EMBO/sd-character-level-ner huggingface dataset:
Figure 2H1299 cells were transfected with Flag-USP36 or the indicated deletion mutants and assayed by IB. FL, full length. Schematic diagram of USP36 indicating that the N-terminus of USP36, which contains the USP catalytic domain, is required for its SUMO E3 activity. Catalytic residues C131 and H382 are shown. H1299 cells transfected with WT USP36, the C131A, or H382A mutant were assayed by IB to detect SUMOylated proteins. H1299 cells transfected with WT USP36 or the indicated mutants and V5-SUMO2 were assayed by IB using anti-V5 antibody to detect total protein SUMOylation. H1299 cells transfected with WT USP36 or the indicated mutants and His-SUMO2 were subjected to Ni2+-NTA agarose beads pull down (PD) followed by IB using anti-SUMO2/3 antibody.
Figure 2A. Scheme of fate-mapping in Cx3cr1CreER:R26-yfpmice that were either treated at six weeks of age (for B) or at postnatal day 1 (for C) and analyzed at 6 days, 2, 4 and 35 weeks for B and 6 days, 6 and 35 weeks for C. B and C. Flow cytometry quantifications of YFP+ MΦ in the brain, liver and kidney at the indicated time points after TAM application in Cx3cr1CreER:R26-yfpmice. Data are summarized from two independent experiments (7-8 mice each) and represented as mean ± s.e.m. D. CCR2 expression on F4/80hi and CD11bhi MΦ in the liver or kidney of adult Ccr2RFP/WT mice, respectively. Grey area depicts fluorescence signal in MΦ of non-transgenic litter mates. Representative cytometric graphs are shown. Four mice were investigated showing similar results. E. Flow cytometry analysis of MΦ populations in the liver or kidney in adult mice lacking Ccr2 or Nr4a1. Each symbol represents one mouse. Data are summarized from three independent experiments and represent mean ± s.e.m. Significant differences were determined by an unpaired t test. n.s.: not significant. *P < 0.05, **P < 0.01.
Figure 4A. Control and OMA1 KO HCT116 cells were cultured in normoxia or in hypoxia (1% O2) for 24 or 48 hours. The whole cell lysates were analyzed by Western blotting with antibodies against HIF-1α, HK2, LDHA, PKM2, GPI, OMA1, or β-Actin. The relative protein levels were further evaluated by densitometry analysis using ImageJ software and were presented as bar graphs in the right panels. Error bars are presented as mean ± SD by a two-way ANOVA (n = 3 independent experiments). *p<0.05, **p < 0.01, ***p < 0.001. B. Control or OMA1 KO Hela cells were untreated (-) or CoCl2-treated (+) for 24 hours with 10 mM MG132 or vehicle added for the last 4 hours. Cell lysates were assessed by Western blotting with antibodies against HIF-1α or β-Actin. Relative protein levels were further evaluated by densitometry analysis. Error bars indicate the mean ± SD by a two-way ANOVA (n = 3 independent experiments). *p<0.05, **p < 0.01. C. Dihydroethidium (DHE) staining of optimum cutting temperature (OCT) covered tissues from the colons of WT and OMA1-/- mice; DAPI was used as counterstain. DHE was shown in red color, while DAPI in blue color (representative data from three independent experiments). D. Quantification of DHE staining. Error bars are presented as mean ± SD (n=3 biological replicates), statistical significance was assessed by a two-way ANOVA, **p < 0.01, ***P< 0.001. E. Control or OMA1 KO HCT116 cells cultured in normoxia or hypoxia (1% O2) for 12 hours were stained with mitoSOX, and then analyzed by confocal microscopy (representative data from three independent experiments). F. The fluorescence intensity of mitoSOX was further analyzed by ImageJ software. Bars indicate the mean ± SD of three independent experiments by a two-way ANOVA, ***p < 0.001. G-I. Control or OMA1_KO HCT116 cells were cultured in normoxia or hypoxia for 12 hours, and were treated with DMSO, NAC (10 mM) or Rotenone (1 μM) for 12 hours at the same time. The whole cell lysates were analyzed by Western blotting with antibodies against HIF-1α, and Tubulin (G). The glucose uptake (H) and lactate production (I) were measured using microplate reader, and the values were normalized to the protein concentration. Error bars indicate the mean ± SD (n = 3 independent experiments), statistical significance was assessed by two-way ANOVA. N.S., not significant, *P < 0.05, ***P< 0.001.
Figure 3A Lysis of SSAN by AMSIN. Vacomycin was used as a control. B Inhibition zone-based assay for synergy between AMSIN and HNP1&2 or lysozyme C (abbreviated as hLZc) to the two oral Streptococcus (SSAN and SSAL). Inset (upper panel, boxed), a representative example of the inhibition-zone test showing the synergy. Peptide doses used here were 0.05 nmol/well for HNP1&2 and hLZc at which no visible inhibition zones were observed for both of them. At the same dose, AMSIN produced a small inhibition zone. For synergetic observation, two peptides were applied to the same well according to the doses indicated. Mean ± SD of three biological replicates is displayed. P values were obtained by Student's t-test (***P<0.001 Note: the nanomolar concentration of HNP1&2 was calculated from the average molecular weight of HNP1 and HNP2. C The FIC index assay for synergy between AMSIN and HNP1&2 or hLZc to SSAL and SSAN. The dashed line represents the cutoff of the FIC index (0.5) for synergy.
mediated-transcriptional activity, cell cycle arrest and apoptosis.Figure 6A p21 promoter luciferase reporter plasmid was cotransfected with the indicated siRNAs into U2OS cells. Luciferase activity was determined as described in Experimental Procedure. B U2OS cells were transfected with siRNAs as indicated. Seventy-two hours later, cells were harvested after treated with actinomycin D for 12 hours. Relative mRNA levels for the indicated genes were analyzed by RT-qPCR. C-E U2OS cells (C and D) and H1299 cells (D) transfected with the indicated siRNAs were treated with actinomycin D and harvested at the indicated time points. Cell cycle profiles were determined by FACS. Results from three independent experiments are shown as mean ± SEM. (two-tailed Student's t test) F U2OS cells were transfected with the indicated siRNAs. Cells were irradiated with 50 J/m2 UV. Forty-eight hours later, apoptotic cells were determined. Quantification of apoptotic cells is presented. (two-tailed Student's t test) G U2OS cells transfected with the indicated siRNAs were plated. Cell numbers were determined as described in Experimental Procedures at the indicated time points. H Colony formation assay were performed with U2OS cells transfected with the indicated siRNAs. The right panel shows the quantification of formed colonies. **p<0.01, ***p<0.001 (two-tailed Student's t test)
Therapeutic effects of AAV-mediated gene delivery of Nurr1 + Foxa2 in the mouse PD model.Figure 5A Experimental scheme. As shown, PD model mice were generated by IP injection of MPTP, and AAVs expressing Nurr1 and Foxa2 (NF-AAV) or AAVs expressing empty vector (control, C-AAV) were stereotaxically injected 3 days prior to the initial MPTP treatment. B-E Cytoprotective and behavioral effects of NF expression were assessed in PD mice unilaterally injected with NF-AAVs on the right side of midbrains. C-AAVs were injected into the left midbrains of the same mice. TH+mDA neuron numbers in the midbrain (B) and TH+fiber intensities in the striatum (C) of the NF-AAV-injected right sides were compared with those of the corresponding left sides after 8 weeks. Insets of (B), high-powered images of the areas indicated by red arrowheads in the C-AAV (left) and NF-AAV-injected (right) sides. TH+cell numbers, *P = 0.000078, n = 7, paired Student's t-test. The sectioned striatal tissues were immunofluorescently stained for TH, and TH+fiber intensities were estimated from mean fluorescence intensities (MFI). n = 56 microscopic fields at a magnification of 200× each for the left and right striatum. *P = 0.000003, paired Student's t-test. Behavioral asymmetry in the mice unilaterally injected with NF-AAVs (right side) was assessed using the apomorphine-induced rotation test (D) after 4 and 8 weeks. The cylinder test (E) was carried out after 8 weeks to determine left forelimb movement. As a control, the behaviors were tested in PD mice bilaterally injected with C-AAV. *P = 0.028 (rotation test), 0.043 (cylinder test); one-way ANOVA followed by Bonferroni post hoc test. F-L Behaviors of the PD mice injected with NF-AAVs bilaterally at both sides of the midbrain were assessed using the pole test (J), beam test (K), and locomotor test (L). *P = 0.004 (pole), 0.044 (beam), and 0.045 (locomotor); one-way ANOVA followed by Bonferroni post hoc test. Shown in (F-I) is a representative TH-stained midbrain (F, H) and striatum (G, I) of mice bilaterally injected with C-AAVs (F, G) and NF-AAVs (H, I). M, N Sustained mDA neuroprotective effects. Shown in (M) is a confocal image of TH/HA-stained cells 6 months after NF-AAV injection (Foxa2 gene tagged with HA). Inset, z-stacked image of the boxed area along the y-axis (right) and x-axis (lower). TH+ cells in the right (NF-AAV) and left (C-AAV injected) midbrains were counted 6 and 12 months after AAV injection (N). *P = 0.000019 (6M), 0.000066 (12M), n = 6-7, paired Student's t-test. O, P NF-AAV-mediated mDA neuroprotective effects were further assessed with the experimental schedule (O), in which AAV injections were subjected after three consecutive MPTP injections. Shown in (P) is TH+cell counts of the left and right sides of the midbrains. *P = 0.000012 (D14), 0.000036 (1M), 0.000027 (2M), n = 6-7, paired Student's t-test.
Figure 2A. The structure of the sE-linker* trimer is displayed as ribbons colored by domains as in Figure 1. The previously determined structure of the TBEV sE401v trimer (PDB 1URZ, [15]) is superposed in light grey with fusion loops (FL) in orange, highlighting the disordered domain II tip in the new structure. The two subdomains of domain II, base and tip, are indicated. Disulfide bonds are shown as dark grey sticks. The residues of the stem are displayed as sticks color-coded according to atom type, with carbon atoms green. B. Close-up view of the sE-linker* trimer with one subunit colored as in (A) and the others in grey. The segment connecting helix αA to the η3 3/10 helical turn (see Appendix Fig S1) of the adjacent subunit is shown as sticks colored according to atom-type with carbon atoms light grey. Broken lines mark polar interactions of the stem with surrounding residues (with distances ranging between 2.7Å and 3.4Å). An open arrow marks the C-terminal end of the sE401v trimer structure reported previously (PDB 1URZ). C. For comparison, the same region of the stem is represented for the DENV-1 sE structure (PDB 4GSX, [19]), using the same color scheme.
Gata1 and 2 are dispensable for cardiac repression, but essential for the emergence of HS/PCs from hemogenic endotheliumFACS analysis of day 7 EBs with markers CD41 and c‐Kit shows efficient generation of HS/PCs from WT, but not SclKO or Gata12KO cells.Figure 6FACS analysis of day 7 EBs with markers CD41 and c‐Kit shows efficient generation of HSPCs from WT, but not SclKO or Gata1,2KO cells. Average of six independent experiments with SEM is shown. FACS analysis of day 5.25 EBs with markers CD41, Flk1, Tie2 and CD31 shows the generation of hemogenic endothelial cells from WT and Gata1,2KO cells, but not SclKO cells. Average of five independent biological experiments with SEM is shown. Assessment of the developmental potential of day 4.75 EB CD41−CD31+Tie2+endothelial cells on OP9 for 14 days shows the generation of CD45+ hematopoietic cells from WT cells but not Gata1,2KO or SclKO cells, and troponin T+cardiomyocytes from SclKO cells, but not WT or Gata1,2KO cells. Average of four independent experiments with SEM is shown. Heatmaps show gene expression differences in subsets of hematopoietic genes between Gata1,2KO and WT or SclKO day 4.75 CD41−CD31+Tie2+endothelial cells. Cardiac derepression is observed only in SclKO cells. *designates non‐functional transcripts.
Figure 3A. Schematic drawing of MDI protein and deletion mutants. These proteins were expressed under the nanos-gal4 driver, and tested for their ability rescue the hatching rate and mtDNA level of eggs from mdi1 females (progeny column) or for localization and recruitment of partner protein Larp to mitochondria in cultured cells (localization column). N= 3 x >100 eggs / genotype for hatching rate. The relative mtDNA level was the average of 3 biological repeats. B. Representative images of S2 cells expressing GFP fusions (green) of the MDI proteins diagramed in (A). Staining with MitoTracker Red (red) to label mitochondria shows an overlapping signal (yellow), indicating proper localization to mitochondria for all fusion proteins except MDIΔMTS. C. Submitochondrial localization of MDI. Mitochondria from cultured cells expressing an MDI-myc fusion protein were kept intact or subjected to swelling to disrupt their outer membranes, and digested or not with Protease K (PK). Protein extracts were subjected to western blot analysis. Tom20, cytochrome C (cyt.C) and SOD2 were used as markers of the outer membrane, inter-membrane space and matrix, respectively. MDI behaved like an outer-membrane protein. D. Western blots of MDI protein in wt and mdi knockout (mdi-ko) cells confirming the mdi-ko cells completely lacked MDI protein. Actin was used as loading control. E. Representative images of wt or mdi-ko cells expressing a Larp-GFP fusion protein and stained with MitoTracker Red to label mitochondria. The majority of Larp localized to mitochondria in wt cells (overlapping red and green signal), but diffused into the cytoplasm of mdi-ko cells. F. Representative images of mdi-ko cells co-expressing Larp-GFP with MDI or the deletion mutants diagramed in (A) fused to the mCherry. Of the 4 MDI deletions that localized to mitochondria, 2 (MDIΔR and MDIΔTudor) did not allow Larp-GFP to localize to mitochondria. The localization of Larp in mdi1 flies expressing MDI or MDI deletion mutants, and the hatching rate and mtDNA level in their eggs were determined. The result was summarized in (A).
Zinc modulates bacterial colonization and composition as a mechanism to protect against TNF.Figure 6(A-B) C57BL/6J mice received control or 25 mM ZnSO4 drinking water for 7 days and were injected i.p. with 50 µg TNF, solved in 200 µL sterile PBS, per 20 g bodyweight or with PBS only (N=5). 18h after TNF challenge, spleen and MLNs were isolated and lysates prepared and plated on TSA plates. Colonies were counted and expressed as bacterial colonies per ml of lysate. 40 colonies per plate were prepared for Maldi Tof identification and the relative amounts of bacterial species found on the plates are mentioned underneath the figure. Data are shown as means +/- SD. p-value was analyzed by means of a Student's t-test (unpaired, two-tailed). (C-D) C57BL/6J mice received control or 25 mM ZnSO4 drinking water for 7 days. Then semi-solid ileum slurry was isolated, weighed, mixed, diluted and normalized and was plated on TSA plates. After randomly picking 110 and 102 colonies from plates of H20 treated mice and ZnSO4 treated mice resp., their identity was determined by MALDI-TOF and expressed in a pie diagram. Chi2-tests were performed. (E) Impact of zinc concentration in bacterial TSA plates on the growth of colonies of 4 bacterial species commonly found in spleen and MLNs of mice injected with TNF (see A-B). Pure colonies of Staphylococcus nepalensis (orange), Staphylococcus sciuri (red), Eschericchia coli (purple) and Enterococcus faecalis (blue) were grown in LB medium, then diluted and plated on TSA plates containing increasing concentrations of ZnCL2. 24h later, the amounts of colonies were counted, and the amounts found in the 0 mM ZnCl2 condition expressed as 100%. Data are shown as mean +/- SD pooled from three independent experiments. Monocolonization experiment. Mice (N=5 all groups) were treated with H20 or ZnSO4 for 7 days, after which the ZnSO4 was removed and replaced by H20. 12h and 24h later, the mice were transplanted with PBS, 108 Staphylococcus sciuri or 108 S. nepalensis (in 100 ul), and 3h later a lethal dose of TNF (35 µg/mouse) was injected, and 24h later body temperature (insert in F) and lethality (F) recorded and estimated by one-way ANOVA and Chi2-tests resp. Survival significance was tested towards the H20 control group. Monocolonization experiment. Mice (N=5 all groups) were treated with H20 or ZnSO4 for 7 days, after which the ZnSO4 was removed and replaced by H20. 12h and 24h later, the mice were transplanted with PBS, 108 Staphylococcus sciuri (in 100 ul), and 3h later a lethal dose of TNF (35 µg/mouse) was injected In a second experiment (G-I) (N=4 all groups) with S. sciuri colonization, Zbp1 and Stat1, two key ISRE genes, and Mt2, a Zn-induced gene, were measured in ileum biopsies by RT-qPCR.
Figure 7Anti-Krt5 (red) and anti-P63 (green) immunofluorescence of isolated hDASCs. Scale bar, 90 μm. The mRNA expression levels of LL-37 was measured by qPCR for WT- and LL-37-hDASCs. n=5. Error bars, S.E.M. H&E staining of native rat lung tissue and decellularized scaffold showed the absence of nuclei following decellularization. Scale bar, 40 μm. Ex vivo biomimetic culture of LL-37-hDASCs recellularized lungs with constant media perfusion. Direct fluorescence image showing GFP-labeled LL-37-hDASCs before (left) and 7 days after (right) being engrafted onto the decellularized scaffold. Scale bar, 100 μm (right panel) and 1000 μm (right panel) Immunofluorescence of recellularized lung with indicated antibodies. Blue color indicates nucleus DAPI staining. Scale bar, 50 μm. The recellularized lung by LL-37-hDASCs displayed growth inhibitory effect on PAO1 and E.coli. Initial dose of bacteria was 2× 104 CFU. Culture duration, 24h. n=6. Error bars, S.E.M. *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001.
Figure 4A Two-colored IFA of ISP1/DHHC2 and ISP3/DHHC2 at zygotes of the triple tagged strain isp1::3V5/isp3::6HA/dhhc2::4Myc (TTS). Right graph shows overlay of fluorescence peaks along cell periphery. Scale bar = 5 μm. B Western blot of DHHC2, ISP1, and P28 (plasma membrane protein) of the TTS zygotes treated with PBS, trypsin (Try), or heat-inactivated (HI) trypsin. Upper panel shows the PPM and IMC structures. C Knockdown of dhhc2 expression. Upper panel shows the promoter swap strategy applied in TTS strain, generating the dhhc2kd mutant with endogenous dhhc2 promoter replaced with clag promoter. Western blot confirming decreased expression of DHHC2 in dhhc2kd zygotes. Three replicates performed. D Two-colored IFA of DHHC2 and P28 proteins in the TTS and dhhc2kd zygotes. Scale bar = 5 μm. Bottom panel, quantifications of DHHC2 fluorescence. Values are means ± SD (n: the number of cells analyzed). Mann-Whitney test, ****, P<0.0001. E Western blot of DHHC2, ISP1 and ISP3 in the TTS and dhhc2kd zygotes. Two replicates performed. F Palmitoylation of ISP1 and ISP3 in the TTS and dhhc2kd zygotes using Acyl-RAC method. NH: NH2OH. Right panel indicates quantification of protein band intensity from three replicates, values are means ± SD. G IFA of ISP1 and ISP3 in zygotes of TTS and dhhc2kd. Scale bar = 5 μm.  H Giemsa staining of zygote/ookinete from in vitro cultured TTS and dhhc2kd parasites. Scale bar = 5 μm. I Time-course analysis of in vitro ookinete differentiation. Values are means ± SD from three independent tests. J IFA of DHHC2 expression in the dhhc2::6HA and dhhc2::6HA;∆isp1/3 zygotes. Scale bar = 5 μm. K Western blot of DHHC2 in zygotes of dhhc2::6HA and dhhc2::6HA;∆isp1/3. L IFA of DHHC2 and ISP1 in zygotes of dhhc2kd parasites complemented with 4Myc tagged WT PyDHHC2 of P. yoelii, PfDHHC2 of P. falciparum, or PyDHHC2 with PAT catalytic deficient mutation C128A. Scale bar = 5 μm. M Western blot of DHHC2 in the complemented dhhc2kd parasites as indicated. N In vitro differentiation to mature ookinetes of the complemented parasites. Values are means ±SD from three independent tests. two-tailed t test, ns: not significant. O Model of DHHC2-mediated palmitoylation of ISP1 and ISP3 in zygote.
LifeAct structure, F-actin interaction and mutations.Figure 2A. Surface representation of F-actin bound to lifeAct as indicated. B & C. Cartoon representation of lifeAct (red) in closer look with n+1 and n+2nd actin monomers (surface representation in magenta and blue), key interacting residues are highlighted. C. Confocal images of U2OS cells transiently expressing lifeAct-GFP wild type and mutants of lifeAct residues interacting with F-actin, cells were additionally stained with SiR-actin to confirm the actin filaments. The line scan as indicated with yellow line on the cells shows the extent of lifeAct (green) and SiR-actin (magenta) co-staining of actin structures. Scale bar = 5µm. D. Binding affinities calculated from titration data of co-sedimentation assays of lifeAct-14 (1.2µM) and lifeAct-17 (2.2µM) as indicated. Data points for each concentration were averaged from 3 independent experiments, error bars represent S.D between independent experiments.
Figure 10COPI relocates from Golgi to LD surface or relocates to the surface of ER tubules via retrograde transport. The interaction between COPI and TRAPPII allows the GEF activity of TRAPPII to activate Rab18. Rab18-GTP becomes LD associated and promotes the formation of ER-LD bridge so that lipid metabolizing enzymes can access the content of LD.
a) Top, FAK−/− cells were sparsely plated and then treated with 3-MA (for 48 h), chloroquine or dasatinib (both for 24 h).figf6(a) Top, FAK−/− cells were sparsely plated and then treated with 3-MA (for 48 h), chloroquine or dasatinib (both for 24 h). Cells were then left at 37 °C for 1 week to allow colony formation, fixed and then stained with crystal violet. Bottom, the number of colonies formed was then quantified and normalized against untreated controls. Data are presented as mean±s.d. and significance is P0.001 for 3-MA, P0.01 for chloroquine and P>0.5 for dasatinib (n=3). (b) Top, wild-type FAK and FAK−/− cells were transiently transfected with two individual Atg5 shRNAs (1 and 2), selected in puromycin and then the clonogenic assay was carried out as described above. Bottom, quantification; data are presented as mean±s.d. and significance is P>0.1 for Atg5 A and B in wild-type FAK cells and P0.01 for Atg5 A and P0.05 for Atg5 B in FAK−/− cells (n=3). (c) Cells were also stained with TUNEL. The positive control was DNase1-treated cells and the negative control lacked TUNEL reaction mix. Quantification is shown and data are presented as mean±s.d. and significance is P0.005 for 3-MA and P0.001 for Atg5 siRNA (n=3). (d) FAK−/− cells were untreated or treated with dasatinib, 3-MA, chloroquine or bafilomycin A (BFA) for 24 h, or were transfected with scrambled or Atg5 siRNA for 72 h and then immunoblotted with an anti-PARP antibody. Uncropped images of blots are shown in Supplementary Fig. S9.
Fig.Figure 6A. Direct contacts to the supplementary miRNA-target duplex shown. Dashed lines indicate potential salt-linkages. B. Protein shown as semi-transparent surface representation with miRNA and target colored by temperature factor. Scale (Å2) shown at bottom.