EMBO/soda-nel huggingface dataset:
Degradation of WTAP Blocks Antiviral Responses by Reducing the m6A Levels of IRF3 and IFNAR1 mRNA (2021)
Figure 6

Figure 6. The protein abundances of IRF3 and IFNAR1 are decreased in virus-infected or nucleic acid analog-treated cells.

A Immunoblot analyses to detect IRF3 and IFNAR1 protein expression in A549 cells stimulated with poly (I:C) or poly (dA:dT) at the indicated time points. Data are representative of 3 independent biological experiments.

B Immunoblot analyses to detect IRF3 and IFNAR1 protein expression in A549 and L929 cells infected with VSV-eGFP at the indicated time points. Data are representative of 3 independent biological experiments.

C, D qRT-PCR analyses of the mRNA abundance of IFNAR1 and IRF3 in A549 cells transfected with poly (I:C)-LMW (C) or infected with VSV-eGFP (D) for 16 hrs, followed by treatment with Act D (5 μg/mL) for the indicated times. n = 3 independent biological replicates, and error bars represent standard deviations. Data were compared using Student's t-test.

E qRT-PCR analyses of the proportion of IRF3 and IFNAR1 mRNAs in polysome fractions from A549 cells transfected with poly (I:C)-LMW for 16 hrs. n = 3 independent biological replicates, and error bars represent standard deviations. Data were compared using Student's t-test.

F Relative luciferase activities of IFNAR1-3' UTR or IRF3-5' UTR with wild-type (WT) m6A sites in 293T cells that were transfected with poly (I:C)-LMW or infected with VSV-eGFP for 24 hrs. Renilla luciferase activity was measured and normalized to firefly luciferase activity. n = 3 independent biological replicates, and error bars represent standard deviations. Data were compared using Student's t-test.

G Immunoblot analyses of IRF3 and IFNAR1 in shCtrl and shWTAP A549 cells, followed by infection with VSV-eGFP at the indicated time points. Data are representative of 3 independent biological experiments.

H, I Immunoblot analysis of the protein abundance of IRF3 and IFNAR1 in A549 cells transfected with poly (I:C)-LMW or infected with VSV-GFP for 16 hrs and then treated with PR-171 or DMSO for additional 4 and 8 hrs. Representative images from 3 independent biological experiments are shown (H), and the relative levels of IFNAR1 (left) and IRF3 (right) are presented as the mean ± SD (I).

Expanded View Figure legends
A brain microvasculature endothelial cell-specific viral vector with the potential to treat neurovascular and neurological diseases (2016)
Figure 5

Figure 5 - AAV-BR1-mediated transduction of brain endothelial cells in vivo

C57BL/6 mice (age 8 weeks) were injected with AAV-BR1 harboring an eGFP reporter gene under control of the CAG promoter. Images show representative examples of n = 6 mice.

A Representative images from cerebellum, olfactory bulb, striatum, cerebral cortex, and the spinal cord, 14 days after vector injection. BR1-eGFP-transduced cells (green) were positive for the endothelial marker CD31 (red). Scale bars represent 250 µm.

B Higher magnification confocal images. The endothelial marker CD31 (red) colocalizes with vector-mediated eGFP expression (green). Scale bars represent 50 µm (upper panel) or 10 µm (lower panel).

C CD13 staining of pericytes. The vector-mediated eGFP expression pattern (green) does not colocalize with CD13 (red). Scale bars represent 250 µm (upper panel) or 10 µm (lower panel).

D Aquaporin 4 staining of astrocyte end-feet. The vector-mediated eGFP expression (green) does not colocalize with aquaporin 4 (red). Scale bars represent 100 µm (upper panel) or 10 µm (lower panel).

E Expression of eGFP in primary brain endothelial cells prepared from C57BL/6 mice, 14 days after injection with AAV-BR1-eGFP. Scale bars represent 500 µm (upper panel) or 100 µm (lower panel).
A brain microvasculature endothelial cell-specific viral vector with the potential to treat neurovascular and neurological diseases (2016)
Figure 6

Figure 6 - AAV-BR1-iCre-mediated gene recombination in Ai14 Cre reporter mice

Two weeks after vector injection (1.8 x1011 genomic particles/ animal), into 16-weeks old mice, Cre-mediated gene recombination driven by the CAG promoter was observed mainly in brain endothelial cells (lower panel, red). No recombination was observed in control animals without BR1-iCre-virus injection (upper panel). CD31 (green) was used as a marker for brain endothelial cells. Panels show representative examples of n = 3 animals. Scale bars represent 250 µm.
A brain microvasculature endothelial cell-specific viral vector with the potential to treat neurovascular and neurological diseases (2016)
Figure 7

Figure 7 - Therapeutic use of AAV-BR1: Normalizing endothelial cell survival and blood-brain barrier permeability in neonatal incontinentia pigmenti mice (Nemo-/+) with AAV-BR1-NEMO

BR1-mediated expression of NEMO or eGFP was driven by the CAG promoter.

A Immunostaining of cerebral microvessels. Treatment with AAV-BR1-NEMO normalized string vessels (white arrows, highlighted in white square inset) in Nemo-/+ mice as compared to Nemo-/+ mice treated with the AAV-BR1-eGFP control vector at postnatal day 8 (P8). The upper left panel shows the staining in untreated wild type control mice. String vessels were identified as capillaries that have lost CD31-positive (green) endothelial cells but stain for the basement membrane protein collagen IV (red). Scale bars represent 200 µm. The lower right panel summarizes quantitative analysis of string vessels in Nemo-/+ and control mice (NemoFl or wild type) at P0 (n=3 animals/ group) and at P8 (n=6 animals/ group, *p = 0.0125). String vessels in the cerebral cortex were quantified as percentage of total vessel lengths.

B Quantification of active caspase 3-positive endothelial cells at P8 (n= 5 animals/ group, *p = 0.0201).

C Albumin in brain tissue as indicator for BBB leakage. In Nemo-/+ mice treated with AAV-BR1-NEMO, less albumin was found in brain tissue, indicating less BBB leakage. Representative Western blot with albumin from P8 Nemo-/+ mice treated with AAV-BR1-NEMO or AAV-BR1-eGFP control vector, respectively, as well as untreated wild type (WT) control mice. Right panel: quantitative analysis of the Western blots (n = 4 animals/ group, *p = 0.0283)

D Body weight of vector-treated mice. WT control or Nemo-/+ mice at P8 treated with AAV-BR1-NEMO or AAV-BR1-eGFP (n= 18 animals/ group, *p = 0.0038).

Data information: Data are shown as mean +SEM or as plotted individual points with bars representing the mean. Differences between vector-treated Nemo-/+ mice were analyzed by unpaired t-test.
A brain microvasculature endothelial cell-specific viral vector with the potential to treat neurovascular and neurological diseases (2016)
Figure 8

Figure 8 - Normalizing endothelial cell survival and blood-brain barrier permeability by intravenous injection of AAV-BR1-NEMO in a conditional murine incontinentia pigmenti model (NemobeKO mice)

BR1-mediated expression of NEMO or eGFP was driven by the CAG promoter. All animals were at the age of 8-12 weeks.

A Representative immunostainings of cerebral microvessels. String vessels (white arrows, highlighted in white square inset) were significantly reduced in NemobeKO mice treated with AAV-BR1-NEMO compared to NemobeKO mice treated with AAV-BR1-eGFP control vector. NemoFl mice served as control animals. Scale bars represent 200 µm.

B Quantification of string vessel lengths in the cerebral cortex as percentage of total vessel lengths. NemobeKO and NemoFl mice were treated with AAV-BR1-NEMO or AAV-BR1-eGFP control vector (n=9 NemoFl animals + BR1-eGFP, 10 NemobeKO animals + BR1-eGFP, 9 NemoFl animals + BR1-NEMO, and 13 NemobeKO animals + BR1-NEMO), ****p < 0.0001.

C Total vessel length measured as total CD31-positive vessels. Vessels were restored in NemobeKO mice treated with AAV-BR1-NEMO compared to the AAV-BR1-eGFP injected mice. NemoFl mice served as a control (n= 9 NemoFl animals +BR1-eGFP, 10 NemobeKO animals + BR1-eGFP, 9 NemoFl animals + BR1-NEMO, and 13 NemobeKO animals + BR1-NEMO), ***p = 0.0007, **p = 0.0038 (NemoKO:eGFP vs. NemoFl:NEMO ), **p = 0.0014 (NemoKO:NEMO vs. NemoKO:eGFP).

D IgG and albumin Western blots of brain lysates. Less leakage in the BBB was detected in NemobeKO mice treated with AAV-BR1-NEMO than in NemobeKO mice injected with AAV-BR1-eGFP (the right panel indicates the quantified gel intensity under the various treatment conditions (n= 5 animals per group). NemoFl mice served as controls, ****p < 0.0001.

E Quantitative immunoglobulin staining of coronal brain sections of NemoFl and NemobeKO mice. Ig extravasation was significantly reduced in AAV-BR1-NEMO-treated mice (n= 9 NemoFl animals + BR1-eGFP, 10 NemobeKO animals + BR1-eGFP, 9 NemoFl animals + BR1-NEMO, and 13 NemobeKO animals + BR1-NEMO), ****p < 0.0001.

F Effect of AAV-BR1 vector on BBB permeability. No vector (left) or empty AAV-BR1 vector (right) was injected i.v. to wild type mice and BBB permeability was assessed by extravasation of the fluorescent tracer sodium fluorescein (n=7 animals per group). No significant difference was detected (n.s.).

Data information: Data are shown as mean +SEM. Data were analyzed by two-way ANOVA followed by Bonferroni’s post test (A-E) or by Student’s t-test (F).
Metabolic regulation of pluripotency and germ cell fate through α−ketoglutarate (2018)
Figure 2

Figure 2. Oxidative metabolism promotes naïve pluripotency.

(A) Investigating the effect of sustained oxidative mitochondrial metabolism on the transition from naïve ESCs to primed EpiLCs through supplementation of the glycolysis inhibitors 2-deoxy-D-glucose (2-DG) and dichloroacetate (DCA), respectively.

(B) Flow cytometry analysis of Rex1-GFPd2 cells following addition of 10mM 2-DG during the 48h EpiLC induction. Representative GFP intensity distributions are depicted. Average proportions of Rex1-GFPd2 positive (GFP+) cells are quantified from two independent biological replicates. Error bars represent ± SE. ***, p=0.0006 (unpaired 1-tailed Student's t-test).

(C) Expression analysis by qRT-PCR of naïve pluripotency and epiblast marker genes in bulk 48h cells after 10mM 2-DG treatment. Relative expression levels, normalized to control culture conditions, are shown. Graphs represent averages from triplicate (duplicate for Klf4 and Tfcp2l1) independent biological experiments. Error bars denote ± SE. *, p ≤ 0.05; **, p ≤ 0.01 (unpaired 1-tailed Student's t-test, see Appendix Table S3 for exact p-values).

(D) Colony-forming ability following 10mM 2-DG supplementation during EpiLC stimulation. Representative images of alkaline phosphatase (AP)-stained colonies are displayed. Scale bar, 250 um. The average colony formation, normalized to control culture conditions, quantified from two independent biological replicates, is shown. Error bars signify ± SE. *, p=0.0424 (unpaired 1-tailed Student's t-test).

Data information: See also Figure EV2.
Metabolic regulation of pluripotency and germ cell fate through α−ketoglutarate (2018)
Figure 3

Figure 3. αKG maintains naïve pluripotency.

(A) Pseudotime expression profiles for the αKG-regulating enzymes Idh2 and Dlst during the transition from naïve to primed pluripotency. Tricarboxylic acid (TCA) cycle enzymes and metabolites produced within the TCA cycle are illustrated.

(B) Representative flow cytometry profiles of Rex1-GFPd2 cells following 4mM dm-αKG supplementation during the EpiLC induction are depicted. Graphs show average fractions of Rex1-GFPd2 positive (GFP+) cells from six independent biological assays. Error bars indicate ± SE. ***, p=1.241E-05 (unpaired 1-tailed Student's t-test).

(C) qRT-PCR analysis of naïve pluripotency regulators and epiblast marker genes following EpiLC stimulation in the presence of 4mM dm-αKG. Expression data are normalized to control culture conditions and represent averages from five biological replicates in bulk 48h cells. Error bars denote ± SE. ***, p ≤ 0.005 (unpaired 1-tailed Student's t-test, see Appendix Table S3 for exact p-values).

(D) Colony forming ability succeeding 4mM dm-αKG treatment during the 48h EpiLC induction. Characteristic images of AP-stained colonies are shown. Scale bar, 250 um. Colony formation is normalized to control-treated cells and quantified from quadruplicate experiments. Error bars signify ± SE. *, p=0.0283 (unpaired 1-tailed Student's t-test).

(E) Representative super-resolution images of TOM-20 immune-labelled mitochondria in ESCs following 48h culture in 2i/Lif/KSR media, and EpiLC-inducing conditions in the presence of 4mM dm-αKG and DMSO, respectively, are displayed. Scale bar, 3um.

(F-H) 10-day culture of Rex1-GFPd2 cells in N2B27/Lif/KSR with 4mM dm-αKG and DMSO, respectively, with passaging every 2.5-days. (F) Characteristic bright field images of Rex1-GFPd2 cells after 10 days of culture in 2i/Lif/KSR and N2B27/Lif/KSR, in the presence of dm-αKG and DMSO, respectively. Scale bar, 10um. (G) Flow cytometer-based quantification of Rex1-GFPd2 positive (GFP+) cells. Representative GFP intensity distributions are displayed. The average fractions of GFP+ cells are measured from duplicate experiments. Error bars denote ± SE. *, p=0.0477 (unpaired 1-tailed Student's t-test). (H) qRT-PCR analysis of naïve pluripotency and differentiation markers in bulk cells harvested at 2.5 day-intervals during the 10-day culture in N2B27/Lif/KSR with dm-αKG or DMSO. Expression data are normalized to time-matched ESCs in 2i/Lif/KSR-culture conditions and are averaged over two independent biological experiments. Error bars indicate ± SE. *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.005 (unpaired 1-tailed Student's t-test, see Appendix Table S3 for precise p-values).

(I) Model illustrating the IDH2-mediated production of αKG in the mitochondrial TCA cycle during oxidative metabolism in ESCs in naïve pluripotency conditions.

Data information: See also Figure EV3, EV4.
Metabolic regulation of pluripotency and germ cell fate through α−ketoglutarate (2018)
Figure 4

Figure 4. αKG promotes PGC fate.

(A) FACS analysis of Prdm1-GFP positive (GFP+) cells in day-4 embryoids specified in the presence of 4mM dm-αKG and PGC cytokines. Representative flow cytometer profiles are depicted. Graphs show the average fractions of GFP+ cells from duplicate experiments. Error bars denote ± SE. *, p=0.0526 (unpaired 1-tailed Student's t-test). P1-GFP, Prdm1-GFP.

(B) FACS analysis of Prdm1-GFP positive (GFP+) cells in day-2 embryoids aggregated under addition of LIF (10ng ml-1) and BMP4 (500ng ml-1), dm-αKG (4mM), or DMSO. Representative flow cytometer profiles are displayed. Average fractions of GFP+ cells are calculated from duplicate assays. Error bars denote ± SE. *, p=0.0526. P1-GFP, Prdm1-GFP.

(C) FACS analysis of Prdm1-GFP positive (GFP+) cells in day-4 PGCLC aggregates specified from 4mM dm-αKG-treated (t=48h to t=72h) EpiLCs. Representative flow cytometer profiles are depicted. The average fractions of GFP+ cells, quantified from triplicate experiments, are shown. Error bars signify ± SE. *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.005 (unpaired 1-tailed Student's t-test, see Appendix Table S3 for precise p-values). P1-GFP, Prdm1-GFP.

(D) Transcript analysis by qRT-PCR of PGC specifiers, demethylating enzymes, mesoderm, endoderm, and ESC regulators in FACS-sorted day-4 Prdm1-GFP embryoids induced from 4mM dm-αKG-treated 72h EpiLCs. Expression levels are normalized to Prdm1-GFP negative cells from control embryoids. Graphs represent averages from triplicate experiments. Error bars indicate ± SE. *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.005 (unpaired 1-tailed Student's t-test, see Appendix Table S3 for precise p-values). +, Prdm1-GFP positive cells; -, Prdm1-GFP negative cells.

(E) Western blot analysis for H3K9me2, H3K27me3, and DNMT3b in 4mM dm-αKG-treated EpiLCs. H3 is used as a loading control. 48h, dm-αKG and DMSO, respectively, supplementation from t=24h to t=48h during the EpiLC differentiation. 72h, dm-αKG and DMSO, respectively, supplementation from t=48h to t=72h during the EpiLC differentiation.

(F) ChIP-qPCR analysis of H3K9me2 and H3K27me3 in putative enhancer regions of genes associated with the naïve pluripotent state (Esrrb, Arid5b) and PGC fate (Tfap2c), respectively, in naïve ESCs and at t=48h following EpiLC induction in the presence of 4mM dm-αKG and DMSO, respectively. Graphs show enrichment of H3K9me2, H3K27me3, and IgG control, respectively, relative to DMSO-treated EpiLCs. Averages represent triplicate independent experiments. Error bars signify ± SE. *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.005 (unpaired 1-tailed Student's t-test, see Appendix Table S3 for precise p-values).

(G) Model illustrating the extension of the transient developmental competence for the PGC fate through αKG.

Data information: See also Figure EV5, EV6.

Expanded view Figure legends
Accumulation of autophagic vacuoles and cardiomyopathy in LAMP-2-deficient
mice (2000)
figf1

a, Northern blot analysis of lamp-2 expression. Total RNA
was hybridized using a lamp-2 cDNA2 probe and a murine
glyceraldehyde-3-phosphate dehydrogenase probe. b, Western blot analysis
of LAMP-2 expression. In control liver and kidney extracts the glycosylated
LAMP-2 molecules were detected. In LAMP-2-/- tissues no LAMP-2
product was found. c, Increased mortality in LAMP-2 deficient outbred
(C57B6/Jx129SV) mice. Forty-two control (closed circles) and LAMP-2-deficient
mice (open circles) were monitored over 300 days. d, Loss of weight
in early dying (n = 7 males) and surviving LAMP-2-deficient
mice (n = 11 males) in comparison with control mice (
n = 12 males) monitored for 60 days after birth.
Accumulation of autophagic vacuoles and cardiomyopathy in LAMP-2-deficient
mice (2000)
figf2

a, Ultrastructure of autophagic vacuoles (arrowheads) in pancreatic
acinar gland cells from a 6.5-month-old LAMP-2-deficient mouse. b,
Autophagic vacuoles containing glycogen (g), cytosol and cellular constituents
in a hepatocyte of an 8-day-old LAMP-2-/- mouse. m, mitochrondria;
bc, bile canaliculus. c, Accumulation of numerous early autophagic
vacuoles in cultured LAMP-2-/- hepatocytes. Arrow, large late
autophagic vacuole containing partially degraded material. Insert, magnification
of the region indicated by an asterisk with typical early autophagic vacuoles
containing undigested cellular constituents. Scale bars: a, 3.5 µm;
b, 0.2 µm; c, 1.3 µm; insert, 0.9 µm.
Accumulation of autophagic vacuoles and cardiomyopathy in LAMP-2-deficient
mice (2000)
figf3

a, Vacuole in a fibre of the soleus muscle. b, Ultrastructure
of a vacuole in a cardiomyocyte. Scale bars: a, 1 µm;
b, 1.5 µm. c, Typical original twitch recordings of
developed force of heart muscles from control (thin line) and LAMP-2-deficient
(thick line) mice. d, Average values of developed force. Preparations
of LAMP-2 deficient mice had a significantly lower baseline developed force
at a stimulation frequency of 4 Hz and 10 Hz; P  0.01.
Accumulation of autophagic vacuoles and cardiomyopathy in LAMP-2-deficient
mice (2000)
figf4

a, Volume density of early (Avi) and late (Avd) autophagic vacuoles
from four independent cultures. Examples of Avi and Avd are shown
in b and c, respectively. Bars represent 0.4 µm.
d, Labelling density of cathepsin-D as estimated in two independent experiments.
e, Labelling density of LAMP-1. f, Degradation of long-lived
proteins followed over 24 h in amino acid and fetal-calf-serum-containing
medium. g, Effect of amino-acid (AA) deprivation and 3-methyladenine
(MA) on protein degradation during 4 h of chase. The error bars in
a, d, e and g represent s.e.m.
Menin-regulated Pbk controls High fat diet-induced compensatory beta cell proliferation (2021)
Figure 2

Figure 2. Pbk kinase activity is crucial for HFD-induced beta cell compensatory proliferation

A. A diagram for generation of Pbk K64K65 → AA KI mice using CRISPR/Cas9 system.

B. Target region in exon5 of murine Pbk locus. Mutant nucleotides for inducing KK→AA mutation were highlighted with blue. Two mismatched bases with silence mutations that generate a novel Mwol site are labelled in yellow. The amplicon and enzyme cutting size are indicated.

C. PCR genotyping of tail genomic DNA for PbkKI/KI mice using RFLP. Genomic fragments from exon 5 were amplified by PCR and digested with Mwol.

D. DNA sequence confirming the mutation from K64K65 to AA in PbkKI/KI mice from the tail genomic DNA compared with PbkWT/WT mice.

E. IPGTT (glucose at 2 g/kg of body weight, i.p.) on 11-week-old male PbkKI/KI mice and PbkWT/WT mice (n=4 for each group). All mice were starved overnight (16h) before testing.

F. Islet number in each pancreas section on 11-week-old male PbkKI/KI and PbkWT/WT mice (n=4 for each group). *P = 0.0443 (two-tailed unpaired student's t-test).

G. Beta cell mass comparison between 11-week-old male PbkKI/KI and PbkWT/WT mice (n=4 for each group). **P = 0.0015 (two-tailed unpaired student's t-test).

H. In vitro perifusion studies on the islets isolated from 11-week-old male PbkKI/KI and PbkWT/WT mice (n=3 for each group).

I. HFD feeding schedule for PbkKI/KI and PbkWT/WT mice. 7-week- old male PbkKI/KI and PbkWT/WT mice were fed with HFD (n=8 for each group) for 12 weeks.

J. Persistent measurement of GTT for PbkKI/KI or PbkWT/WT mice on HFD (n=4 for each group) or chow diet (n=2 for each group) at various times, i.e., 0, 3rd, 5th, 8th, and 12th week during HFD-feeding. The AUC data of GTT at various time points were shown. *P = 0.0294, (Two way ANOVA). ns, not statistically significant difference.

K, L. Representative images of double immuno-staining for insulin (green) and BrdU (red) of pancreas from PbkWT/WT (K) or PbkKI/KI (L) mice with 5 weeks HFD feeding. Islet area was circled with white dashed line. Nuclei were labeled by DAPI (blue). Scale bar: 50 μm.

M. Quantification of BrdU and insulin dual positive cells (n=4 per group). Four mice for each group, and 5-10 islet images per mouse were analyzed. *P = 0.0220 (two-tailed unpaired student's t-test).

N. Beta cell mass comparison between PbkKI/KI and PbkWT/WT mice (n=4 per group). **P = 0.0010 (two-tailed unpaired student's t-test).

O. Quantification of Pbk expression positive beta cells. Four mice for each group, 10 islet images per mouse were analyzed. ns, not statistically significant difference (unpaired two-tailed student's t-test).

Data information: Data are represented as mean ± SEM.