001; NS, non-significant. results were repeated for three times with similar results.Cell viability in cell pellets were examined using a cell viability assay through checking ATP levels inside cells (I). The percentage of cells undergoing pyroptosis was calculated through checking released LDH in the supernatants (J). Student's t-test was used and data were shown as means±SD of three technical replicates. *, P<0.05; **, P<0.01; ***, P<0.001; NS, non-significant. Results were repeated for three times with similar results. Data information: Student's t-test was used and data were shown as means±SD of three technical replicates. *, P<0.05; **, P<0.01; ***, P<0.001; NS, non-significant. results were repeated for three times with similar results.
Figure 2(B, C) THP-1 cells stably expressing the indicated SARS-CoV-2 genes were stimulated with 10 μM nigericin for 30 min following pretreatment with 1 μg/ml LPS for 3 h. Supernatants were collected for an ELISA assay to determine supernatant IL-1β levels (B) and cell pellets were subjected to a cell viability assay to verify the percentage of viable cells through checking cellular ATP levels (C). For B-C, Student's t-test was used and data were shown as means±SD of three technical replicates. NS, non-significant. Data information: control vector used here was empty vector corresponded to the nucleocapsid-containing vector. Student's t-test was used and data were shown as means±SD of three technical replicates. **, P<0.01; NS, non-significant. Similar results were observed for at least three times.(D-F) THP-1 cells stably expressing the nucleocapsid gene or control vector were stimulated with 10 μM nigericin for 30 min following pretreatment with 1 μg/ml LPS for 3 h. Supernatants and cell pellets were collected and immunoblotted with antibodies against the indicated proteins (D). The percentage of viable cells was determined through checking cellular ATP levels in cell pellets (E). The percentage of cells undergoing pyroptosis was calculated through checking released LDH in the supernatants (F). For E, F, Student's t-test was used and data were shown as means±SD of three technical replicates. **, P<0.01; NS, non-significant. Data information: control vector used here was empty vector corresponded to the nucleocapsid-containing vector. Student's t-test was used and data were shown as means±SD of three technical replicates. **, P<0.01; NS, non-significant. Similar results were observed for at least three times.
Figure 3(D) GSDMD+/+ and GSDMD-/- THP-1 cells were transfected with plasmids encoding nucleocapsid for 24 h, followed by cell fixation, permeabilization and stain with antibodies against nucleocapsid and GSDMD. The nucleus was counterstained with DAPI. Scale bar: 5 μm.(E, F) THP-1 (E) or human CD14+ monocytes (F) were transfected with plasmids encoding FLAG-tagged nucleocapsid for 36 h, followed by pretreatment of 1 μg/ml LPS for 3 h and 10 μM nigericin stimulation for 30 min. Cells were lysed and immunoprecipitated with antibody against FLAG or isotype IgG. Input and precipitates were immunoblotted as indicated.(I) GST-GSDMD and control GST were mixed with HEK293T cell lysate containing FLAG-tagged full-length nucleocapsid or truncated nucleocapsid lacking amino acids 70-160 and 290-360, followed by a GST-pulldown assay. Input and precipitates were immunoblotted as indicated.
Figure 4(A, B) THP-1 cells stably expressing nucleocapsid or control vector were stimulated with 10 μM nigericin for 30 min following pretreatment with 1 μg/ml LPS for 3 h. Cells were either immunoblotted with antibodies against the indicated proteins (A) or stained with propidium iodide (PI) (B, left). Scale bar: 20 μm. The percentage of PI positive cells was calculated (B, right). Student's t-test was used and data were shown as means±SD of three technical replicates. ***, P<0.001; NS, non-significant. Results were repeated for three times with similar results. Data information: control vector used here was empty vector corresponded to the nucleocapsid-containing vector. Student's t-test was used and data were shown as means±SD of three technical replicates. **, P<0.01; ***, P<0.001; NS, non-significant. Similar results were observed for at least three times.(H, I) THP-1 cells stably expressing full-length nucleocapsid or mutant lacking amino acids 70-160 and 290-360 were stimulated with 10 μM nigericin for 30 min following pretreatment with 1 μg/ml LPS for 3 h. Cells were either immunoblotted with antibodies against the indicated proteins (H). Cells were also stained with PI and the percentage of PI positive cells was calculated (I). Student's t-test was used and data were shown as means±SD of three technical replicates. **, P<0.01; NS, non-significant. Results were repeated for three times with similar results. Data information: control vector used here was empty vector corresponded to the nucleocapsid-containing vector. Student's t-test was used and data were shown as means±SD of three technical replicates. **, P<0.01; ***, P<0.001; NS, non-significant. Similar results were observed for at least three times.
Figure 5(A) GST or GST-GSDMD, p20 (C285A)-His, p10-His and increasing amount of His-nucleocapsid were incubated for a GST-pulldown assay, followed by immunoblotting with antibodies against the indicated protein tags. Data information: Experiments were repeated for three times with similar results.(G, H) Wild-type GSDMD and GSDMDsusti. (G) or mCherry-GSDMD-C (H) were purified from HEK293T cells and incubated with recombinant p20/p10 complex and His-tagged nucleocapsid in a cleavage buffer, followed by immunoblotting with antibodies against the indicated protein tags or antibody against GSDMD C-termimus. Data information: Experiments were repeated for three times with similar results.
Figure 6GSDMD-/- THP-1 cells rescued with wild-type or linker substituted GSDMD were transfected with plasmids encoding nucleocapsid or control vector and then stimulated with 10 μM nigericin for 30 min following pretreatment with 1 μg/ml LPS for 3 h. Cells were immunoblotted with antibodies against the indicated proteins (A)(E, F) GSDMD-/- THP-1 cells rescued with wild-type or linker substituted GSDMD were infected with SARS-CoV-2 at an MOI of 1 for 1h, followed by washing away extracellular viruses and further culture for 3 h. Otherwise, cells were stimulated with 10 μM nigericin for 30 min following pretreatment with 1 μg/ml LPS for 3 h. Supernatants were subjected to an ELISA assay to determine IL-1β levels (E). Otherwise, cell viability in cell pellets was examined using a cell viability assay through checking ATP levels inside cells (F). For E, F, Student's t-test was used and data were shown as means±SD of three technical replicates. **, P<0.01; ***, P<0.001; NS, non-significant. Results were repeated for three times with similar results.(G) Sorted CD14+ monocytes from donors were infected with SARS-CoV-2 at an MOI of 1 for 1h, followed by washing away extracellular viruses and further culture for 3 h. Cells were immunoblotted with antibodies against the indicated proteins. CD14+ monocytes from healthy donors were treated with 10 μM nigericin for 30 min following pretreatment with 1 μg/ml LPS for 3 h and served as a positive control. Data information: experiments were repeated for three time with similar results.
Figure 1(B) Lineage tracing of the anagen matrix from β-actin-CreER:Confetti, 7 days after induction with either a high dose of tamoxifen (TM) (top) or low dose (bottom) for single cell clonal lineages.(C) Images of skin sections at various stages of the hair cycle, immunostained for Gata6 (red), CD34 or K14 (green) and DNA (blue).(D) images (D') for Gata6 inducible knockout (iKO) and wild type (WT) mice over time, showing the loss of Gata6 (red) from the hair follicle. Scale bars: 30 m.
Figure 2(B) Representative images of hematoxylin staining of skin sections from the telogen induced mice sacrificed at 2, 4, and 10 days (d) post TM induction. (C) Images of hematoxylin staining of skin section from the anagen induced mice. Quantification shown in Figure EV2D. Scale bars: 30 m.
Figure 3(B-G) Gata6 WT and iKO skin after 3 days after TM induction and 12 hours after BrdU labeling or 5 days after TM induction and 2 days after BrdU labeling stained with markers as indicated in corresponding color.
Figure 4(A) Hair follicles exhibit immunostaining signal for DNA damage marker H2A.X (red) and apoptosis marker Caspase3 (green) as early as one day after Gata6 iKO induction. Top panel shows a section of UV irradiated epidermis as a positive control for DNA damage. Note that Caspase 3 is not expressed in the H2A.X cells of the epidermis but is found in some iKO hair follicle matrix cells. Right panels show single color images as indicated at top (B) Quantification (average SD) of the percentage of apoptotic cells per matrix as determined by counting of Caspase3+ cells (n=50 follicles from 3 mice per group) (C) Quantification (average SD) of the percentage of DNA damaged cells per matrix as determined by counting of H2A.X+Caspase3- cells (n=50 follicles from 3 mice per group).(D) Regional distribution of apoptosis was analyzed by dividing the matrix into four compartments as shown in the legend (left) and counting the fraction of Caspase3+ cells per each matrix compartments (right; n=50 follicles from 3 mice per group).(E) Proliferation in the matrix compartments shown in (D) was assessed by BrdU labeling for 1 hour, followed by staining, and quantification (F) (interior cells labeled with solid bars, exterior cells labeled with striped bars). Unpaired t-test: *P-value < 0.05. n=20 follicles from 2 mice per group.
Figure 5(A) Colony formation of WT and TM induced Gata6 iKO keratinocytes stained with hematoxylin. Primary keratinocytes were isolated from induced iKO newborn mice, plated on feeder cells, and stained with hematoxylin at day 7 after plating (top). Phase contrast images of cells are shown below. Scale bars: 30 m. (B) Quantification of colony formation from (A), average SD from n=3 mice in each genotype.(C) Western blot of WT and iKO cultured keratinocytes after 0 or 6 hours of tamoxifen induction, blotted with actin and Gata6 antibodies, show a decrease in Gata6 protein levels after 6 hours.(D) Phase contrast images of WT and Gata6 iKO keratinocytes 3 days after induction with TM (n > 3 per genotype).(E-H) Images and quantification of DNA damage in cultured keratinocytes based on staining for DNA damage marker H2A.X (E and G) and comet assay (F and H). Arrowheads in (F) indicate DNA damaged cell nuclei with comet shape. Quantification of H2A.X (G) represents average SD from at least 300 cells per condition from 3 experiments using different cell lines . *Unpaired t-test P-values: 6h=0.01 and 24h=0.004. (H) Quantification of comet assay represents average SD (n=3).(I-J) FACS cell cycle profiles and quantification show that Gata6 iKO keratinocytes show decreased proliferation and G2-M arrest.(I-J) FACS cell cycle profiles and quantification show that Gata6 iKO keratinocytes show decreased proliferation and G2-M arrest. (J) Quantification of FACS cell cycle analysis represent average SD (n=3 per genotype). *Unpaired t-test P-values: G1 = 0.03, S = 5x10-4, G2-M = 7x10-4.(K) Quantification of the frequency of H2A.X+ staining in keratinocytes within S phase (EdU+) or another cell cycle stage (EdU-) following a 20 minute EdU pulse labeling (average SD, n=5). *Unpaired t-test P-values WT=0.1, iKO= 0.05. Mann-Whitney U test P-values: WT=0.09, iKO=0.03.(L-M) Cultured keratinocytes were made quiescent by gradual serum depletion. (L) Quantification of EdU+ cells in proliferative and serum depleted quiescent cells (average SD, n=4). (M) Quantification of H2A.X+ keratinocytes during proliferative and quiescent conditions (average SD, n=4). *Unpaired t-test P-value = 0.04. Mann-Whitney U test P-value=0.06.
Figure 6(A) Heatmap of differentially expressed genes (adjusted P-value < 0.05 and fold change > 1.5) from RNA-seq (n=3 different WT and iKO cell lines).(B) qRT-PCR validation of candidate genes selected from RNA-seq analysis using RNA from cultured cells (average SD, n=4). *Unpaired t-test P-values: Edaradd=0.03, Apcdd1=0.005, Gls2=7x10-5, Ppargc1b=3x10-4, Rab17=2x10-5, Foxn1=0.03. Mann-Whitney U test P-values: Edaradd=0.06, Apcdd1=0.03, Gls2=0.03, Ppargc1b=0.03, Rab17=0.03, Foxn1=0.03. (C) Table summarizing selection of candidate genes from (B). Genes were selected based on known function or skin phenotype related to Gata6 iKO phenotype.(D) Staining of ROS by DHE. LY83583 treated cells served as positive control with elevated ROS. (E) Quantification of fold change in ROS at 6 and 24 hours after TM compared to WT, based on fluorescence intensity of DHE (average SD, n=2).(F) Analysis of transcription factor binding site overrepresentation (oPOSSUM) in differentially expressed genes from RNA-seq. Points represent transcription factors plotted as Z.score of the frequency of transcription factor binding site occurrences in the promoters of downregulated genes compared to the background rate of occurrences in all gene promoters and Fisher P-value of the proportion of downregulated gene promoters containing each transcription factor binding site compared to the frequency in all gene promoters.(H) qRT-PCR of selected genes found to be overlapping between Gata6 iKO and Eda transgenic analyzed in Gata6 WT and iKO keratinocytes (average SD, n=4). *Unpaired t-test P-values: Mcm3=0.001, Mcm6=0.02, Mcm10=0.04, Exo1=0.02, Pold1=0.02. Mann-Whitney U test P-values: Mcm3=0.03, Mcm6=0.03, Mcm10=0.03, Exo1=0.03, Pold1=0.06.
Figure 7(B) qRT-PCR confirmation of the change in Edaradd mRNA expression in-vivo from mouse whole skin (average SD, 1d n=3, 2d n=4). *Unpaired t-test P-values: 1d = 0.02, 2d = 0.05. Mann-Whitney U test P-values: 1d=0.06, 2d=0.03.(C) ChIP-qPCR for Gata6 at three conserved binding sites within the Edaradd promoter. GAPDH served as a negative control. Binding site 1, 2, and 3 are located 50 bp, 1 kb, and 1.5 kb upstream of the transcription start site, respectively (average SD, n=4). *Unpaired t-test P-values GAPDH=0.4, 1=0.002, 2=0.8, 3=0.2. Mann-Whitney U test P-values: GAPDH=0.3, 1=0.06, 2=0.9, 3=0.2.(D) Western blot from whole skin lysate. Note decrease of Edaradd and activated pNF-B in the iKO.(E-F) Staining for Edaradd and quantification show decrease in Edaradd protein levels in the matrix of Gata6 iKO. *Unpaired t-test P-value = 3.5x10-14. n=100 follicles from 3 mice per genotype.(G-H) Staining for pNF-B and quantification show decrease of active NF-B in the Gata6 iKO matrix. *Unpaired t-test P-value = 2.2x10-16. n=50 follicles from 3 mice.(I) Rescue of Gata6 iKO keratinocyte growth by transfection with pGata6 or pEdaradd. Cell plates of keratinocytes are stained with Rhodanile Blue (n=3 cell lines each).(J) Quantification of H2A.X in cells stably transfected with pMock, pGata6, or pEdaradd plasmids, followed by induction with TM for 6 hours (average SD, n=4 with > 500 cells counted per condition). *Unpaired t-test P-values = 2x10-4 (iKO pGata6 vs pMock), 2x10-4 (iKO pEdaradd vs pMock).(K) qRT-PCR analysis of Mcm10 expression in pMock, pGata6, and pEdaradd WT and iKO expressing cells (average SD, n=4). *Unpaired t-test P-values: 8x10-4 (iKO pGata6 vs pMock), 0.03 (iKO pEdaradd vs pMock). Mann-Whitney U test P-values: 0.03 (iKO pGata6 vs pMock), 0.03 (iKO pEdaradd vs pMock).
Figure 1E. RT-qPCR of Axin2 in bEnd.3 cells treated with NDP (200 ng/ml), isotype control or F4L5.13 (1200 ng/ml) and transfected with control, Tspan12 or Fzd4 siRNAs. Data are presented as mean ± SD, n=3 technical replicates. Data is representative of two independent experiments. F. Time course of phosphorylated Disheveled-3 (p-DVL3) and ßcatenin protein levels in bEnd.3 cells treated with 30nM of F4L5.13 or NDP. Histogram represents the ratio of the DVL3 phosphorylation levels over total DVL3 protein and ßcatenin levels over ß-Tubulin measured by densitometry of independent experiments. Data are presented as mean ± SEM, n=4-6 (*p<0.05 as compared with NT). Significance was calculated by one-way ANOVA with Bonferroni's multiple comparisons test (*p<0.05 as compared with NT).
Figure 2A. Volcano plot of gene expression changes in bEnd.3 cells following 24h of treatment with 30nM F4L5.13. Genes with significant changes in expression level are highlighted in blue and genes co-regulated by treatment with 30nM NDP for 24h are highlighted in red. Statistical analysis was performed using the DESeq2 R package and adjusted p-value (Wald test) threshold less than 0.1 was used. Labelled genes are associated with enriched GO biological processes indicated below.