EMBO/soda-nel huggingface dataset:
Long-lived macrophage reprogramming drives spike protein-mediated inflammasome activation in COVID-19 (2021)
Figure 3

Figure 3: S-protein stimulated macrophages from convalescent individuals secrete IL-1β and show distinct gene expression signatures.

A Subgroup analysis of IL-1β secretion of macrophages after stimulation with S-protein and nigericin from COVID-19 patients with severe (red/black; n=23) or mild (white/red; n=21) disease. Student's t-test with Welsh corrections was used to calculate statistical differences. Graph shows mean ± SEM.

B CD14+ cells of two convalescent individuals were isolated sequentially at d18, d26 and d56 after confirmation of SARS CoV2 infection by PCR. Following M-CSF differentiation, macrophages were stimulated with LPS (red triangle) or S-Protein (red circles) and IL-1β concentration (pg/ml) was quantified upon incubation with nigericin. Blue dashed line indicates mean IL-1β level of SC-naïve individuals stimulated with S-protein and nigericin.

C Quantification of IL-1β concentration (pg/ml) in the supernatant of primary macrophage cultures from convalescent COVID-19 patients (SC-conv) (n = 4; red bars) or SARS-CoV-2 naïve individuals (SC-naïve) (n = 4; blue bars) stimulated with S-protein (1 µg/ml) and nigericin. Students t-test with Welsh corrections was used to calculate statistical differences as indicated. Graph shows mean ± SEM. *p<0.05.

D Venn diagram showing number of differentially expressed genes (DEGs) in healthy/naïve (SC-naïve; blue) and convalescent COVID-19 patient-derived macrophages (SC-conv; red) upon stimulation with LPS (left) or S-Protein (right). Circle sizes of the venn diagram correspond to the number of genes.

E KEGG-gene enrichment analysis, based on RNA-Seq data, showing activated signaling pathways in S-protein stimulated macrophages compared to unstimulated in convalescent COVID-19 patient-derived macrophages (SC-conv). Padj values are indicated (color code) and size of the circles represent number of DEGs (count). P-values are calculated using Wald test and p-adjusted values using the FDR/Benjamini-Hochberg approach.

F Volcano plot showing DEGs in S-protein stimulated macrophages from convalescent COVID-19 patients (SC-conv) compared to unstimulated cells. Negative log10 adjusted p-values are plotted against the log2 fold-change. Select genes are labeled. P-values are calculated using Wald test and p-adjusted values using the FDR/Benjamini-Hochberg approach.

G Heat-map indicating DEG patterns comparing LPS or S-protein stimulation of SC-conv macrophages. Z-score is indicated in a color score.

Data information: RNA-Seq analyses included data with padj <0.05 and log2-fold change of ≤-1 and ≥1.
Long-lived macrophage reprogramming drives spike protein-mediated inflammasome activation in COVID-19 (2021)
Figure 4

Figure 4: TLR-2 is involved in IL-1β secretion of S-protein stimulated macrophages.

A Mean fluorescence intensity (MFI) comparing surface TLR2 expression on healthy/naïve (SC-naïve; blue; n = 7) and COVID-19 patient (SC-conv; red; n = 10) derived macrophages. Students t-test with Welsh corrections was used to calculate statistical differences as indicated.

B IL-1β concentration (pg/ml) in supernatants of COVID-19 patient derived macrophages pretreated with DMSO (n = 44), KINK-1 (n = 11) and MMG-11 (n = 18) and subsequent stimulation with S-protein and nigericin. For statistical analysis, one-way ANOVA was used.

C IL-1β concentration (pg/ml) in supernatants of COVID-19 patient derived macrophages pretreated with DMSO (n = 44), KINK-1 (n = 11) and MMG-11 (n = 19) and subsequent stimulation with LPS and nigericin. For statistical analysis, one-way ANOVA was used.

D IL-1β concentration (pg/ml) in supernatants from macrophages of SC naïve (blue; n = 7) and COVID-19 patients (red; n = 11). Macrophages were pre-treated with DMSO (SC-naïve n = 7; COVID-19 n = 11), KINK-1 (SC-naïve n = 6; COVID-19 n = 7) and MMG-11 (SC-naïve n = 4; COVID-19 n = 4) for 2h and then stimulated with zymosan (4h) and nigericin (2h) as indicated. For statistical analysis, two-way ANOVA with tukey post hoc test was used.

E IL-1β concentration (pg/ml) in supernatants from COVID-19 patients which were stimulated for 2h with a blocking anti-TLR2 monoclonal antibody in different concentrations as indicated. Subsequently, macrophages were stimulated with LPS (1 ng/ml: n = 6; 10 and 100 ng/ml: n = 7) and S-Protein (1 ng/ml: n = 12; 10 and 100 ng/ml: n = 13) for 4h and both groups for 2h with nigericin. For statistical analysis, one-way ANOVA was used.

Data information: Graphs show mean ± SEM. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001.
Long-lived macrophage reprogramming drives spike protein-mediated inflammasome activation in COVID-19 (2021)
Figure 5

Figure 5: Transcriptomic changes are associate with innate immune responses and redox-status of stimulated macrophages.

A Heat-map indicating DEG patterns comparing SC-conv and SC-naïve macrophages stimulated with the S-protein. Z-score is indicated in a color score.

B BP enrichment analysis, based on RNA-Seq data, showing activated signaling pathways upon S-protein stimulation in SC-conv macrophages compared to SC-naïve macrophages. Padj values are indicated (color code) and circle size corresponds to the number of DEGs contributing to the pathways (count).

C Heat-map indicating DEG patterns comparing SC-conv and SC-naïve macrophages without any stimulation. Z-score is indicated in a color score.

D Volcano plot showing DEGs in unstimulated COVID-19 patient-derived macrophages (SC-conv) compared to macrophages from SARS-CoV-2 naïve individuals (SC-naïve). Negative log10 padj values are plotted against the log2 fold-change. Selected genes of interest are labeled.

E IL-1β concentration (pg/ml) in supernatants of SC-naïve individuals (n = 7) which were pre-treated for 24h with FCCP (1µg/ml) and H2O2 (10µg/ml). Macrophages were then incubated with LPS or S-Protein (4h) and nigericin (2h) as indicated in the figure. Control cells were left unstimulated or stimulated with LPS or S-protein without pre-treatment with FCSP or H2O2 (n = 6). For statistical analysis, Brown-Forsythe and Welsh ANOVA test was used.

Data information: RNA-Seq analyses included data with padj <0.05 and log2-fold change of ≤-1 and ≥1. P-values for the DEGs were calculated using Wald test and p-adjusted values using the FDR/Benjamini-Hochberg approach. For the pathways and GO analysis p-values were calculated by hypergeometric distribution and the adjusted p-value was calculated with the Benjamini-Hochberg method. Graphs show mean ± SEM. *p<0.05.
Long-lived macrophage reprogramming drives spike protein-mediated inflammasome activation in COVID-19 (2021)
Figure 6

Figure 6: Epigenetic immune memory in stimulated macropghages.

A Volcano plot showing differentially expressed miRNAs from unstimulated COVID-19 patient-derived macrophages (SC-conv) compared to those from naïve individuals (SC-naïve). Negative log10 adjusted p-values are plotted against the log2 fold-change. Genes of interest are indicated. For miRNA analyses data with a padj value of smaller 0.05 and log2-fold change of ≤-0.85 (blue) and ≥ 0.85 (red) were included.

B Average profile plot showing distribution of histone modifications identified in CUT&RUN experiments displayed as normalized read counts per million around known transcription start site (TSS) of known genes. Color code for H3K4me3 (SARS-CoV-2 naïve/green; COVID-19/red) and for H3K27ac (SARS-CoV-2 naïve/yellow; COVID-19/purple).

C GO biological process analysis performed on differentially enriched loci of H3K27ac peaks (enhancer) showing associated pathways from SC-conv monocytes compared to SC-naïve monocytes.

D KEGG enrichment analysis performed on differentially enriched loci of H3K27ac peaks (enhancer) showing involved pathways from SC-conv monocytes compared to SC-naïve monocytes. Adjusted p-values are indicated (color code) and size of the circles corresponds to the number of detected genes associated with each pathway (count).

E CNET-plot (derived from analysis shown in D) illustrating genes involved in tuberculosis and phagosome KEGG pathways.

F Representative gene loci (TLR2 (top) and S100A7A/8/9/12 (bottom) H3K27ac peaks) comparing SC-naïve (blue) and SC-conv (red) monocytes. Red peaks reach higher values than blue peaks (representative examples of four samples for each condition).

G Quantification of IL-1β concentrations (pg/ml) in the supernatant of primary macrophage cultures from patients with active/untreated tuberculosis (n = 6; red/black bars) or healthy/SARS-CoV-2 naïve individuals (n = 3; blue bars) or the same patients after 6 months of anti-tuberculous treatment; n = 6; white/red bars) stimulated with LPS or S-protein (0.1 µg/ml) for 4h and subsequent incubation with nigericin for 2h. For statistical analysis, two-way ANOVA with tukey post hoc test was used.

Data information: P-values for the DEGs were calculated using Wald test and p-adjusted values using the FDR/Benjamini-Hochberg approach. For the pathways and GO analysis p-values were calculated by hypergeometric distribution and the adjusted p-value was calculated with the Benjamini-Hochberg method. Graphs show mean ± SEM. *p<0.05; **p<0.01
N6-methyladenosine Modification of lncRNA Pvt1 Governs Epidermal Stemness (2021)
Figure 1

Figure 1. Mettl14 regulates skin tissue homeostasis and wound healing.

(A) Mettl14 cKO leads to perinatal lethality. Newborn cKO pups are smaller with tight and shiny skin.

(B) Immunohistochemistry confirms loss of Mettl14 expression in skin in cKO animals.

(C) H/E staining of newborn skin sections from WT and Mettl14 cKO mice.

(D) Expression of p63 in skin epidermis was examined by immunofluorescence staining in WT and cKO mice.

(E) Number of p63-positive cells in WT and cKO skin was quantified and shown as box and whisker plots. The plot indicates the mean (solid diamond within the box), 25th percentile (bottom line of the box), median (middle line of the box), 75th percentile (top line of the box), 5th and 95th percentile (whiskers), 1st and 99th percentile (solid triangles) and minimum and maximum measurements (solid squares). n=6 (biological repeats), P<0.05 (Student's t-test).

(F) Skin stratification in WT and cKO skin was determined by immunofluorescence staining with different antibodies as indicated. Krt14: Keratin 14; Krt10: Keratin 10; β4: β4-integrin; DAPI for nucleus staining. The dashed line denotes the basement membrane that separates dermis and epidermis (Epi).

(G) Wound healing as monitored by wound size 8 days post injury. n=3; P<0.01 (Student's t-test). Error bar represents S.D. (standard deviation).

(H) Histological staining of skin sections at the wound edges. Halves of wound sections are shown. Note significant reduction of HPE (hyperproliferative epidermis) in cKO skin. Es: eschar. Dotted lines denote epidermal boundaries.

(I) Quantification of Ki67-positive cells present in wound HPE. The plot indicates the mean (open circles within the box), 25th percentile (bottom line of the box), median (middle line of the box), 75th percentile (top line of the box), 5th and 95th percentile (whiskers), 1st and 99th percentile (solid triangles) and minimum and maximum measurements (solid squares). n=19, P<0.01 (Student's t-test).
N6-methyladenosine Modification of lncRNA Pvt1 Governs Epidermal Stemness (2021)
Figure 2

Figure 2. Loss of Mettl14 impairs epidermal stemness.

(A) EdU staining of WT or Mettl14 cKO skin after pulse-chase labeling. Skin samples were counterstained with antibody against β4-integrin. Note reduced EdU label-retaining cells in cKO skin epidermis. Arrows indicate Edu-positive cells.

(B) Label-retaining cells in WT or Mettl14 cKO skin were quantified and shown as box plots. The plot indicates the mean (solid diamond within the box), 25th percentile (bottom line of the box), median (middle line of the box), 75th percentile (top line of the box), 5th and 95th percentile (whiskers), 1st and 99th percentile (solid triangles) and minimum and maximum measurements (solid squares). n=6 (biological repeats), P<0.05 (Student's t-test). n=19, P<0.01 (Student's t-test).

(C) Number of holoclones derived from WT and Mettl14 cKO skin was quantified and shown as box and whisker plots. The plot indicates the mean (solid diamond within the box), 25th percentile (bottom line of the box), median (middle line of the box), 75th percentile (top line of the box), 5th and 95th percentile (whiskers), 1st and 99th percentile (solid triangles) and minimum and maximum measurements (solid squares). n=6 (biological repeats), P<0.05 (Student's t-test). n=24, P<0.01 (Student's t-test).

(D) Morphology of primary keratinocytes isolated from WT or Mettl14 cKO skin.

(E) Expression of Mettl14 and p63 in WT and KO cells was determined by immunoblots with respective antibodies. Immunoblot for GAPDH was used as loading control.

(F) Proliferation of WT and Mettl14 null cells in vitro was quantified and shown as dot plots. n=3, P<0.01 (Student's t-test) for Day 6, 9, and 12, and P<0.05 (Student's t-test) for Day 3. Error bar represents S.D.

(G-H) CFE (colony formation efficiency) of WT and Mettl14 null cells was determined in vitro. Results were quantified and shown as bar graph (F). n=19, P<0.01 (Student's t-test). Error bar represents S.D.

(I) Fluorescence microscopy demonstrates different survival capability of WT and Mettl14 inducible KO cells with or without Tamoxifen (TAM) treatment.

(J) Ratio of WT and Mettl14 inducible KO cells in the co-culture model was quantified and shown as dot plots. n=8, P<0.01 (Student's t-test) for KO cells with TAM treatment compared with WT cells or KO cells without TAM stimulation at both Day 7 and 14. Error bar represents S.D.
N6-methyladenosine Modification of lncRNA Pvt1 Governs Epidermal Stemness (2021)
Figure 3

Figure 3. Pvt1 regulates epidermal stemness and tissue homeostasis.

(A) Profiling of RNA methylome by m6A-seq shows significant changes of lncRNA modifications upon epidermal differentiation in vitro.

(B) Mutations in the predicted m6A modification sites can significantly reduce Pvt1 methylation.n=3. Error bar represents S.D.

(C) Deletion of endogenous Pvt1 by an inducible Cas9 (iCas9) system can significantly reduce Pvt1 RNA level (quantification from RT-PCR, left panel) and MYC protein level (quantification from immunoblot, right panel) expression. n=3, P<0.01 (Student's t-test). Error bar represents S.D.

(D) CFE of WT, Pvt1 inducible KO, and Pvt1 inducible KO cells rescued with WT or mutant Pvt1 was quantified and presented as bar graph. n=3, **: P<0.01 (Student's t-test). Error bar represents S.D.

(E) Skin organoids derived from WT or Pvt1 inducible KO cells were grafted to nude mice. The regenerated skin was analyzed by H/E staining.

(F) Epidermal thickness of WT and Pvt1 KO skin grafts was quantified and presented as box and whisker plots. The plot indicates the mean (solid diamond within the box), 25th percentile (bottom line of the box), median (middle line of the box), 75th percentile (top line of the box), 5th and 95th percentile (whiskers), 1st and 99th percentile (solid triangles) and minimum and maximum measurements (solid squares). n=6 (biological repeats), P<0.05 (Student's t-test). n=18, P<0.01 (Student's t-test).

(G) Number of p63-positive cells in WT and cKO skin was quantified and shown as bar graph. n=5, P<0.01 (Student's t-test). Error bar represents S.D.

(H) Fluorescence microscopy demonstrates different survival capability of WT and Pvt1 inducible KO cells with or without Doxycycline (Dox) treatment.

(I) Ratio of WT and Pvt1 inducible KO cells in the co-culture model was quantified and shown as dot plots. n=8, P<0.01 (Student's t-test) for KO cells with Dox treatment compared with WT cells or KO cells without Dox stimulation at both Day 7 and 14. Error bar represents S.D.
N6-methyladenosine Modification of lncRNA Pvt1 Governs Epidermal Stemness (2021)
Figure 4

Figure 4. Pvt1 methylation regulates epidermal stemness through its interaction with MYC.

(A) Interaction between Pvt1 and MYC was determined by immunoprecipitation followed with RT-PCR. Note significant decrease in Pvt1 and MYC interaction upon FTO treatment (left panel), calcium shift-induced differentiation (middle panel, Hi: high calcium), and Pvt1 methylation site mutations (right panel). n=3, P<0.01 (Student's t-test). Error bar represents S.D.

(B) Immunoblots show decreased MYC protein level in differentiated keratinocytes and Mettl14 KO cells.

(C-E) Undifferentiated WT and Mettl14 KO cells (C); or undifferentiated WT and Pvt1 KO cells (D); or WT cells before and after calcium shift-induced differentiation (E) were treated with cycloheximide. Cell lysates were collected at 0, 30, 60, 90, 120, 180, and 300 minutes post treatment and subjected to immunoblotting with different antibodies as indicated.

(F) Skin organoids derived from Mettl14 KO cells or KO cells rescued with exogenous expression of MYC were grafted to nude mice. The regenerated skin was analyzed by H/E staining.

(G) Epidermal thickness of KO and KO skin rescued with MYC expression was quantified and presented as box and whisker plots. The plot indicates the mean (solid diamond within the box), 25th percentile (bottom line of the box), median (middle line of the box), 75th percentile (top line of the box), 5th and 95th percentile (whiskers), 1st and 99th percentile (solid triangles) and minimum and maximum measurements (solid squares). n=6 (biological repeats), P<0.05 (Student's t-test). n=18, P<0.01 (Student's t-test).

(H) Number of p63-positive cells in KO and KO skin rescued with MYC expression was quantified and shown as bar graph. n=5, P<0.01 (Student's t-test). Error bar represents S.D.

(I) Fluorescence microscopy demonstrates different survival capability of WT and Mettl14 KO cells with inducible expression of MYC.

(J) Ratio of WT and Mettl14 KO cells with inducible expression of MYC in the co-culture model was quantified and shown as dot plots. n=8, P<0.01 (Student's t-test) for KO cells without Dox treatment (no MYC expression) compared with WT cells or KO cells with Dox stimulation (exogenous MYC expression) at both Day 7 and 14. Error bar represents S.D.
A novel cytoprotective function for the DNA repair protein Ku in regulating p53 mRNA translation and function (2016)
Figure 1

Figure 1. Ku downregulation increases p53 expression. Treatment of HCT116 cells with siRNA targeting Ku70 (A-I), ASF (C) (positive control for γ-H2AX activation [29]), XRCC4 (F) or control (A-I) combined or not with a treatment with doxorubicin (Doxo) (positive control for phosphorylation of ATM and p53) (B), wortmannin (WTN) (D), NU-7441 (E,F), MG132 (H) or cycloheximide (CHX) (I) followed by western blot analysis (A-F, H-I) or RT-qPCR analysis (G). (A) The basal levels of p53 normalized to GAPDH were arbitrarily set at 1.0 and the fold change is shown in (ii) (n=4). In RT-qPCR analysis (G), mRNA levels were standardized against HPRT mRNA (n=3). The two panels of Fig. 1C are partion of the same gel. Statistical analysis by unpaired t-test (*P<0.05; **P<0.01). All error bars reflect SEM.
A novel cytoprotective function for the DNA repair protein Ku in regulating p53 mRNA translation and function (2016)
Figure 2

Figure 2. Ku interacts with the p53 mRNA. (A) RNA affinity chromatography using the p53 5’UTR (5’p53) or a portion of the p47 5'UTR (5'p47) (depicted in (i)) and HeLa cytoplasmic extracts (CE), followed by western blot analysis (ii). (B) IP of in cellulo RNA-protein complexes (RIP) in HCT116 cells, followed either by RT-qPCR analysis. Bar plots represent the mean and the data of two independent experiments (i) or by western blot analysis (ii). The relative p53 mRNA levels for each IP sample were normalized to the corresponding IP IgG and to the corresponding input sample and were plotted relatively to the HPRT mRNA. (C) UV cross-linking of recombinant (Ku70/Ku80) with the in vitro transcribed 5’p53 with alpha 32P-GTP or alpha 32P-UTP (i). UV cross-linking of recombinant Ku70/Ku80 with the in vitro transcribed 5’p53 and the Ctrl (5’p53 antisense) with alpha 32PGTP (ii). (D,E) RNA affinity chromatography using the different RNA constructs depicted in (i), as described in A.
A novel cytoprotective function for the DNA repair protein Ku in regulating p53 mRNA translation and function (2016)
Figure 3

Figure 3. Ku regulates p53 mRNA translation and function. (A) Non polysomal (NP) and polysomal (P) fractions were extracted from HCT116 cells transfected with the siRNA Ku70 and quantitative RT-qPCR was performed using specific primers for p53 and HPRT mRNAs. The p53 mRNA levels in P and NP fractions were normalized to the input (n=3) (B) Western blot from H1299 (p53-null) cells treated with siRNA Ku70 or control (ii), followed by transfections of RNA constructs depicted in (i). p53 protein levels from the WT and the ΔB2 reporters depicted in (i) normalized to firefly luciferase (transfection control reporter) (iii, n=3). (C) AnnexinV/PI staining and flow cytometry experiments performed as in (B) to assess cell death (n=3). (D) RT-qPCR analysis of Bax, TP53I3, BTG2, GADD45α and p21 after siRNA-mediated Ku or control depletion in HCT116 p53 +/+ or HCT116 p53 -/- (n=3). Relative mRNA levels were standardized against GAPDH mRNA. Statistical analysis by unpaired t-test (*P<0.05; **P<0.01; *** P<0.001 NS, non significant). All error bars reflect SEM.
A novel cytoprotective function for the DNA repair protein Ku in regulating p53 mRNA translation and function (2016)
Figure 4

Figure 4. Ku acetylation relieves repression of p53 mRNA translation. (A) Western blot analysis after treatment with Ku70 or control siRNA, followed by incubation with DMSO, etoposide (ETO) or bleomycin (BLEO) for 16 h (i). p53 levels after Ku70 depletion normalized to siRNA control for each condition (ii, n=3) (B) Western blot analysis as in A, except that tricostatin A (TSA) was used after Ku/control knockdown (n=4). (C) Western blot in cells treated either with DMSO, TSA, or ETO for 16 or 24 h. (D) RNA affinity chromatography using the 5’p53 RNA and total extracts from cells treated with TSA, ETO or BLEO for 24 h, followed by western blot analysis of Ku70 quantified and normalized to the input (ii, n=3) (E) RNA affinity chromatography using the 5’p53 RNA and total extracts from cells transfected or not with PCAF encoding plasmid for 48 h, followed by western blot analysis. (F) RNA affinity chromatography using the 5’p53 RNA or a dsDNA and total extracts from cells transfected with HA-tagged Ku70 wild-type (WT) or mutated (Mut6E) Ku70 [25] for 48 h, followed by western blot analysis. (G) RNA affinity chromatography as in F, but with two additional Ku mutants (K282/338Q and K282/338R), followed by western blot of Ku70 quantified and normalized to the input and to the loading control, PTB (ii, n=3) All the experiments were performed with HCT116 cells. GAPDH: loading control in A, B, C. Statistical analysis by unpaired t-test (*P<0.05; **P<0.01; *** P<0.001; NS, non significant). All error bars reflect SEM.