EMBO/soda-nel huggingface dataset:
Physiologically relevant miRNAs in mammalian oocytes are rare and highly abundant. (2021)
Figure 1

Figure 1 Most abundant miRNAs in mammalian oocytes.

(A) miRNA abundance in RNA-sequencing samples from murine, porcine, and bovine oocytes Gad et al., 2019Garcia-Lopez et al., 2015Graf et al., 2014(; ; ). Each graph shows on the y-axis reads per million of 19-32 nt reads for miRNAs and on the x-axis miRNAs ordered by name.

(B) Schematic depiction of miRNA precursors. Precursor sequences and predicted basepairing were adopted from miRbase miRNA annotations Kozomara et al., 2019(). miR-10b secondary structures in the mirRbase contained two different basepairing versions in the loop, the alternative folding is displayed in Fig. EV1A.

(C) qPCR quantification of ssc-miR-205 and bta-miR-10b miRNAs in murine (M.m.), porcine (S.s.), and bovine (B.t.) oocytes, respectively. Shown are a mean values from three independent experiments. Error bars = SD. Detailed information concerning miRNA quantification is provided in the Methods section.
Physiologically relevant miRNAs in mammalian oocytes are rare and highly abundant. (2021)
Figure 2

Figure 2 Endogenous miRNA activity is detected in bovine and porcine oocytes.

(A) bta-miR-10b and ssc-miR-205 miRNAs efficiently repress microinjected NanoLuc luciferase reporters carrying a perfectly complementary (1x perfect) miRNA binding site or four partially complementary (bulged) miRNA binding sites. See Fig. EV2a for overview of reporter design. Error bars = SD. Each data point represents a value obtained from five microinjected oocytes. Data were collected in two microinjection sessions, each microinjection session used a new batch of in vitro transcribed reporter mRNAs.

(B) De-repression of predicted ssc-miR-205 targets. In three independent microinjection experiments, ssc-miR-205 was inhibited by a microinjected antisense oligonucleotide inhibitor and target levels were assessed 24 hours after microinjection by RT-qPCR. Error bars = SD.

(C) Inhibition of ssc-miR-205 by a microinjected antisense oligonucleotide inhibitor results in reduced development to the blastocyst stage. Shown is development of microinjected oocytes to the blastocyst stage relative to water-injected oocytes. Five independent experiments (represented by individual data points) were performed, ~50 porcine oocytes were microinjected in each group in each experiment. Difference between let-7a and miR-205 inhibitors effects is statistically significant (two-tailed paired t-test p-value = 0.016). Error bars = SD.

Data information: In panels A-C, asterisks indicate statistical significance (p-value) of one-tailed t-test, (*p < 0.05, **p < 0.01, ***p < 0.001).
Physiologically relevant miRNAs in mammalian oocytes are rare and highly abundant. (2021)
Figure 4

Figure 4 Selected features of ssc-miR-205.

(A) ssc-miR-205 turnover. Fully-grown transcriptionally quiescent oocytes were cultured for indicated periods of time and selected miRNAs were quantified by RT-qPCR in four (ssc-let-7a), five (ssc-miR-22) or six (ssc-mir10b and ssc-miR-205) independent experiments. Error bars = SD.

(B) Comparison of mean relative changes of miRNA level at 20 h of culture (see Fig. EV4A for 40 h data). ssc-miR-205 in oocytes is less than ssc-miR-10b, ssc-miR-22, or ssc-let-7a. Error bars = SD. Asterisks indicate statistical significance (p-value) of one-tailed paired t-test, (*p < 0.05, **p < 0.01).

(C) Treatment by sodium periodate followed by qPCR does not support 2'-OH modification of either ssc-miR-205 or bta-miR-10b miRNA. Shown are relative levels of the sodium periodate-treated samples to non-treated samples, which were set to one. The experiment was performed three times. Error bars = SD. Efficiency of periodate treatment was confirmed using non-methylated and methylated miR-221 RNA oligonucleotides (EV4B).

(D) 3′ tailing of small RNAs in murine, bovine, and porcine oocytes Gad et al., 2019Garcia-Lopez et al., 2015Graf et al., 2014(; ; ). Shown are percentages of miRNAs tailed at 3′ end with mono- and oligonucleotides.

Expanded View Figures
Human isotype-dependent inhibitory antibody responses against Mycobacterium tuberculosis (2016)
Figure 1

Figure 1. Anti-MTB serum antibody response in peripheral blood of TB patients

(A) Serum IgG ELISA reactivity with MTB (H37Rv) whole cell lysate, purified cell membrane fraction and secreted culture filtrate proteins (CFP) for TB patients and healthy donors (HD). ELISA graphs (left; TB: black lines; n=25; HD: red dotted lines; n=2) and area under the curve (AUC) values for all tested samples (right; TB: n=25; HD: n=17) are shown. Median is shown. (B) AUC values for the IgA serum ELISA response against MTB whole cell lysate, purified cell membrane fraction and secreted culture filtrate proteins (CFP) of TB patients (n=25) and healthy donors (HD; n=9). Median is shown. (C) Flow cytometric gating strategy and frequency of circulating plasmablasts (CD19+CD27++CD38+) for one representative TB patient (TB24) and HD (left). Frequency of circulating CD19+CD27++CD38+ plasmablasts of all CD19+ B cells in peripheral blood of TB patients (n=24) and HD (n=8). Dashed line indicates threshold for detectable plasmablast populations (right). Median and SEM are shown. (D) Expression of CD19 and activation markers (CD86, CD84 and CD24) in plasmablasts and memory B cells as measured by flow cytometry. (E) Plasmablast response of patient TB7 (top) and IgG serum response against MTB cell lysate of patient TB7, TB19 and TB39 (bottom) and one HD at the indicated time points before and after treatment onset. (F) Dots indicate AUC values for the anti-MTB cell lysate IgG serum ELISA response (y-axis) versus frequency of circulating plasmablasts (x-axis) for individual TB patients. All data are representative of two independent experiments. Spearman correlation and P-value are shown. (A and B) P-values were determined using Mann-Whitney test; **p<0.01; ****p<0.0001; ns, not significant.
Human isotype-dependent inhibitory antibody responses against Mycobacterium tuberculosis (2016)
Figure 2

Figure 2. Somatic hypermutation level and isotype distribution of single-cell-sorted plasmablasts and antigen-specific memory B cells

(A) Gating strategy, phenotype and frequency of circulating plasmablasts (CD19+CD27++CD38+) isolated by flow cytometric cell sorting from 3 TB patients (TB7, TB24, TB33) in comparison to one representative HD. Boxes indicate sort gates. The plasmablast frequency is indicated. (B) Absolute number of somatic hypermutations (SHM) in the IGHV, IGKV and IGLV segments of IgA and IgG plasmablast antibody genes sequenced from TB7, TB24 and TB33. The absolute number of sequences analyzed is indicated below the graph. Geometric means with SEM are indicated in grey. SHM means of historic data from sorted CD27+IgA+ or CD27+IgG+ cells from peripheral blood of HD are indicated in red for comparison (Berkowska et al, 2015a; Tiller et al, 2007). (C) Isotype distribution of plasmablast and memory B cells from TB7, TB24 and TB33. PB, plasmablasts (CD19+CD27++CD38+); M, memory B cells (CD19+CD27+). (D) Gating strategy, phenotype and frequency of HBHA-reactive memory B cells (CD19+CD27+HBHA+) in peripheral blood of one representative TB patient (TB29), health-care worker (HCW) 2 and HD, respectively. (E) Dots indicate frequency of HBHA-reactive memory B cells out of all CD27+ memory B cells in individual TB patients (n=23) and HCWs (n=7). Mean and SEM are indicated. P-value was determined using Wilcoxon-Mann-Whitney test; ****p<0.0001. (F) Dots indicate the frequency of resting memory B cells (CD19+CD27+CD10-) out of all B cells in peripheral blood of individual TB patients compared to HD. (G) Anti-HBHA serum IgG ELISA reactivity for TB patients and HCW (black lines) compared to two representative HD (red lines). Dashed line indicates the threshold OD405nm for positive reactivity (left). Asterisks indicate serum responses of donors selected for single sorting of HBHA-reactive memory B cells. Data are representative of two independent experiments. (H) Absolute number of somatic hypermutations (SHM) in the IGHV (IGHA, IGHG and IGHM), IGKV or IGLV segments of sorted anti-HBHA memory cells from TB patients and HCWs. Geometric means with SEM are indicated in grey. For comparison, red lines indicate the historic SHM means for randomly sorted CD27+IgA+, CD27+IgG+ or CD27+IgM+ cells from peripheral blood of HDs (Berkowska et al, 2015a; Tiller et al, 2007; Tsuiji et al, 2006). (I) Number and size of clonally expanded B cell clusters among HBHA-reactive memory B cells from two TB patients (TB35, TB29) and HCWs (HCW1, HCW 2). Cells in clusters are indicated in grey, single cells are indicated in white. No B cell clusters were shared between donors. (J) IgA, IgG and IgM isotype distribution of single-cell-sorted HBHA-reactive memory cells. Mean and SEM are indicated.
Human isotype-dependent inhibitory antibody responses against Mycobacterium tuberculosis (2016)
Figure 4

Figure 4. The surface-exposed virulence factors LAM and HBHA are targets of the human plasmablast and memory B cell response to MTB

(A) Binding of antibodies TB24PB037 and TB33PB123 to LAM purified from MTB as determined by Western Blot. The negative isotype control (mGO53) and a commercially available anti-LAM antibody (positive control) are shown for comparison. (B) Anti-BCG specificity of the LAM-reactive antibody TB24PB037 (black) compared to E.coli as determined by ELISA with whole bacteria (left) and flow cytometry (right). (C) Anti-LAM ELISA for TB24PB037 or negative control antibody at the indicated concentrations. The mean ±SD of the absorbance was calculated. (D) Antibody TB24PB037 binds to MTB-LAM but not to LAM from M. smegmatis. ELISA performed as described in (C). (E) Representative HBHA ELISA for antibodies cloned from HBHA-reactive memory B cells of patient TB35 and HCW1. Dashed red line indicates the threshold OD405nm for positive reactivity. Green line indicates negative control antibody (mGO53; (Wardemann et al, 2003)). (F) Reactivity to HBHA was confirmed by Western Blot for a selected set of antibodies with HBHA ELISA reactivity. (G) Fluorescence microscopy shows BCG- and MTB-reactivity of representative anti-HBHA antibodies compared to a non-reactive isotype control antibody. Scale bar 10 μm (top/bottom). (A-G) All data are representative of two independent experiments.
Human isotype-dependent inhibitory antibody responses against Mycobacterium tuberculosis (2016)
Figure 5

Figure 5. IgA and IgG isotype-dependent functional differences of monoclonal antibodies on MTB infection of human lung epithelial cells

Relative bacterial counts (CFU) compared to the medium control (100%) in human lung epithelial A549 cells 1 h after infection with MTB, pre-incubated with recombinant MTB-reactive IgA1, IgA2 or IgG1 antibodies and non-reactive isotype control as indicated. (A) Representative results for individual MTB-reactive plasmablast antibodies. Mean and SEM are indicated. (B) Direct comparison of all tested MTB-reactive IgA (n=12) and IgG (n=26) antibodies. Mean and SEM are indicated. (C) Representative results for individual HBHA-reactive memory B cell antibodies from TB patients and HCW. (D) Same as in (B) but with HBHA-reactive antibodies as shown in (C). (D) Direct comparison all tested HBHA-reactive IgA (n=6) and IgG (n=7) antibodies. (E) Direct comparison of the indicated MTB-reactive antibodies expressed as IgA and IgG. Mean and SEM are indicated. P-values were determined using non-parameric t-test, * p<0.1; **p<0.01, ns: p>0.5. (A-E) Data are representative of at least two independent experiments with three technical replicates each. (B, D) P-values were determined using Wilcoxon-Mann-Whitney test, *** p<0.001; ****p<0.0001.
Human isotype-dependent inhibitory antibody responses against Mycobacterium tuberculosis (2016)
Figure 6

Figure 6. Fc-receptor expression on human lung epithelial cells

(A) Human lung epithelial A549 cells and primary bronchial epithelial cells (hAECB) lack FcγR (CD16, CD64 and CD32) and FcαR (CD89) cell surface expression as analyzed by flow cytometry. Human THP1 cells serve as positive control. (B) The neonatal FcR (FcRn) is expressed in both A549 cells and human THP1 cells as determined by Western Blotting. Data are representative of at least two independent experiments. (C) Absolute bacterial counts (CFUs) in human THP1 macrophages 1 h after infection with MTB pre-incubated with the LAM-reactive IgA1 (left) or IgG1 (right) antibody TB24PB037 or the respective isotype control antibody as indicated. Mean and SEM are indicated. (A-C) Data are representative of three independent experiments.
Specific ACE2 Expression in Cholangiocytes May Cause Liver Damage After 2019-nCoV Infection (2020)
Figure 1.

single cell analysis of published liver cell atlas. A) liver cell atlas visualized by tSNE, colored by cluster number. Cluster number information is provided by the authors. B) liver cell atlas visualized by tSNE, colored by major cell type compartments. Clusters were collapsed into major cell types based on their canonical markers. C) The dotplot showing cell type marker expression of all cell types. D) The violin plot of ACE2 gene expression of all major cell types.
Specific ACE2 Expression in Cholangiocytes May Cause Liver Damage After 2019-nCoV Infection (2020)
Figure 2.

single cell analysis of the in-house cohort of healthy livers. A) Cell type annotations of all four patients by the tSNE plot. Hepatocyte-cholangiocytes cluster includes potential stem-like cells capable of differentiating into both cell types. B) tSNE plot colored by patients C) Heatmap of marker genes of 16 cell types identified. The red box highlights the cell type specific expression of ACE2. D) The violin plot of ACE2 gene expression across all cell types.
Human CST complex protects stalled replication forks by directly blocking MRE11 degradation of nascent-strand DNA (2020)
Figure 1

Figure 1. CST localizes at stalled forks.

Scheme of SIRF assay. Nascent DNA was pulse-labelled with EdU. Upon fork stalling, proteins located at stalled forks are in close proximity to EdU. EdU was then biotinylated using click reaction, followed by incubating with biotin and target protein antibodies. PLA amplification was used to visualize the localization of target proteins at EdU-labeled stalled forks.

B, C. Detection of CST at stalled and active forks with SIRF assay in U2OS (B) and HCT116 (C) cells. To detect CST at stalled forks, cells were pulse-labeled with EdU for 8 min, followed by 4 mM HU treatment for 3 h. To detect CST at active forks, SIRF was performed without HU treatment. Representative SIRF images of CTC1, STN1 and PCNA on normal or stalled forks are shown. Scale bars: 10 µm. Three independent experiments were performed and scatter plots from one experiment are shown in the main figure. Scatter plots of two additional biological replicates are included in Appendix Figure S1C (U2OS cells) and Appendix Figure S1D and S1E (HCT116 cells). Relative SIRF fluorescence intensity was calculated by normalizing to untreated samples and then to their respective EdU-SIRF signals. Mean value in each sample is labeled in the graph. N: the number of cells analyzed in each sample. P values were calculated by Mann-Whitney test. *** P<0.001. Red lines indicate the mean values. Error bars: SEM.
Human CST complex protects stalled replication forks by directly blocking MRE11 degradation of nascent-strand DNA (2020)
Figure 2

Figure 2. CST suppresses the accumulation of ssDNA at stalled forks and protects nascent-strand DNA from MRE11 degradation.

Western blot showing STN1 knockdown. shLUC (siRNA targeting luciferase) was used as the control.

CST deficiency causes ssDNA accumulation upon fork stalling. HeLa cells with STN1 knockdown were incubated with 10 µM BrdU for 48 h, followed by 2 mM HU treatment for 3 h, and subsequently stained with BrdU antibody under a non-denaturing condition to detect ssDNA. N, the number of cells analyzed in each condition. Scale bars: 20 µm. Relative BrdU fluorescence intensity was quantitated by Image J. P values were calculated by one-way ANOVA analysis with post hoc Tukey. Error bars, SEM, *** P<0.001, ** P<0.01. Three independent experiments were performed. Results from two additional biological replicates are included in Appendix Figure S2A.

Scheme of DNA fiber analysis and representative fiber images.

DNA fiber analysis of U2OS cells with STN1 knockdown and its RNAi-resistant Flag-STN1 WT re-expression with and without mirin treatment. Results from biological replicates and corresponding representative fiber images are included in Appendix Figure S2B.

DNA fiber analysis of BJ cells with STN1 knockdown by two siRNA sequence with and without mirin treatment. Results from biological replicates are included in Appendix Figure S2C.

DNA fiber analysis of HCT116 cells with STN1 knockdown with and without mirin treatment. Results from biological replicates are included in Appendix Figure S2D.

DNA fiber analysis of U2OS cells showing that SMARCAL1-mediated fork reversal is needed for nascent-stand degradation observed in CST-deficient cells. Results from biological replicates are included in Appendix Figure S2E.

DNA fiber analysis of U2OS cells showing that ZRANB3-mediated fork reversal is needed for nascent-strand degradation observed in CST-deficient cells. Results from biological replicates are included in Appendix Figure S2F.

Data information: For all DNA fiber experiments in this study, at least two independent treatments and fiber experiments were performed to ensure reproducibility. Results from one experiment are shown in the main figure. Scatter plots indicate IdU/CIdU tract length ratios for individual replication forks. N, the number of DNA fibers analyzed in each condition. P values were calculated by one-way ANOVA analysis with post hoc Tukey. *** P<0.001, ** P<0.01, * P<0.05. Red lines indicate mean values. Error bars: SEM. Western blots showing protein knockdown in DNA fiber assays are included.
Human CST complex protects stalled replication forks by directly blocking MRE11 degradation of nascent-strand DNA (2020)
Figure 3

Figure 3. CST deficiency increases MRE11 localization at stalled forks, leading to ssDNA accumulation.

SIRF detection of MRE11 at stalled forks in STN1-deficient U2OS cells. Representative SIRF images of MRE11 at normal or stalled replication forks are shown. Scale bars: 10 µm. Two independent experiments were performed. Results from the biological replicate are shown in Appendix Figure S3A. P values were calculated by one-way ANOVA analysis with post hoc Tukey, *** P<0.001. Red lines indicate mean values. Error bars: SEM.

ssDNA accumulation analysis in STN1-deficient U2OS cells with and without mirin treatment. Representative images of native BrdU staining are shown. Scale bars: 20 µm. Three independent experiments were performed. Results from two additional biological replicates are shown in Appendix Figure S3C. P values were calculated by one-way ANOVA analysis with post hoc Tukey, *** P<0.001. Red lines indicate mean values. Error bars, SEM.
Human CST complex protects stalled replication forks by directly blocking MRE11 degradation of nascent-strand DNA (2020)
Figure 4

Figure 4. CST binding to 5' overhangs directly blocks MRE11 degradation.

Coomassie blue stained SDS-PAGE gel (15%) of purified human MRE11.

(i) Scheme shows the nuclease activity of MRE11 in degrading 5' Cy3-labeled substrates (25 nt + 60 nt with phosphorothioate bonds on both ends). (ii) The 5' Cy3-labeled substrates (80 nM) were incubated with the indicated concentration of MRE11 with or without the Mn2+ cofactor at 37°C for 20 min. Reactions were stopped by SDS and proteinase K and resolved on 27% denatured polyacrylamide gel. (iii) The results are graphed, and error bars represent the standard deviation (± SD) calculated from at least three independent experiments.

Coomassie blue stained SDS-PAGE gel (15%) of purified human CST complex (CTC1 wild- type and ∆700N).

DNA binding activity of wild-type CST complex and Δ700N mutant complex. The 5' Cy3- labeled substrates (80 nM) were incubated with the indicated concentrations of CST. Samples were analyzed by 0.8 % agarose gel.

MRE11 degradation analysis. 5' Cy3-labeled substrates (80 nM) were pre-incubated with indicated concentrations of CST wild-type (i) or ∆700N mutant variant (ii) at 37°C for 5 min. Reactions were completed by adding MRE11 (200 nM) for an additional 20 min incubation and then stopped by SDS and proteinase K. Samples were resolved in 27% denatured polyacrylamide gel. The results are graphed and error bars represent the standard deviation (± SD) calculated from at least three independent experiments.
Human CST complex protects stalled replication forks by directly blocking MRE11 degradation of nascent-strand DNA (2020)
Figure 5

Figure 5. The ssDNA binding ability of CST is required for protecting nascent strands from degradation.

Deletion of the N-terminal 700 aa of CTC1 abolishes CTC1 localization at telomeres. U2OS stably expressing vector (V), WT Myc-CTC1 and Myc-∆700N were treated with or without HU and co-stained with Myc (red) and TRF2 (green) antibodies. Boxed areas are amplified in inserts to indicate CTC1/TRF2 colocalization (yellow).Scale bars: 10 μm. Quantification of percent of cells with >3 CTC1/TRF2 colocalization foci were from three independent experiments. In each experiment, >150 cells from each sample were analyzed. P values were calculated by one-way ANOVA analysis with post hoc Tukey from three independent experiments, *** P<0.001. Error bars: SEM.

∆700N does not affect CST complex formation. HEK293T cells were co-transfected with Myc- CTC1 or Myc-∆700N, His6-STN1 and HA-TEN1. Co-IP was performed with Myc antibody to pulldown His6- STN1 and HA-TEN1. Full-length Myc-CTC1 was prone to degradation during immunoprecipitation.

∆700N retains RAD51 interaction. HEK293T cells were co-transfected with Myc-CTC1 or Myc-∆700N, His6- STN1, HA-TEN1 and Flag-RAD51 or vector control (V), and treated with HU (2 mM, 16 h). Co-IP was performed with Flag antibody to pulldown Myc-CTC1.

DNA fiber analysis of CTC1 depleted U2OS cells with RNAi-resistant WT or ∆700N re-expression. Two independent experiments were performed. Results from the biological replicate are included in Appendix Figure S4B. Mean values in each sample are listed at the top of the graph. P values were calculated by one-way ANOVA analysis with post hoc Tukey. N, the number of cells analyzed in each condition, *** P<0.001. Red lines indicate the mean values. Error bars: SEM.