EMBO/soda-nel huggingface dataset:
The 18S rRNA m6A methyltransferase METTL5 promotes mouse embryonic stem cell differentiation (2020)
Figure 4

Figure 4. Depletion of Mettl5 affects the translation efficiency of Fbxw7.

A Volcano plot depicting down-regulated (left) and up-regulated (right) genes in Mettl5 KO mESCs compared with WT mESCs in Ribo-seq. Significantly altered genes are defined as those ≥1.5-fold change and P value < 0.05 (shown in red dots). Blue dots represent genes with <1.5-fold change and P value < 0.05. Green dots represent genes with ≥1.5-fold change and P value ≥ 0.05. Black dots represent genes with ＜1.5-fold change and P value ≥ 0.05. P values were inferred from two-sided Wald tests (DESeq2) and P value adjustments were made for multiple comparisons. Examples of up- or down-regulated or non-significantly changed genes are annotated in the graph.

B Track plot showing translational changes of Fbxw7 in Mettl5 KO mESCs compared with controls.

C RT-qPCR analysis of Fbxw7 mRNA levels in WT and Mettl5 KO cells during the indicated time points course of EB induction. Data are represented as mean ± SD from three biological replicates. Student's t test, two-tailed. ns, not significant.

D Western blot analysis showing FBXW7 expression in control and Mettl5 KO cells during the indicated time points of EB induction. GAPDH was used as a loading control.

E RT-qPCR analysis showing Fbxw7 mRNA levels in Day7 KO, KO+HA-MT5WT, and KO+HA-MT5Mut EBs. Data are represented as mean ± SD from three biological replicates. Student's t test, two-tailed. ns, not significant.

F Western blot analysis showing FBXW7 expression in Day7 KO, KO+HA-MT5WT, and KO+HA-MT5Mut EBs. GAPDH was used as a loading control.
The 18S rRNA m6A methyltransferase METTL5 promotes mouse embryonic stem cell differentiation (2020)
Figure 5

Figure 5. FBXW7 induction partially rescues the defects in Mettl5 KO mESCs while c-MYC induction mimics the differentiation block.

­­­A Western blot analysis showing dynamic expression patterns of FBXW7 and c-MYC during the indicated time points of EB induction. α-TUBULIN was used as a loading control.

B Western blot analysis showing FBXW7 and c-MYC expression in control, Mettl5 KO (KO-6B) and KO+HA-MT5WT Day7 EBs, respectively. α-TUBULIN was used as a loading control.

C Schematic diagram depicting induction of FBXW7 expression using rtTA-dependent system in Mettl5 KO mESCs. Reverse tetracycline-controlled trans-activators (rtTA) can recognize the TET response element (TRE) and express FBXW7 at the presence of a derivative of tetracycline-doxycycline (Dox).

D RT-qPCR analysis of the expression of pluripotency and lineage-specific markers in Day7 FBXW7-induced EBs at the presence or absence of 10 ng/ml Dox. Data are represented as mean ± SD from three biological replicates. Student's t test, two-tailed. *, P < 0.05; **, P < 0.01; ***, P<0.001.

E Immunostaining analysis of the expression of pluripotency marker POU5F1, lineage-specific markers FOXA2, T and NESTIN in Day7 FBXW7-induced EBs at the presence or absence of 10 ng/ml Dox. Scale bars represent 100 μm.

F Schematic diagram depicting induction of c-MYC expression using rtTA-dependent system in WT mESCs.

G RT-qPCR analysis the expression of pluripotency and lineage-specific markers in Day7 c-MYC-induced EBs at the presence or absence of 10 ng/ml Dox. Data are represented as mean ± SD from three biological replicates. Student's t test, two-tailed. *, P < 0.05; **, P < 0.01; ***, P<0.001.

H Immunostaining analysis of the expression of pluripotency marker POU5F1, lineage-specific markers FOXA2, T and NESTIN in Day7 c-MYC-induced EBs at the presence or absence of 10 ng/ml Dox. Scale bars represent 100 μm.
Dynamics of Staphylococcus Aureus Cas9 in DNA target Association and Dissociation (2020)
Figure 1

Figure 1. The interaction sites and strengths between dSaCas9 and DNA. A. Cartoon illustrating the single-molecule DNA unzipping experiment used for the detection of the interactions between dSaCas9 and DNA and the definition of the DNA sequence.

B. Representative traces of the forward DNA unzipping in the presence of dSaCas9/sgRNA showing the force versus the number of unzipped base pairs. The naked DNA unzipping signatures are also presented for comparison (gray). Insets: zoomed-in view of the regions with increases in force. The dashed lines illustrate the interaction sites between dSaCas9 and the DNA. Black arrows indicate the force peaks of the pre-PAM interaction.

C. Representative traces of the reverse DNA unzipping in the presence of dSaCas9/sgRNA showing the force versus the number of unzipped base pairs. Insets: zoom-in view of the regions with increases in force. The dashed lines illustrate the interaction sites between dSaCas9 and the DNA. Black and blue arrows indicate the force peaks of the pre-PAM and post-PAM interactions, respectively.

D. Positions and strengths of the two interactions between dSaCas9 and DNA. The black and blue triangles indicate the pre- and post-PAM interactions, respectively. In total, 27 and 34 traces were collected in the forward and reverse unzipping assays, respectively. pre-PAM interactions (black) were detected in both the forward and reverse DNA unzipping assays. The error bars represent the SD.

E. Exonuclease III footprinting of dSaCas9 on the DNA target with Cy5 labeled on the 5' end of the nontarget strand.

F. Exonuclease III footprinting of dSaCas9 on the DNA target with Cy5 labeled on the 5' end of the target strand.
Dynamics of Staphylococcus Aureus Cas9 in DNA target Association and Dissociation (2020)
Figure 2

Figure 2. DNA-tracking motors were stalled by DNA-bound dSaCas9.

A. DNA unwinding by DnaB was initiated from the upstream side of the PAM in the presence of dSaCas9 (n = 10). Representative traces show the number of unwound base pairs versus the time under an assisting force of 12 pN in the absence (black) or presence (red) of the prebound dSaCas9. For clarity, the traces have been shifted along the time axis. The dashed lines indicate the expected dSaCas9 binding positions.

B. DNA unwinding by DnaB was initiated from the downstream side of the PAM in the presence of dSaCas9 (n = 11). Representative traces show the number of unwound base pairs versus the time under an assisting force of 12 pN in the absence (black) or presence (red) of the prebound dSaCas9.

C. DNA unwinding by BLM was initiated from the upstream side of the PAM in the presence of dSaCas9 (n = 12). Representative traces show the number of unwound base pairs versus the time under an assisting force of 12 pN in the absence (black) or presence (red) of the prebound dSaCas9.

D. DNA unwinding by BLM was initiated from the downstream side of the PAM in the presence of dSaCas9 (n = 14). Representative traces show the number of unwound base pairs versus the time under an assisting force of 12 pN in the absence (black) or presence (red) of the prebound dSaCas9.

E. Phi29 DNAP strand-displacement synthesis was initiated from the upstream side of the PAM (n = 15). Representative traces show the number of unwound/synthesized base pairs versus the time under an assisting force of 12 pN in the absence (black) or presence (red) of the prebound dSaCas9.

F. Phi29 DNAP strand-displacement synthesis was initiated from the downstream side of the PAM (n = 19). Representative traces show the number of unwound/synthesized base pairs versus the time under an assisting force of 12 pN in the absence (black) or presence (red) of the prebound dSaCas9.
Dynamics of Staphylococcus Aureus Cas9 in DNA target Association and Dissociation (2020)
Figure 4

Figure 4. Effects of mismatches on SaCas9 activity.

A. DNA binding fractions by dSaCas9 at the fully matched (target) and partially matched DNA target positions, as revealed by the reverse DNA unzipping assays (n = 15, 16, 22, 26 and 46 from left to right). The subscript "MM" represents the mismatches of the DNA sequence with the sgRNAs.

B. Positions and strengths of the pre-PAM interactions (n7-22MM = 21, n11-22MM = 41, n15-22MM = 39) and post-PAM interactions (n7-22MM = 21, n11-22MM = 25, n15-22MM = 45) between dSaCas9/sgRNA and mismatched DNA targets. The error bars represent the SD.

C. Schematic depiction of the stopped-flow experiment. 2-AP was substituted at position +18 of the nontarget strand. The 2-AP fluorescence signals as a function of time are shown for four sgRNAs. The solid lines are double-exponential fits of the data

D. A summary of fitted values of rates.

E. Cleavage activities of SaCas9 on DNA targets with sgRNA containing bases mismatched to DNA. The reactions were quenched at five time points (0, 15, 30, 45, and 60 min). The experiment was performed in triplicate, and a representative gel is shown.
Dynamics of Staphylococcus Aureus Cas9 in DNA target Association and Dissociation (2020)
Figure 5

Figure 5. The dissociation of SaCas9 after DNA cleavage.

A. Representative traces of the reverse DNA unzipping in the absence (gray) and presence (blue) of wild-type SaCas9 showing the force versus number of base pairs unzipped. In total, 23 traces were collected in this assay. The dashed line shows the expected interaction sites between dSaCas9 and DNA. The blue arrow indicates the force peak of the post-PAM interaction.

B. Representative traces of the forward DNA unzipping in the absence (gray) and presence (black) of wild-type SaCas9 showing the force versus number of base pairs unzipped. In total, 49 traces were collected in this assay. The dashed line shows the expected interaction sites between dSaCas9 and DNA.

C. Confocal images of fluorescently labeled λ DNA with dSaCas9 (n = 11) and wild-type SaCas9 (n = 16) bound to its DNA target. DNA-bound SaCas9 was visualized by labeling the 5' end of the crRNA with Cy3. The λ DNA molecule was either suspended between two microspheres held by two optical traps under 5 pN or stretched by laminar flow. The fluorescence intensity of Cy3 alongside the image is shown to indicate the on-target binding of SaCas9.

D. DNA unwinding by DnaB was initiated from the downstream side of the PAM after the association of the SaCas9 protein (n = 11). Representative traces show the number of unwound base pairs versus time under an assisting force of 12 pN. For clarity, the traces have been shifted along the time axis. The dotted lines indicate the expected SaCas9 binding positions.

E. Phi29 DNAP-mediated strand-displacement synthesis was initiated from the downstream side of the PAM after the association of the SaCas9 protein (n = 13). Representative traces show the number of unwound/synthesized base pairs versus time under an assisting force of 12 pN.

F. DNA unwinding by BLM was initiated from the downstream side of the PAM after the association of the SaCas9 protein (n = 23). Representative traces show the number of unwound base pairs versus time under an assisting force of 12 pN.
Autophagy proteins regulate innate immune responses by inhibiting the release of mitochondrial DNA mediated by the NALP3 inflammasome (2010)
figf1

(a) Immunoblot analysis of caspase-1 and IL-1β in lysates (Lys) and supernatants (Sup) of peritoneal macrophages obtained from Map1lc3b−/− mice and Becn1+/− mice and the corresponding wild-type littermate mice, then left unstimulated (−) or stimulated with LPS alone (LPS) or LPS followed by 30 min of ATP treatment (L + A). p10, mature form of caspase-1. Right margin, molecular size in kilodaltons (kDa). (b) Enzyme-linked immunosorbent assay (ELISA) of cytokine secretion by peritoneal macrophages stimulated with LPS, followed by treatment for 1 h with ATP. (c) Immunoblot analysis of caspase-1 and IL-1β in lysates (left) and ELISA of cytokines in supernatants (right) of Map1lc3b+/+ and Map1lc3b−/− BMDMs incubated with LPS, followed by 30 min (immunoblot) or 1 h (ELISA) of ATP treatment. (d) ELISA of IL-1β secretion by LPS-primed Map1lc3b−/− mice and Becn1+/− BMDMs incubated for 1 h with z-YVAD-fmk (z-YVAD; 10 μM) or vehicle (dimethylsulfoxide (DMSO)), followed by stimulation for 1 h with ATP. *P 0.05 (Student's t-test). Data are representative of three or four experiments (mean and s.d. in b–d).
Autophagy proteins regulate innate immune responses by inhibiting the release of mitochondrial DNA mediated by the NALP3 inflammasome (2010)
figf2

(a,b) Transmission electron microscopy of morphological changes in mitochondria (arrows) in Map1lc3b−/− BMDMs (a) or Becn1+/− BMDMs (b) left untreated or incubated for 6 h with LPS (10 ng/ml) and then stimulated for 30 min with ATP (1 mM). Outlined areas (middle) enlarged at right. Scale bars, 500 nm. (c) Flow cytometry of Map1lc3b−/− and Becn1+/− macrophages left unstained or labeled with MitoSOX. (d) Flow cytometry of macrophages left untreated or stimulated with LPS and ATP; cells were stained for 15 min with MitoTracker Deep Red and MitoTracker Green before ATP treatment. Data are representative of three experiments.
Autophagy proteins regulate innate immune responses by inhibiting the release of mitochondrial DNA mediated by the NALP3 inflammasome (2010)
figf3

(a) ELISA of cytokines in supernatants (left) and immunoblot analysis of caspase-1 and IL-1β in lysates (right) of J774A.1 macrophages exposed to EtBr (0, 10, 100 or 500 ng/ml; wedges) and left unstimulated or stimulated with LPS alone or with LPS and ATP. (b) Immunoblot analysis of caspase-1 and ASC in lysates of J774A.1 or ρ0 J774A.1 macrophages (treated with 100 ng/ml of EtBr) incubated with LPS, followed by stimulation for 0, 15 or 30 min with ATP. (c) Cytokine secretion by J774A.1 or ρ0 J774A.1 macrophages incubated with various combinations of LPS and ATP (below graph). (d) Cytokine secretion (right) and immunoblot analysis of caspase-1 and IL-1β in lysates (left) of LPS-primed macrophages incubated for 1 h with rotenone (+; 5 μM) or DMSO (−) before ATP treatment. (e) ELISA of IL-1β secretion by J774A.1 or ρ0 J774A.1 macrophages incubated with LPS and rotenone (or DMSO), followed by ATP. (f) Cytokine secretion by peritoneal macrophages preincubated for 1 h with Mito-TEMPO and then stimulated with various combinations of LPS and ATP. (g,h) ELISA of IL-1β secretion (left) and immunoblot analysis for caspase-1 in lysates (right) of Map1lc3b+/+ and Map1lc3b−/− (g) or Becn1+/+ and Becn1+/− (h) peritoneal macrophages incubated for 1 h with Mito-TEMPO (500 μM), followed by LPS and ATP. *P 0.05 (Student's t-test). Data represent three experiments (mean and s.d. in a,c,d–h).
Autophagy proteins regulate innate immune responses by inhibiting the release of mitochondrial DNA mediated by the NALP3 inflammasome (2010)
figf4

(a) ELISA of cytokine secretion by LPS-primed Map1lc3b+/+ and Map1lc3b−/− BMDMs incubated with cyclosporine A or DMSO, followed by stimulation for 1 h with ATP. (b) ELISA of cytokine secretion by LPS-primed Becn1+/+ or Becn1+/− BMDMs incubated with cyclosporine A (10 μM) or DMSO, followed by ATP stimulation. (c) Quantitative PCR analysis of cytosolic mtDNA in LPS-primed BMDMs incubated for 1 h with rotenone (5 μM) or vehicle (DMSO) before ATP stimulation. (d) Quantitative PCR analysis of cytosolic mtDNA in macrophages preincubated for 1 h with Mito-TEMPO (500 μM) and then incubated with LPS, followed by ATP stimulation. (e) Quantitative PCR analysis of cytosolic mtDNA in LPS-primed BMDM incubated with cyclosporine A (10 μM), plus either rotenone (5 μM) or DMSO, followed by ATP stimulation. (f) Quantitative PCR analysis of cytosolic mtDNA in LPS-primed Map1lc3b−/− and Becn1+/− BMDMs stimulated for 30 min with ATP. The amount of cytosolic mtDNA is presented relative to the amount in untreated control cells, set as 1. *P 0.05 (Student's t-test). Data are representative of three experiments (mean and s.d.).
Autophagy proteins regulate innate immune responses by inhibiting the release of mitochondrial DNA mediated by the NALP3 inflammasome (2010)
figf5

(a) ELISA of cytokines in supernatants of BMDMs incubated for 4 h after delivery of 3 μg DNase I, lactate dehydrogenase LDH (Control) or heat-inactivated DNase I (HI DNase I) and then stimulated with LPS, followed by stimulation for 1 h with ATP. (b) Quantitative PCR analysis of cytosolic mtDNA in BMDMs transfected as in a and then stimulated with LPS, followed by stimulation for 15 min with ATP. (c) ELISA of IL-1β secretion by Aim2+/+ and Aim2−/− BMDMs primed for 4 h with LPS (200 ng/ml), then transfected for 6 h with 1 μg mtDNA (through the use of liposomes as the vehicle), followed by stimulation for 1 h with ATP. *P 0.05, versus Aim2−/− cells treated with LPS and ATP (Student's t-test). (d) ELISA of cytokine secretion by LPS-primed Aim2−/− BMDMs transfected for 6 h with mtDNA, poly(dA:dT) or mtDNA predigested by DNase I (+ DNase; far left), followed by stimulation for 1 h with ATP. *P 0.05 (Student's t-test). Data are representative of three experiments (mean and s.d.).
Autophagy proteins regulate innate immune responses by inhibiting the release of mitochondrial DNA mediated by the NALP3 inflammasome (2010)
figf6

(a) Cytokine secretion by LPS-primed Map1lc3b+/+ and Map1lc3b−/− (a) or Becn1+/+ and Becn1+/− (b) peritoneal macrophages incubated for 15 min with glybenclamide (Glyb; 100 μM (a) or 50–100 μM (b)), Bay 11-7082 (Bay; 12 μM) or DMSO (–), followed by ATP. (c) Cytokine secretion by LPS-primed wild-type (WT) and NALP3-deficient (NALP3-KO) macrophages incubated with rotenone, followed by stimulation for 1 h with ATP. (d) Quantitative PCR analysis of cytosolic mtDNA in wild-type and NALP3-deficient BMDMs left unstimulated (–) or stimulated with LPS, followed by treatment for 15 or 30 min with ATP (left), and immunoblot analysis of cytosolic cytochrome c and activation of caspase-1 in the cells at left (right). (e) Quantitative PCR analysis of cytosolic mtDNA in LPS-primed wild-type, NALP3-deficient or ASC-deficient (ASC-KO) BMDMs stimulated for 15 min with ATP. *P 0.05, NALP3-deficient or ASC-deficient versus wild-type treated with LPS and ATP (Student's t-test). (f) Flow cytometry of untreated macrophages (control) or of LPS-primed macrophages left unstained or stained with MitoSOX, then stimulated for 15 min with ATP. (g) Flow cytometry of untreated macrophages (control) or of macrophages stimulated with LPS and ATP, stained with MitoTracker Deep Red. Numbers above bracketed lines indicate percent cells with loss of mitochondrial membrane potential. (h) Flow cytometry of LPS-primed wild-type, ASC-deficient and NALP3-deficient peritoneal macrophages stimulated for 30 min with ATP and stained with annexin V. Data represent three experiments (mean and s.d. in a–e).
Autophagy proteins regulate innate immune responses by inhibiting the release of mitochondrial DNA mediated by the NALP3 inflammasome (2010)
figf7

(a) ELISA of serum IL-1β and IL-18 at 24 h after intraperitoneal injection of LPS (12 mg per kg body weight) into male Map1lc3b+/+ and Map1lc3b−/− mice. (b) Survival of Map1lc3b+/+ mice (n = 11) and Map1lc3b−/− mice (n = 10) after LPS challenge (100 mg per kg body weight), assessed over a period of 2 weeks. (c,d) ELISA of serum IL-1β and IL-18 in male Map1lc3b+/+ and Map1lc3b−/− mice (c) and Becn1+/+ and Becn1+/− mice (d) at 24 h after CLP or sham laparotomy. In a,c,d, each symbol represents an individual mouse; small horizontal lines indicate the mean. P values, unpaired two-tailed Student's t-test (a,c,d) or log-rank test (b). Data are representative of two experiments.
Clipped histone H3 is integrated into nucleosomes of DNA replication genes in the human malaria parasite Plasmodium falciparum (2019)
Figure 1

Figure 1. P. falciparum nucleosomal histone H3 is proteolytically processed in a stage-specific manner between amino acid 21 and 22. A. Immunoblot analysis of nuclear and cytoplasmic extracts prepared from parasites synchronized at ring (R), trophozoite (T) and schizont (S) stages with anti-histone H3 C-terminus antibodies identifies three bands: full-length histone H3, an intermediate form (H3-int) and a fully processed form (H3p). Anti-histone H4 and anti-PfAldolase antibodies served as loading controls for the nuclear and cytoplasmic fraction, respectively. B. Immunoblot assays of mononucleosomes with anti-histone H3 C-terminus or anti-histone H4 antibodies. The right panel shows a mononoucleosome preparation separated on a denaturing polyacrylamide gel and stained with Coomassie brilliant blue (C.B.). C. Immunoblot analysis of a schizont-stage histone preparation with antibodies against the indicated post-translational modification (PTM) in the N-terminal tail of histone H3 (upper panel). D. Results from the Edman degradation analysis of full length PfH3 or processed PfH3p; the arrow indicates the position of histone H3 clipping.
Clipped histone H3 is integrated into nucleosomes of DNA replication genes in the human malaria parasite Plasmodium falciparum (2019)
Figure 2

Figure 2. A Cathepsin C-type protease in schizont-stage nuclear extracts may mediate P. falciparum histone H3 processing. A. Schematic representation of the in vitro proteolysis assay developed in this study. N-terminally GST-tagged PfH3 was incubated with schizont-stage nuclear extracts at 37°C for 3 h and analyzed by immunoblotting with anti-GST antibodies. GST-tagged PfH2A served as a control. B. GST-PfH3 or GST-PfH2A were incubated with increasing amounts of nuclear extracts, the reactions resolved using denaturing gel electrophoresis and analyzed by immunoblotting with anti-GST antibodies. C. GST-PfH3 was incubated with 25 μg of nuclear extracts and the indicated protease inhibitor. The resulting products were analyzed by denaturing gel electrophoresis and immunoblotting with anti-GST antibodies. (-) indicates a reaction without nuclear extract and protease inhibitor. D. Target profile of the protease inhibitors used in part C.
Clipped histone H3 is integrated into nucleosomes of DNA replication genes in the human malaria parasite Plasmodium falciparum (2019)
Figure 3

Figure 3: Ectopically expressed Histone H3p localizes to the nucleus during parasite asexual development and incorporates into nucleosomes. A. Indirect immunofluorescence assays were performed to determine the localization of ectopically expressed PfH3p-HA in ring (R), trophozoite (T) and schizont (S) stages of P. falciparum asexual growth. PfH3p-HA was detected using anti-HA antibodies (green) and endogenous histone H3 with anti-histone H3 N-terminal antibodies (red). DAPI (blue) was used to stain the nucleus. Scale bar = 5 μm. B. Nuclei isolated from wild-type (WT) or PfH3p-HA-expressing (WT + PfH3p-HA) schizont stage parasites were treated with 4 U/ml of micrococcal nuclease (MNase) for the indicated amounts of time, the DNA purified and migrated on a 2% agarose gel, and stained with ethidium bromide. Mononucleosomes purified after 10 minutes of MNase treatment were separated using denaturing polyacrylamide gel electrophoresis and either stained with Coomassie Brilliant blue (C.B.) or visualized by immunoblotting with anti-HA (α-HA) or anti-C-terminal histone H3 (α-H3c) antibodies. C. Co-immunoprecipitation (IP) experiments of purified mononucleosomes obtained from wild-type (WT) or transfected (WT + PfH3p-HA) schizont stage parasites were performed with either anti-HA antibodies or mouse IgG. Immunoprecipitated products (right panel) were analyzed by immunoblotting using anti-HA or anti-histone H4 antibodies.