EMBO/soda-nel huggingface dataset:
Infancy-onset diabetes caused by de-regulated AMPylation of the endoplasmic reticulum chaperone BiP (2022)
Figure 5

Figure 5:

Expressed at the endogenous FICD locus, the p.(Arg371Ser) mutation leads to defective clearance of AMPylated BiP in ER stressed cells

A On the left is a schema of the Chinese Hamster Ovary cell FICD locus. Exons depicted as blue boxes, introns as lines. Forward (F) and reverse (R) primers used to genotype the locus and the guide RNA that directs the Cas9 nuclease (CRISPR guide) are depicted by arrows. A schema of the repair template is provided below, with the Arg371Ser mutation, the silent BamHI site and silent PAM-destroying features noted. To the right is the sequence of the locus before and after recombination and a sequencing trace of the locus in the mutant FICDR371S derivative cell line.

B On the left are immunoblots of AMPylated proteins detected with pan-AMP, BiP and eIF2α (a loading control) reactive antibodies in lysates of cells of the indicated genotype. The cells were untreated or exposed to the protein synthesis inhibitor, cycloheximide (Cx, 100 µg/mL) for the indicated time. In the panel on the right are immunoblots of samples immunoprecipitated with a BiP-specific antiserum from the lysates of the samples shown on the left.

C Immunoblots as in B in lysates of cells of the indicated genotype. The cells were untreated or exposed to the protein synthesis inhibitor, cycloheximide (Cx, 100 µg/mL) and/or thapsigargin (Tg) for the indicated periods prior to harvest.

D Quantification of ratio of the BiP-AMP to total BiP signal (expressed in arbitrary units) of repeats of the experiment shown in C. Mean ± SD of the BiP-AMP signal normalized to total BiP signal from the same samples are depicted, (P values by two tailed t-test, n = 6, biological replicates).

Source data are available online for this figure.
Infancy-onset diabetes caused by de-regulated AMPylation of the endoplasmic reticulum chaperone BiP (2022)
Figure 6

Figure 6:

Compromised secretion by deregulated FICD depends on residues specifying BiP AMPylation

A Representative immunoblot of AMPylated proteins in FICD∆ CHO cells transiently transfected with expression plasmids encoding the indicated FICD enzymes (replicated three times). Note that the hyperactive E234G mutant bearing the K124E-H131A mutations that compromise its ability to interact with BiP, is nonetheless consistently auto-AMPylated.

B Schema of the assay used to measure the effect of FICD on secretion.

C Bar diagram of the amount of alkaline phosphatase [normalised to cytoplasmic luciferase (a transfection marker)] secreted from FICD∆ CHO cells co-expressing the indicated FICD enzymes. The K124E-H131A mutations that interfere with engagement of BiP as a substrate reverse the secretion defect imposed by the hyperactive FICD enzymes [Mean ± SD, P values by ANOVA with Šídák's multiple comparisons test, n = 8-16 (biological replicates)].
MicroRNA-574 Regulates FAM210A Expression and Influences Pathological Cardiac Remodeling (2020)
Figure 1

Figure 1. miR-574-5p and miR-574-3p expression is induced in human and mouse failing hearts.

(A) miRNA profiling identified miR-574-5p as the commonly upregulated miRNA in the hearts of aged mice, MI mice, and patients; and miR-574-3p is induced in exercised hearts and MI patients.

(B) Sequence and location of miR-574 gene in the human genome.

(C) miR-574-5p and miR-574-3p were highly expressed in the explanted failing hearts from chronic HF patients (n=17) versus donor non-failing hearts (n=6).

(D) miR-574-5p and miR-574-3p were induced in the hearts of mice with isoproterenol (ISO) infusion (4 weeks; n=10-12).

(E) miR-574-5p and miR-574-3p were induced in the hearts of mice under transverse aortic constriction (TAC) surgery (4 weeks; n=8-10).

Data information: Data were presented as mean ± SEM. P values were calculated by Mann-Whitney test (C) and unpaired two-tailed Student t test (D, E).
MicroRNA-574 Regulates FAM210A Expression and Influences Pathological Cardiac Remodeling (2020)
Figure 2

Figure 2. miR-574 gene knockout augments ISO-induced pathological cardiac remodeling.

(A) H&E of hearts from WT and miR-574-/- mice with or without isoproterenol (ISO) treatment for 4 weeks. Mice were between 8-10 weeks old females.

(B) Ratio of HW/TL (heart weight/tibia length) in WT and miR-574-/- mice (n=8/7/8/6 for 4 groups of WT, Veh.; WT, ISO; KO, Veh.; KO, ISO).

(C) WGA (wheat germ agglutinin) staining in WT and miR-574-/- mice. Cross-sectional area (CSA) of CMs was measured and quantified (n≥500 cells). Scale bar: 50 μm. In the violin plot, black line shows median value for the group and peach dashed lines represent two quartile lines in each group.

(D) Picrosirius red staining in WT and miR-574-/- mice (n=6 per group). Scale bar: 1 mm.

(E) RT-qPCR of fetal cardiac genes in WT and miR-574-/- mice with ISO treatment (n=3 per group). ANF, atrial natriuretic factor; BNP, B-type natriuretic peptide; Myh6/Myh7, myosin heavy polypeptide 6/7; Col1a2/Col3a1, procollagen, type I, α2; type III, α1.

(F) TUNEL assay for heart tissue sections from WT and miR-574-/- mice under ISO versus vehicle treatment (n=6 per group). Scale bar: 10 μm.

Data information: Data were presented as mean ± SEM. P values were calculated by unpaired two-tailed Student t test (E), Kruskal-Wallis test with Dunn's multiple comparisons test (C), or two-way ANOVA with Tukey's multiple comparisons test (B, D, F).
MicroRNA-574 Regulates FAM210A Expression and Influences Pathological Cardiac Remodeling (2020)
Figure 3

Figure 3. miR-574 gene knockout augments TAC-induced pathological cardiac remodeling.

(A) H&E staining of hearts from WT and miR-574-/- mice 4 weeks after transverse aortic constriction (TAC) surgery.

(B) Ratio of HW/TL in WT and miR-574-/- mice (n=8 per group).

(C) WGA staining of hearts from WT and miR-574-/- mice 4 weeks after TAC surgery (n≥500 cells). Scale bar: 20 μm. In the violin plot, black line shows median value for the group and peach dashed lines represent two quartile lines in each group.

(D) Picrosirius red staining of hearts from WT and miR-574-/- mice under TAC surgery (n=8 per group). Scale bar: 1 mm.

(E) RT-qPCR of fetal cardiac genes in WT and miR-574-/- mice with TAC surgery (n=4 per group).

(F) Echocardiography measurement of cardiac functions of hearts from WT and miR-574-/- mice 4 weeks post TAC (n=8 per group). FS, fractional shortening.

Data information: Data were presented as mean ± SEM. P values were calculated by Kruskal-Wallis test with Dunn's multiple comparisons test (C) or two-way ANOVA with Tukey's multiple comparisons test (B, D-F).
MicroRNA-574 Regulates FAM210A Expression and Influences Pathological Cardiac Remodeling (2020)
Figure 5

Figure 5. Identification of Fam210a as a common target of miR-574-5p/3p in vivo.

(A) Volcano curve analysis of dysregulated genes in miR-574-/- versus WT heart. The Padj is the P-values adjusted for multiple testing with correction using the Benjamini-Hochberg procedure. The dots above the dashed line show Padj<0.05.

(B) Heatmap of significantly upregulated genes in miR-574-/- mice at baseline analyzed by RNA-Seq. P60 male mice, n=3 per group, Padj<0.05. The Padj is the P-values adjusted for multiple testing with correction using the Benjamini-Hochberg procedure.

(C) Gene Ontology analysis of enriched pathways of upregulated genes in RNA-Seq. The top three pathways are listed with enriched gene sets.

(D) Identification of Fam210a as a target of miR-574-5p and miR-574-3p. Seed sequence-bearing target genes were predicted by TargetScan.

(E) Expression of Fam210a mRNA in the heart of miR-574-/- and WT mice from 3 biological replicates.

(F) Expression of FAM210A protein in the heart of miR-574-/- and WT mice. Protein intensity was quantified in the right panel (n=6 per group).

(G) Seed sequences of miR-574-5p and miR-574-3p in Fam210a mRNA 3'UTR (TargetScan) and their mutants.

(H) Dual luciferase reporter assays with co-transfection of miR-574-5p mimics and FLuc-Fam210a 3'UTR bearing wild type and mutant seed sequences. The assays were performed in three biological replicates.

(I) Dual luciferase reporter assays with co-transfection of miR-574-3p mimics and FLuc-Fam210a 3'UTR bearing wild type and mutant seed sequences. The assays were performed in three biological replicates.

Data information: Data were presented as mean ± SEM. P values were calculated by unpaired two-tailed Student t test (E, F).
MicroRNA-574 Regulates FAM210A Expression and Influences Pathological Cardiac Remodeling (2020)
Figure 6

Figure 6. FAM210A is a conserved mitochondrial protein located in the mitoplast.

(A) The schematic of FAM210A protein domain composition and evolutionary conservation of FAM210A protein sequence from 10 representative species analysed by AlignX software. The yellow colour shows completely conserved residues across all the species at a given position; the blue colour indicates consensus residues derived from a block of similar residues at a given position while the green colour stands for the consensus residues derived from the occurrence of greater than 50% of a single residue at a given position. The green letters suggests weak similarity to consensus residues at a given position.

(B) The expression level of FAM210A mRNA in 15 human organs from the GTEx Portal database. TPM: transcripts per million of reads. The central band shows the median value and the box indicates the first and third quartiles. The sample size of human samples used for quantification in GTEx Portal database (from testis to pancreas): n=361/429/432/555/803/240/432/226/406/578/241/202/85/328/755.

(C) Cellular localization of FAM210A in primary mouse ACMs detected by IF. Scale bar: 30 μm.

(D) Cellular localization of recombinant FAM210A in human AC16 CM cells. FAM210A-3xFlag expression plasmid was transfected into AC16 cells for 48 hrs. Scale bar: 40 μm.

(E) Immunoblot detection of FAM210A in isolated cytoplasmic and mitochondrial fractions from mouse hearts. VDAC1 and tubulin are used as fraction controls. Cyto: cytoplasm; Mito: mitochondria. A representative image is shown from three biological replicates.

(F) Sub-organelle localization of FAM210A in isolated mouse cardiac mitochondria. TOM20 and ATP5B are used as the outer membrane (OM) and mitoplast (MP) markers, respectively.
MicroRNA-574 Regulates FAM210A Expression and Influences Pathological Cardiac Remodeling (2020)
Figure 7

Figure 7. FAM210A interacts with EF-Tu and regulates mitochondrial encoded protein expression.

(A) Antibody-based affinity purification of endogenous FAM210A from purified murine cardiac mitochondria coupled with mass spectrometry analysis.

(B) Top list of FAM210A-interacting proteins in IP-MS (immunoprecipitation-mass spectrometry) analysis. PSM: peptide spectrum match.

(C) Confocal imaging of co-localization of FAM210A-3xFLAG and endogenous EF-Tu by immunostaining. Scale bar: 40 μm.

(D) Left panel: IP-IB validates FAM210A-interacting proteins using pre-immune IgG and FAM210A antibodies. Right panel: IP-IB validates FAM210A-interacting proteins in HEK293T cell lysates subjected to IP using pre-immune IgG and anti-FLAG antibodies. A representative image is shown in three biological replicates.

(E) Mapping of interacting domains in FAM210A for binding EF-Tu.

(F,G) Protein expression of mitochondrial ETC component genes was measured in AC16 cells after following treatments in comparison with control cells: (F) siRNA knockdown of FAM210A. (G) FAM210A overexpression. Quantitative data were shown in Figure EV4A, B.
MicroRNA-574 Regulates FAM210A Expression and Influences Pathological Cardiac Remodeling (2020)
Figure 8

Figure 8. miR-574 regulates mitochondrial protein expression through targeting FAM210A.

(A) miR-574-5p and miR-574-3p overexpression reduced FAM210A mRNA expression in AC16 cells (n=3 in each group).

(B) miR-574-5p and miR-574-3p inhibition increased FAM210A mRNA expression in AC16 cells (n=3 in each group).

(C,D) Protein expression of mitochondrial ETC component genes was measured in AC16 cells after following treatments in comparison with control cells: (C) miR-574-5p or miR-574-3p overexpression. (D) Transfection of anti-miR-574-5p or anti-miR-574-3p inhibitor.

(E,F) Mitochondrial membrane potential and ATP production in AC16 cells under ISO treatment (10 μM for 24 hr) with overexpression of miR-574-5p or miR-574-3p. >120 cells/group were quantified in (E) and n=4 biological replicates in (F). The dashed line in the violin plot shows medium value for the group and the dotted lines represent two quartile lines in each group.

(G,H) Mitochondrial membrane potential and ATP production in primary mouse ACMs isolated from WT and miR-574-/- mice under ISO treatment. Scale bar: 10 μm. >110 cells/group were quantified in bottom panel of (G) and n=5 in (H).

(I) transfection of miR-574-5p/3p in the absence or presence of FAM210A overexpression. All the Western blot images were provided as representative data from 3-4 biologically replicated experiments. Quantitative data were shown in Figure EV4H.

(J) Increased protein expression of FAM210A and ND1 in miR-574 KO hearts compared to WT hearts upon TAC surgery. N=5 hearts (~110-150 CM cells) were measured per group. The dashed line in the violin plot shows medium value for the group and the dotted lines represent two quartile lines in each group.

(K) The schematic model of the cardioprotective function and mechanism of miR-574-FAM210A axis against cardiac pathological remodeling.

Data information: Data were presented as mean ± SEM. P values were calculated by Kruskal-Wallis test with Dunn's multiple comparisons test (A, B, E), one-way ANOVA with Tukey's multiple comparisons test (F), two-way ANOVA with Tukey's multiple comparisons test (G, H), or one-way ANOVA with non-parametric Kruskal-Wallis test (J)

Extended View Figure Legends
SIRT2-mediated inactivation of p73 is required for glioblastoma tumorigenicity (2018)
Figure 1

Figure 1. Knockdown of SIRT2 using siRNA or treatment with AGK2 induces growth arrest and apoptosis of glioblastoma cells.

A Sphere formation of GB2 cells transfected with an shRNA targeting SIRT2 was analyzed by an In Cell Analyzer 2000. Primary spheres were re-plated to evaluate secondary sphere formation. Bars indicate mean ± s.d. of 10 wells.

B Knockdown of SIRT2 causes a decrease in the sphere formation capacity of GB16. (n = 3).

C mRNA levels of the indicated genes in GB2, GB4 and GB16 cells infected with a lentivirus expressing an shRNA targeting SIRT2 were measured by qRT-PCR. The results were normalized with the values for GAPDH. Bars indicate mean ± s.d. (n = 3-4).

D The sphere formation capacity of CD133-positive and CD133-negative cells sorted by FACS directly from a tumor sample. GB17 was infected with a control (Empty) or shSIRT2-expressing (shS2 #1) lentivirus. Bars indicate mean ± s.d. of 8 wells.

E The sphere formation capacity of CD133-positive and CD133-negative cells sorted by FACS directly from a tumor sample. (Left panel) GB18 was treated with AGK2 (10 μM) or DMSO. (Right panel) Secondary sphere formation of GB18 was examined in the absence of AGK2. Bars indicate mean ± s.d. of 8 wells.

Data information: Statistical significance were evaluated using the likelihood ratio test (for Fig 1B) or unpaired two-tailed t-test. *p < 0.05; **P < 0.01.
SIRT2-mediated inactivation of p73 is required for glioblastoma tumorigenicity (2018)
Figure 2

Figure 2. Knockdown of SIRT2 suppresses the tumorigenicity of GB2 cells.

A Kaplan-Meier overall survival curves of mice transplanted with GB2 cells (Left panel), GB13 cells (Middle panel) or GB16 cells (Right panel) infected with the indicated lentivirus (1 × 104 cells, 7 mice per group). The y-axis indicates the percent survival.

B Mice were transplanted with the indicated number of GB2 cells infected with a control (Empty) or shSIRT2-expressing (shS2 #1) lentivirus. Six weeks after transplantation, mice (3 or 4 animals, see number of dots) were sacrificed and the expression levels of human β-actin mRNA were quantified by qRT-PCR.

C H&E staining of tumors that were developed in mice implanted with GB16 cells that had been infected with a control (Empty) or shSIRT2-expressing (shS2#1) lentivirus. Scale bar, 1 mm. An image with higher magnification is shown on the right (scale bar, 50 μm).

D The effect of AK7 on the deacetylation of acetyl-tubulin (Left panel) and the proliferation of GB2 cells (Right panel). (Left panel) Immunoblotting analysis of the effect of AK7 (20 μM) on deacetylation of acetyl-tubulin/tubulin were performed on day 3 in Right panel. Bars indicate mean ± s.d. (n = 3).

E Ten days after intracranial transplantation of GB2 cells (1.0 × 104 cells), AK7 was intraperitoneally administrated for 4 weeks (15mg/kg, twice/week). After eight weeks, mice (4 or 5 animals, see number of dots) were sacrificed and the expression levels of human β-actin mRNA was quantified by qRT-PCR.

Data information: Statistical significance were evaluated using the log-rank test (for Fig 2A) or unpaired two-tailed t-test. *p < 0.05; **P < 0.01.
SIRT2-mediated inactivation of p73 is required for glioblastoma tumorigenicity (2018)
Figure 4

Figure 4. SIRT2 deacetylates p73 and suppresses its transcriptional activity.

A Cells transfected with FLAG-tagged p73 isoforms were treated with 20 μM AGK2 or DMSO for 6 h and subjected to immunoprecipitation with anti-FLAG antibody followed by immunoblotting with antibody against acetylated lysine or FLAG.

B Three conserved lysine residues are located in the most C-terminal region of p73. Amino acid sequences of p73 of the indicated species and human p53 and p63 are aligned. *, conserved and acetylated lysine residues, black frame, highly conserved residues, gray frame, conserved residues.

C Schematic representation of TAp73α and K3R. K3R is a mutant TAp73α in which three lysine (K) residues in the C-terminal region are replaced with arginine (R).

D Cells transfected with the indicated constructs were treated with 20 μM AGK2 or DMSO, respectively, for 6 h and subjected to immunoprecipitation with anti-FLAG antibody followed by immunoblotting with antibody against acetylated lysine or FLAG.

E HEK293T cells transfected with FLAG-tagged TAp73α were treated with 20 μM AGK2 (pre-AGK2 +) or DMSO (pre-AGK2 -) for 6 h. p73 was purified by immunoprecipitation and incubated with recombinant SIRT2 (10 U), NAD (1 mM) and/or AGK2 (3.5 μM) as indicated.

F qRT-PCR analysis of PUMA and GADD45 mRNA in GB2 cells infected with the indicated lentiviruses. Bars indicate mean ± s.e.m. (n = 4-5).

G Expression of p73 in (F) was determined by immunoblotting with anti-p73 antibody. RFP was used as a control.

H GB glioblastima neurospheres [5 × 104 cells (indicated by the dashed line)] were infected with the indicated lentiviruses. After 96 h, the number of viable cells was counted. Bars indicate mean ± s.d. (n = 4).

I GB2 cells infected with the indicated lentiviruses were intracranially transplanted into immunocompromised mice. After 8 weeks, mice (3 to 5 animals, see number of dots) were sacrificed and the expression levels of human β-actin mRNA were quantified by qRT-PCR.

J SIRT2-mediated inactivation of p73 is critical for the proliferation and tumorigenicity of glioblastoma cells. SIRT2 regulates the transcriptional activity of the tumor suppressor p73 by deacetylating its C-terminal lysine residues.

Data information: Statistical significance were evaluated using unpaired two-tailed t-test. *p < 0.05; **P < 0.01.

Expanded View Figure Legends
Deciphering the Physiological Response of Escherichia coli Under High ATP Demand (2021)
Figure 4

Figure 4: Simulation of the PFK rate with varying ADP/(ATP+ADP) ratios.

Data information: The PFK flux was simulated as single (isolated) reaction with the following fixed metabolite concentrations relevant for the kinetic rate law of the PFK (for the latter see Appendix Supplementary Text, section 1.3): PEP: 0.27 µmol/gDW; F6P: 0.91 µmol/gDW; FBP: 9.74 µmol/gDW ; total concentration ATP+ADP: 2.67 µmol/gDW.
PI(5)P Regulates Autophagosome Biogenesis. (2015)
Figure 1

PI(5)P Regulates Autophagosome Biogenesis

(A and B) Western blot analysis of LC3-II and tubulin levels and quantification of LC3-II/tubulin ratio in HeLa cells treated with carrier alone, or in combination with PI(5)P di-C16 at indicated concentrations for 1 hr, in the absence and presence of 400 nM BAF (treated in combination with lipids). Note that LC3-I is often very faint compared with LC3-II in HeLa cells under the protein extraction conditions we used (see Figure S1C). However, this was not a problem because one should relate LC3-II to tubulin (mean ± SEM).

(C and D) HeLa cells stably expressing GFP-LC3 were treated as in (A) and then left in complete media (basal) or starvation media (HBSS) for 2 hr, then fixed and analyzed on a Cellomics ArrayScan system. Quantification of numbers of GFP-LC3 vesicles per cell in the different conditions is shown in (D) (mean ± SEM).

(E) HeLa cells stably expressing GFP-mRFP-LC3 were treated as in (A) and analyzed on a Cellomics ArrayScan system. Quantification of numbers of autophagic vesicles (AV) or autolysosomes (AL) per cell in the different conditions is shown in the graph (mean ± SEM).

(F and G) Western blot analysis of free ATG12 and ATG5-ATG12 complex levels with anti-HA antibody in HeLa cells transfected with HA-ATG12 and ATG5 and loaded with exogenous lipids. We quantified the ratio of ATG5-ATG12 versus free ATG12 by direct infrared fluorescence detection on an Odyssey Infrared Imaging System to assess ATG5-ATG12 conjugation efficiency (mean ± SEM).

(H) HeLa cells transfected with GFP-PHD3X or GFP-PHD3X Znmut and RFP-LC3 for 16 hr were left in complete media (basal) or starvation media (HBSS) for 1 hr, then fixed and imaged on a confocal microscope. Empty arrowheads indicate PHD3X single-labeled structures, while filled arrowheads indicate PHD3X-LC3- and PHD3X-ATG16-positive structures. Bar, 10 μm.

(I) HeLa cells transfected with GFP-PHD3X and RFP-LC3 for 16 hr were left in HBSS for 1 hr, then fixed and imaged on Elyra superresolution microscope. Final visualization was performed in Volocity 6.3 software using isosurface rendering of selected cropped regions of the data sets. Note that this rendering means that vesicles positive for green and red do not look yellow but have green and red on the surface. Representative cropped regions from different cells are shown. Bar, 1.13 μm. See also Figure S1.
PI(5)P Regulates Autophagosome Biogenesis. (2015)
Figure 2

PI(5)P Synthesized by PIKfyve Regulates Autophagosome Formation

(A) Schematic representation of pathways for PI(5)P and PI(3)P synthesis/turnover and drugs targeting enzymes involved in these pathways.

(B and C) Western blot analysis of LC3-II and tubulin levels and quantification of LC3-II/tubulin ratio in HeLa cells treated with DMSO or increasing concentrations of YM-201636 for 2 hr in the presence or absence of BAF (mean ± SEM).

(D and E) HeLa cells transiently transfected with Strawberry-ATG16L1 were treated with YM-201636 (100 nM, 2 hr) in HBSS, then fixed and stained for endogenous WIPI-2. Bar, 10 μm. (E) Quantification of ATG16L1 and WIPI2 structures per cell, n = 20 cells (mean ± SEM; n = 3 independent experiments; ∗∗∗p < 0.001, t test).

(F) Western blot analysis of free ATG12 and ATG5-ATG12 complex levels with anti-HA antibody in HeLa cells transfected with HA-ATG12 and ATG5 and treated with YM-201636 (100 nM, 2 hr) (mean ± SEM).

(G) HeLa cells transfected with GFP-PHD3X and Strawberry-ATG16L1 for 16 hr were left in HBSS for 1 hr, then fixed and imaged on Elyra superresolution microscope. Final visualization was performed as previously described. Bar, 1.13 μm.

(H and I) Western blot analysis of LC3-II and tubulin levels and quantification of LC3-II/tubulin ratio in cells incubated with YM-201636 (100 nM, 2 hr) and loaded with exogenous PI(5)P for the last 1 hr in the presence or absence of BAF (mean ± SEM). See also Figure S2.