EMBO/soda-nel huggingface dataset:
ANGPTL4 exacerbates pancreatitis by augmenting acinar cell injury through upregulation of C5a. (2020)
Figure 7

Figure 7. Therapeutic effect of the ANGPTL4-neutralizing antibody in AP and SAP.

A Concentrations of C5a in the sera from pancreatitis patients, and correlation between ANGPTL4 and C5a in the sera from pancreatitis patients (n = 90). The box plots show a typical display consisting of a median value depicted by the line in the center of the box; an interquartile range (IQR; 25th to the 75th percentile) depicted by the box; and the maximum (Q3+1.5*IQR) and minimum (Q1-1.5*IQR) values depicted by the whisker. Statistical significance of Mann-Whitney U-tests is indicated (*** P < 0.001). Exact P values are shown in Appendix Table S2. The correlation between C5a and ANGPTL4 is depicted with Pearson correlation coefficient (R). Each dot represents the levels of C5a and ANGPTL4 from an individual patient (n = 90). The immunofluorescent expression of C5a and amylase was observed in pancreatitis and adjacent normal regions in patient tissues. Scale bar represents 50 µm

B Immunofluorescence staining of C5a in pancreas tissues from ANGPTL4 -/- and WT animals and macrophage depletion/WT models with pancreatitis. Scale bar represents 150 µm. Levels of C5a were measured in serum (n = 7).

C After macrophages were activated with LPS (100 ng/ml), the cells were treated with either neutralizing ANGPTL4 or C5a antibody (100 ng/ml) or an isotype control (IgG). ANGPTL4, C5a, TNF-α, and IL-6 levels were measured in culture media (n = 5).

D The neutralizing ANGPTL4 antibody (10 mg/kg) was injected twice (IP) into the AP and SAP models. H&E staining of pancreatic tissues and the concentrations of ANGPTL4, C5a, TNF-α, and amylase in the serum (n = 10, each group). Scale bar represents 50 µm

E Summary for how ANGPTL4 induces the severity of pancreatitis in acinar cells and macrophages.

Data information: Each value in B-D represents the mean ± SEM. (** P < 0.01 and *** P < 0.001). Values of P were calculated using one-way ANOVA with Tukey's post-hoc analysis. Exact P values are shown in Appendix Table S2.
Integrative characterization of the near-minimal bacterium Mesoplasma florum (2020)
Figure 5

Figure 5. Expression levels of M. florum protein-coding genes and enrichment of functional categories.

A) Transcription levels of all M. florum coding sequences (CDS) quantified by RNA-seq. Transcription levels were calculated according to the number of fragments per kilobase per million of mapped reads (FPKM) observed over six replicates. The corresponding numbers of mRNA copies per cell, estimated from the measured M. florum RNA mass, are also indicated. CDS were sorted from least to most transcribed. The transcription level of selected genes of importance are presented. LDH, L-lactate dehydrogenase (peg.600/mfl596); G3PDH, glyceraldehyde-3-phosphate dehydrogenase (peg.583/mfl578); PGK, phosphoglycerate kinase (peg.582/mfl577); L24p and L7/L12, large subunit ribosomal proteins L24p (peg.133/mfl134) and L7/L12 (peg.605/mfl601); S7p, small subunit ribosomal protein S7p (peg.626/mfl623); RNAPβ, RNAPβ', and RNAPα, RNA polymerase subunits β, β', and α (peg.601/mfl597, peg.602/mfl598, and peg.149/mfl150); HPr, phosphotransferase system phosphocarrier protein HPr (peg.570/mfl565); σ70, RNA polymerase sigma factor RpoD (peg.269/mfl270).

B) Transcription level of CDS included in transcription units (TUs) compared to CDS not attributed to any TU (orphan CDS). The median and interquartile range are shown for both groups. The mean rank of each group was compared using a Mann-Whitney test (two-sided, ****p-value <0.0001).

C) Expression levels of all M. florum reference proteins quantified by two-dimensional liquid chromatography-tandem mass spectrometry (2D LC-MS/MS). Abundance was estimated according to the normalized spectral abundance factor (NSAF) calculated for each protein. A NSAF value of 0 was assigned to undetected proteins. The corresponding number of protein molecules per cell (derived from the biomass data) are indicated. Proteins were sorted from least to most abundant. The selected genes of importance presented in panel A are also highlighted.

D) Overall DNA, tRNA, rRNA, mRNA, and protein proportions in terms of intracellular abundances in M. florum.

E) Voronoi diagram illustrating the relative abundance of M. florum reference proteins grouped into different functional categories. Each polygon represents a specific protein weighted by its expression level quantified by 2D LC-MS/MS. Functions were attributed based on the KEGG Orthology (KO) database (Kanehisa et al, 2016a). The unmapped category regroups proteins for which no KO identifier could be assigned, while the unclear category contains proteins with KO numbers matching to unclear functions.

F) As panel E but for mRNA abundances quantified by RNA-seq.
Cancer-specific mutations in p53 induce the translation of Δ160p53 promoting tumorigenesis (2016)
Figure 1

Figure 1. Mutant p53 expresses p53 isoforms.

(A-B) Western-Blotting (WB) of p53-negative lung cancer H1299 cells stably expressing (A) or transiently expressing (B) the indicated constructs. Post-translationally modified Δ133p53 and Δ160p53 isoforms are indicated with *. (C) The same WB membrane containing lysates from R273Hp53-expressing A431 cells was incubated with rabbit polyclonal CM-1 antibody and mouse monoclonal 1801 antibody against the N-terminus of p53. Detection using anti-rabbit IRDye 680LT (red) and anti-mouse IRDye 800CW (green) secondary antibodies confirmed that the Δ160p53 band corresponds to a C-terminal isoform of p53. (D) WB of H1299 cells transiently expressing the indicated constructs. (E) WB of cell lines expressing wild-type (HCT116, U2OS, A549) or mutant (SW480 [R273H/P309S], MDAMB231 [R280K], A431 [R273H], HT29 [R273H] and SKBR3 [R175H]) p53. (F) Cell lines harbouring endogenous wild-type (A549) or R273H mutant (A431) p53 were treated or not with control siRNA (ctl) or siRNA targeting both p53 transcripts (ex6 and ex7), full-length mRNA only (ex2/3) or Δ133p53 mRNA only (in4) before lysis and WB. (G) WB of H1299 cells stably expressing R273Hp53 and treated with DMSO, proteasome inhibitor MG132 or mRNA translation inhibitor cycloheximide (CHX). Shown are representative data of three independent experiments. The numbers in parenthesis indicate the amounts of protein for the indicated bands according to WB quantifications and normalization against α-tubulin or GAPDH and relative to the value indicated in bold and set to 1.0. In WBs for endogenous p53 cell lines the Δ160 lane is used as a marker lane showing Δ160p53 as transiently expressed in p53-null H1299 cells.
Cancer-specific mutations in p53 induce the translation of Δ160p53 promoting tumorigenesis (2016)
Figure 2

Figure 2. Cell survival capacity of mutant R273Hp53 requires Δ160p53 isoform expression.

(A) Cell lines endogenously expressing mutant R273Hp53 (A431 and HT29) were treated with control siRNA (ctl) or siRNA targeting exon 7 of p53, submitted to endoplasmic reticulum (ER) stress by exposure to thapsigargin (Th) and then analysed for apoptosis by incubation with propidium-iodide (PI) and FACS analysis. Data was normalized against si ctl condition, which was set to 100. (B and C) A549 (B) and H1299 (C) lung cancer cell lines stably expressing the indicated constructs were counted with Trypan blue for several days following stress stimuli (over-confluency (O-C) for A549 and thapsigargin (Th) for H1299). Data was normalized against 1st day values. Δ160p53 shows similar pro-proliferative capacities as mutant R273Hp53. On the other hand R273Hp53 lost pro-proliferative functions when deficient for Δ160p53 expression (M160AR273Hp53). Also shown is the WB of A549 cells used in (B) and stably expressing the indicated constructs. (D to G) H1299 cells transfected with the indicated constructs were submitted to ER stress (E and G), DNA-damage by 36h 3 μM etoposide (Eto) treatment (F) or cultured under regular conditions (D). Data was normalized against empty vector (E) or p53 (D) condition, which were set to 100. Cells were then analysed for apoptosis as in (A). Shown are representative data or averages + s.d. of three independent experiments (*P < 0.05, **P < 0.01, ***P < 0.005 and ^P > 0.05 compared to control (“Th + si ctl” or “-” ) or as indicated).
Cancer-specific mutations in p53 induce the translation of Δ160p53 promoting tumorigenesis (2016)
Figure 3

Figure 3. Both mutant p53 and Δ160p53 confer increased adhesion and invasion, disrupt three-dimensional mammary architecture and induce invasive structures.

(A) Adhesion assay shows that MCF10A cells stably expressing Δ133p53, Δ160p53, R273HΔ160p53 or missense mutant R273Hp53 adhere more strongly than control cells. Excluding Δ160p53 expression from the mutant background (M160A/R273Hp53) rescued control-cell phenotype. Scale bar represents 200 μm. (B) WB of MCF10A cells used in (A) and stably expressing the indicated constructs. Shown are representative data of three independent experiments and quantifications. (C and D) MCF10A human breast epithelial cells stably expressing the indicated constructs were cultured in matrigel. Control- and Δ133p53-expressing cells formed regular three-dimensional (3D) mammary acinar structures with hollow lumina but Δ160p53-, R273HΔ160p53 and to a smaller extent mutant R273Hp53-expressing cells formed acini with filled lumen and long invasive structures (n > 30 acini per experiment per condition). (E) H1299 cells stably expressing empty vector (control) or the indicated constructs were assessed by confocal micrographs for invasion further than 30 μm through a Matrigel-fibronectin matrix towards EGF-supplemented growth media. The 30 μm plane is indicated and scale bars represent 100 μm. Quantifications are shown in the right panel. Shown are representative data and/or averages + s.d. of three independent experiments (*P < 0.05 and ***P < 0.005 compared to control or as indicated). Scale bars represent 20 μm (C) and 100 μm (D).
Cancer-specific mutations in p53 induce the translation of Δ160p53 promoting tumorigenesis (2016)
Figure 4

Figure 4. Endogenous R273H-associated pro-oncogenic “gain-of-functions” require Δ160p53 isoform expression.

(A) WB of H1299 cells stably expressing Δ133p53 and treated with control morpholino oligos (Ctl-1 or Ctl-2) or antisense morpholino oligo targeting Δ160p53’s translation initiation (MO) (B) A431 and HT29 cells expressing endogenous mutant R273Hp53 were treated with control morpholinos (Ctl-2 or Ctl-1) or antisense morpholino oligo targeting Δ160p53’s translation initiation site (MO) before lysis and WB. wtp53-expressing A549 cells were used as reference for endogenous wtp53 expression levels. Δ160 lane was used as a marker lane showing Δ160p53 as transiently expressed in p53-null H1299 cells. (C) Shown are the average Δ160p53 protein levels expressed in R273Hp53 versus other mutant p53 cell lines that were treated with control morpholino (Ctl-1) or morpholino targeting 160p53’s translation initiation site (MO). MO efficiently targets 160p53 in endogenous R273Hp53 cell lines. See also figure EV3 for the raw data. (D-E) A431 (D) and HT29 (E) cells endogenously expressing mutant R273Hp53 were treated with control siRNA (si Ctl) or control MO (MO Ctl-2 or MO Ctl-1) or siRNA targeting exon 7 of p53 (si) or MO targeting Δ160p53’s translation initiation site (MO), as indicated, submitted to endoplasmic reticulum stress by exposure to thapsigargin (Th) and then analysed for apoptosis by incubation with propidium-iodide (PI) and FACS analysis. wtp53-expressing A549 cells were similarly control-treated and used as comparison. Data was normalized against A549 cell condition, which was set to 100. (F) A431 and HT29 cells endogenously expressing mutant R273Hp53 were treated with control morpholino Ctl-1 or morpholino targeting Δ160p53’s translation initiation site (MO) and then assessed by confocal micrographs for invasion further than 30 μm through a Matrigel-fibronectin matrix towards EGF-supplemented growth media. The 30 μm plane is indicated and scale bars represent 100 μm. Quantifications are shown in the right panel. (G) Model illustrating how mutations in p53 induce loss of function but also gain of new functions via isoform expression. The semi-transparent boxes represent plausible minimal full-length p53 and short p53 isoforms activities. The numbers in parenthesis indicate the amounts of protein for the indicated bands according to WB quantifications and normalization against α-tubulin or GAPDH and relative to the value indicated in bold and set to 1.0. Shown are representative data or averages + s.d. of three independent experiments (*P < 0.05, **P < 0.01 and ***P < 0.005 compared to A549 (D, E), control (C, F) or as indicated).
Distinct Regulation of Bioenergetics and Translation by Gp I mGluR and NMDAR (2020)
Figure 5

Figure 5:

NMDAR mediated AMPK activation is extracellular Ca2+-dependent.

(A) Representative immunoblots describing changes in the phospho-AMPK and total-AMPK levels at various time points after NMDA treatment in cortical synaptoneurosomes in the presence or absence of extracellular Ca2+. Note in each case phospho and total AMPK levels were normalized individually to their respective tubulin levels before calculating the phospho/total AMPK ratio.

(B) Line graph showing the normalized average value for the synaptic phospho/total ratio of AMPK at various time points after NMDA treatment until 5 minutes of recovery in the presence or absence of extracellular Ca2+. Data presented as mean +/- SEM. Values in all other groups were normalized to the 0 min group for the corresponding experiment and all the data points are represented as a fraction of the initial time point. **p<0.01, ****p<0.0001, n= 6 animals per group. Two-way ANOVA followed by Bonferroni's multiple comparison test.

(C) Representative images depicting intracellular Ca2+ levels through Fluo8 fluorescence (Pseudo-colored) of a cortical neuron plated at low-density before stimulation, 15 sec (immediate) after NMDA treatment, 300 sec (delayed) after NMDA treatment and after ionomycin treatment in the presence of 10mM Ca2+. Scale bar 20μm.

(D) Representative time trace showing the normalized change in Fluo8 fluorescence on NMDA treatment in the presence or absence of extracellular Ca2+ and D-AP5. Values in all other groups were normalized to the unsimulated condition for the corresponding experiment and all the data points are represented as a fraction of the initial time point. Ionomycin treatment in the presence of 10mM Ca2+ was used to calculate fluorescence maximum for a particular cell.

(E) Box plot showing the distribution of ΔF/F0 across multiple neurons on the basal condition, on NMDA treatment in the presence Ca2+, on NMDA treatment in the absence of Ca2+, on NMDA treatment with D-AP5 and on NMDA treatment along with MK-801. The box extends from 25th to 75th percentile with the middlemost line representing the median of the dataset. Whiskers range from minimum to maximum data point. ****p<0.0001, n≥30 neurons per group from 3 independent platings. One-way ANOVA followed by Bonferroni's multiple comparison test.
In vivo generation of a mature and functional artificial skeletal muscle. (2015)
Figure 2

Implanted constructs of Mabs in PF on the surface of TA

A Gross morphology of the PF implant containing Mabs-nLacZ/PlGF immediately after implantation on the surface of underlying TA (arrow).

B, C Artificial muscles (arrows) developed within 8 weeks over the host TA surface (arrowheads), encapsulated by the host perimysium (B) and released after tendon resection of the TA (C).

D The isolated artificial muscle from the TA surface (arrow) is comparable in size to the underlying host TA (arrowhead).

E Stereomicroscopy images revealing blood vessel formation in the artificial muscle. Scale bar: 100 μm.

F X-Gal and H&E staining on artificial muscle section revealing vessel connection with host vasculature by Indian ink, injected into the femoral artery immediately before sacrifice. Scale bar: 20 μm.

G Western blot analysis of protein extracts from artificial muscles (PF) and host TA (Ctrl) reveals remarkable expression of muscle- and vessel-specific proteins (n = 5).

H Densitometric analysis from n = 3 different Western blots gauging the level of expression of muscle- and vessel-specific proteins; presented values are expressed as means ± standard error.

I–L X-Gal and H&E staining of artificial muscle sections at 4 (I, K) and 8 weeks (J, L) after implantation, showing donor origin of muscle cells (nLacZ+ nuclei) and correlation between implant duration and myofibre maturation (I, J images are the results of the collage of several photos). Scale bar: (I, J) 200 μm, (K, L) 100 μm.

M–P Immunofluorescence analysis on adjacent sections revealing laminin organization (green) and myosin heavy chain (MyHC) expression (red) at 4 weeks (M) and 8 weeks (N) after grafting (M, N displayed items are obtained from a multi-photo collage); DAPI staining (blue) shows centrally located nuclei in developing fibres (O) and peripheral nuclei in mature myofibres (P). Scale bar: (M, N) 200 μm, (O, P) 50 μm.

Q Confocal isosurface image of neurofilament (green) and bungarotoxin (red) immunofluorescence in the artificial muscle section shows a mature neuromuscular plaque. Scale bar: 10 μm.

R Immunofluorescence against smooth muscle actin (blue), laminin (green) and VE-Cad (red) superimposed on phase contrast image shows blood vessels (arrows) adjacent to the myofibres in the artificial muscle section. Scale bar: 20 μm.

S X-Gal staining on mature artificial muscle (arrowhead) and the underneath host TA (arrow) revealing no tendon development at the extremity of the artificial tissue; implanted LacZ-positive Mabs are present within the host TA. Scale bar: 200 μm.

Source data are available online for this figure.
In vivo generation of a mature and functional artificial skeletal muscle. (2015)
Figure 3

Structural and functional analysis of artificial muscle and underlying TA control muscle

A Electron microscopy with Bluo-Gal staining reveals a LacZ-positive donor nucleus (arrow) peripheral to transversely sectioned sarcomeres of the artificial muscle. Scale bar: 1 μm.

B Longitudinal EM section of an artificial muscle showing mature sarcomere myofibril organization. Scale bar: 0.5 μm.

C A single fibre from an artificial muscle showing a striation pattern typical of a mature myofibre. Scale bar: 10 μm.

D X-Gal staining of freshly dissected artificial muscle bundle at 8 weeks showing abundant LacZ-positive donor nuclei. Scale bar: 100 μm.

E Immunofluorescence analysis of LacZ-positive nuclei (green) and dystrophin (red) in an individual muscle fibre after functional analysis. Scale bar: 10 μm.

F The same fibre shown in (E) is counterstained with DAPI to show that the interstitial nuclei (blue) are of donor origin. Scale bar: 10 μm.

G–I Specific force (Po/CSA) (G), maximum shortening velocity (Vo) (H), and cross-sectional area (CSA) (I) of single fibres (n = 153) from artificial muscles and control (CTRL) muscles (n = 103).

J Silver-stained SDS–PAGE (8%) gel revealing myosin heavy chain (MyHC)-2B isoform expression in two single fibres of artificial muscles (lanes 1, 2) and a bulk muscle control sample from soleus muscle containing all four adult MyHC isoforms indicated in (K) (lane 3).

K Comassie blue-stained SDS–PAGE (8%) of bulk samples of two artificial muscles (lanes 1, 2) and of a bulk control muscle sample (lane 3), as in (J), showing different MyHC isoform expression.

Data information: In (G–I), values are expressed as means ± standard error, significance was tested using Student's t-test, and P > 0.05 was considered not significant (ns).
m6A-modification of HSATIII lncRNAs regulates temperature-dependent splicing (2021)
Figure 1

Figure 1. Localization of m6A factors within nSBs

A m6A-related proteins identified by HSATIIII-ChIRP-MS (cf. Table EV3 in Ninomiya et al., 2020).

B, C HSATIII lncRNA-dependent localization of m6A-related factors. Control and HSATIII knockdown HeLa cells were exposed to thermal stress (42°C for 2 h and recovery for 1 h at 37°C) and stained by HSATIII-FISH and immunofluorescence using an anti-WTAP antibody (B) or anti-YTHDC1 antibody (C). The nuclei were stained with DAPI. Scale bar: 10 µm.

D Enrichment of m6A RNAs in nSBs. Thermal stress-exposed HeLa cells were stained by HSATIII-FISH and immunofluorescence using an anti-m6A antibody. The nuclei were stained with DAPI. Scale bar: 10 µm.

E, F, G 3-DZA inhibits localization of m6A RNAs (E), YTHDC1 (F) and WTAP (G) in nSBs. HeLa cells were exposed to thermal stress in the presence of 3-DZA (100 µM) and stained as described above. Scale bar: 10 µm.

H, I, J Box plot of relative intensities of m6A (H) , YTHDC1 (I) and WTAP (J) to HSATIII within nSBs in individual nuclei. nSB areas were defined by binarized images of HSATIII-FISH. The mean is indicated with X. The first and third quartiles are the ends of the box, the median is indicated with a vertical line in the box, and the minimum and maximum except for the outliers are the ends of the whiskers. The outliers are indicated with open circles. P-value (Mann-Whitney U-test) is shown above (n=30).

K YTHDC1 accumulation within nSBs was arrested by METTL3 knockdown. HeLa cells were stained as described in Figure 1C. Scale bar: 10 µm.

L Box plot of relative intensities of YTHDC1 to HSATIII within nSBs in individual nuclei (n=30). The mean is indicated with X. The first and third quartiles are the ends of the box, the median is indicated with a vertical line in the box, and the minimum and maximum except for the outliers are the ends of the whiskers. The outliers are indicated with open circles. P-values (Kruskal-Wallis test, followed by Dunn's multiple comparison test) are shown above.
m6A-modification of HSATIII lncRNAs regulates temperature-dependent splicing (2021)
Figure 3

Figure 3. Localization of the m6A-related factors in nSBs during stress recovery

A Schematic of HSATIII-ChIRP-MS at various time points during and after thermal stress exposure.

B Bar graph of relative ChIRP-MS enrichment score. ChIRP-MS enrichment score for each protein was calculated as (the average number of identified peptides at each time point + 0.00001) / (the average number of identified peptides at 37°C + 0.00001) and normalized to the maximum value of each protein.

C Western blot-validation of HSATIII-ChIRP. nSB proteins (YTHDC1, WTAP and SRSF9) were detected by western blotting using specific antibodies (input 1%). HSATIII lncRNAs were detected by semi-quantitative RT-PCR (input 100%). In; input, PD; pulldown by ChIRP.

D-G (D, F) Changes of nSB localization of m6A-related factors at various time points during and after thermal stress exposure. HeLa cells were exposed to thermal stress (42°C for 2 h) and cultured at 37°C for the indicated periods. m6A-related factors were visualized by HSATIII-FISH and IF using anti-WTAP antibody (D) or anti-YTHDC1 antibody (F). Nuclei were stained with DAPI. Scale bar: 10 µm. (E, G) Box plot of relative intensities of WTAP (D) and YTHDC1 (F) to HSATIII within nSB areas in individual nuclei. nSB areas were defined by binarized images of HSATIII-FISH. The mean is indicated with X. The first and third quartiles are the ends of the box, the median is indicated with a vertical line in the box, and the minimum and maximum except for the outliers are the ends of the whiskers. The outliers are indicated with open circles. P-values (Kruskal-Wallis test, followed by Dunn's multiple comparison test) are shown above (n=30).