EMBO/soda-nel huggingface dataset:
Spermidine and resveratrol induce autophagy by distinct pathways converging on the acetylproteome. (2011)
Figure 4.

Convergent alterations in the phosphoproteome status after resveratrol and/or spermidine treatment. (A–C) Human colon carcinoma HCT 116 cells were treated for 2 h with vehicle (Co, control), 100-µM resveratrol (Resv), and 100-µM spermidine (Spd), alone or in combination (Resv + Spd). (A) Representative phosphoprotein arrays are shown. (B) Clustering analysis for the effects on protein kinase phosphorylation. (C) Representative immunoblots of selected kinases whose phosphorylation status was unaffected (PRKAA1, RPS6KB1, and acetyl-CoA carboxylase a [ACACA]) or affected by resveratrol or/and spermidine treatment (PTKB, AKT1, MAPK8, and CDKN1B), validating phosphoprotein array data. (D) Human colorectal carcinoma HCT 116 cells were transfected with a GFP-LC3–encoding plasmid, cultured in complete medium for 24 h, and then treated with either vehicle or the indicated dose of resveratrol or spermidine, alone or in combination, for 2 h. Quantitative data. Bars depict the percentages (means ± SD; n = 3; *, P < 0.05) of cells showing the accumulation of GFP-LC3 in puncta (GFP-LC3vac). GAPDH, glyceraldehyde 3-phosphate dehydrogenase.
Spermidine and resveratrol induce autophagy by distinct pathways converging on the acetylproteome. (2011)
Figure 8.

Autophagy can be efficiently regulated by cytoplasmic (de)acetylation reactions. (A and B) HCT 116 cells were transfected with a GFP-LC3–encoding plasmid, cultured in complete medium for 24 h, and enucleated to obtain cytoplasts. The cytoplasts were treated with either vehicle (Co, control), 100-µM resveratrol (Resv), or 100-µM spermidine (Spd) for 4 h. 1-µM rapamycin (Rapa) was used as a positive control. (A) Representative images of cytoplasts indicative of autophagic activity. Arrows indicate GFP-LC3–transfected cytoplasts, whereas insets show nonenucleated transfected cells. (B) Quantitative data. Bars show the percentages of cells or cytoplasts showing the accumulation of GFP-LC3 in puncta (GFP-LC3vac). (C–E) HCT 116 cells were cotransfected with a plasmid for the expression of RFP-LC3 together with an empty vector (pcDNA3) or plasmids encoding wild-type (WT) SIRT1 or a SIRT1 variant with a mutation in the nuclear localization signal fused to GFP (WT GFP-SIRT1 and mtNLS GFP-SIRT1, respectively) for 12 or 24 h, fixed, and analyzed by fluorescence microscopy. (C) Representative images indicative of 24-h autophagic activity. (D) Quantitative data. Bars depict the percentages of cells exhibiting the accumulation of RFP-LC3 in puncta (RFP-LC3vac). (B and D) Means ± SEM; n = 3; *, P < 0.05. (E) Representative immunoblots for 24-h endogenous LC3 lipidation. The asterisk stands for a nonspecific band. GAPDH, glyceraldehyde 3-phosphate dehydrogenase.
Spermidine and resveratrol induce autophagy by distinct pathways converging on the acetylproteome. (2011)
Figure 9.

Low doses of resveratrol and spermidine synergistically induce autophagy in vitro and in vivo. (A) Human colorectal carcinoma HCT 116 cells were transfected with a GFP-LC3–encoding plasmid, cultured in complete medium for 24 h, and then treated with either vehicle (Co, control) or the indicated dose of resveratrol (Resv) or spermidine (Spd), alone or in combination, for 2 h. (top) Quantitative data. Bars depict the percentages (means ± SD; n = 3; **, P < 0.05) of cells showing the accumulation of GFP-LC3 in puncta (GFP-LC3vac). (bottom) Representative immunoblots showing endogenous LC3 lipidation. (B) Representative immunoblots showing endogenous LC3 lipidation in the presence of bafilomycin A1 (BafA1). (C–E) Transgenic C57BL/6 mice expressing a GFP-LC3 fusion protein were injected with resveratrol and spermidine at the indicated concentrations. 3 h later, mice were killed, and tissues were processed for immunofluorescence microscopy determinations of GFP-LC3vac. (C) Representative images. (D) Quantitative results. Bars represent the percentages (means ± SEM; n = 3; *, P < 0.05; and **, P < 0.01 as compared with the same tissue from untreated animals) of cells exhibiting GFP-LC3vac. (E) Representative immunoblots showing endogenous LC3 lipidation and p62 protein content in WT C57BL/6 mouse tissues. GAPDH, glyceraldehyde 3-phosphate dehydrogenase.
hnRNPLL controls pluripotency exit of embryonic stem cells by modulating alternative splicing of Tbx3 and Bptf (2020)
Figure 2

Figure 2. Seven RBPs are required for ES cells exit-from-pluripotency.

The research strategy to dissect the role of RBPs in regulating exit-from-pluripotency. Firstly, we constructed individual RBP KO ES cells and examined their ability to exit from pluripotency through forming EB and random differentiation by LIF withdrawal assay.

AP staining of 11 RBP-KO ES cell lines maintained under ES cells culture conditions. Scale bar, 200 μm.

EB formation from WT and RBP-KO ES cell lines. WT ES cell-derived EBs formed cystic structures by day 8 of differentiation; most of RBP-KO ES cell-derived EBs were more compact. Scale bar, 600 μm.

Time-course analysis of Oct4 expression and three lineage differentiation markers (Hand1, Brachyury and FGF5) during EB formation. It was quantified by qPCR, the relative expression of genes was calculated by the double delta Ct (ΔΔCt) method, normalized to the expression value at day 0. The relative expression data was represented as the heatmap.

AP staining of WT and 11 RBP-KO ES cell lines after LIF withdrawal. Scale bar, 200μm.

RBP ranking based on ES cell abilities to self-renewal (determined by colony formation (±LIF) and Oct4 expression) and exit-from-pluripotency (determined by AP staining, EB formation and marker genes expression) in the context of RBP KO. The expression value of Oct4, FGF5, Hand1 and Brachyury was represented as the relative level of these genes compared to their expressions in WT ES cells. The quantitation of other phenotypes, such as colony formation, AP staining and EB formation was manually defined.
hnRNPLL controls pluripotency exit of embryonic stem cells by modulating alternative splicing of Tbx3 and Bptf (2020)
Figure 3

Figure 3. hnRNPLL disruption or overexpression influences exit-from-pluripotency of ES cells.

Immunofluorescence staining of Oct4 and hnRNPLL in WT and hnRNPLL-KO ES cells from two clones (H8 and G6). hnRNPLL was not expressed in either of the hnRNPLL-KO clones. DAPI stained the nuclei. Scale bar, 100 μm.

EB formation from WT and the two hnRNPLL-KO ES cell clones. WT ES cell-derived EBs formed cystic structures by day 8 of differentiation; While both hnRNPLL-KO ES cell clones-derived EBs were more compact. Scale bar, 200 μm.

AP staining of WT and both hnRNPLL-KO ES cell clones after LIF withdrawal. Scale bar, 200 μm.

Cumulative distribution showing the relative expression of WT-differentially expressed genes in hnRNPLL-KO ES cells during EB differentiation. Red lines and green lines represent the up- and down-regulated genes during EB differentiation of WT ES cells, while the black line stands for all expressed genes in WT and hnRNPLL-KO ES cells. Kolmogorov-Smirnov test was used to determine the significance. The relative expression ratio was log2-transformed before plotting.

Heatmap of key pluripotency and differentiation associated gene expression during EB formation from WT and hnRNPLL-KO clones (H8 and G6).

Hematoxylin and eosin labeling and Oct4 immunohistochemical staining of teratomas derived from WT and hnRNPLL-KO ES cells (left). Representative Oct4-positive regions are shown. The percentage of Oct4 positive region (%) is plotted (right). The data represent the means ± SD, (n=3, from biological repeats). *P＜0.05; ***P＜0.001, T-test was used to determine the significance. Scale bar, 100 μm.

AP staining of WT and hnRNPLL-overexpression ES cell lines maintained under ES cells culture conditions and colony formation assay. Scale bar, 200 μm. The data represent the means ± SD (n=3 biological repeats).

Relative expression of Oct4 and Sox2 after hnRNPLL overexpression. The data represent the means ± SD (n=3, from biological repeats). ***P＜0.001. T-test was used to determine the significance.
hnRNPLL controls pluripotency exit of embryonic stem cells by modulating alternative splicing of Tbx3 and Bptf (2020)
Figure 4

Figure 4. hnRNPLL-deficient mice are developmental impaired and growth retarded.

Schematic of hnRNPLL knockout mouse, in which its exon two is deleted.

Birth rate of the different hnRNPLL genotypes, the birth rate of hnRNPLL-/- mice was significantly lower than that of WT hnRNPLL+/+ mice. The data represent the means ± SD. T-test was used to determine the significance.

Representative images of hnRNPLL+/+ and hnRNPLL-/- littermates at 3 weeks.

Body weight growth curve of mice at different postnatal days. The data represent the means ± SD (n=5-10). ***P＜0.001. T-test was used to determine the significance.

Survival curve analysis for hnRNPLL+/+, hnRNPLL+/-and hnRNPLL-/-mice (ten mice in hnRNPLL+/+/+/-group and seven mice in hnRNPLL-/-group).

Genotypes and representative images of hnRNPLL+/+ embryo and retarded hnRNPLL-/- embryos at E11.5 which generated from hnRNPLL+/- intercrosses. Scale bar, 1mm.
hnRNPLL controls pluripotency exit of embryonic stem cells by modulating alternative splicing of Tbx3 and Bptf (2020)
Figure 5

Figure 5. hnRNPLL regulates RNA alternative splicing in ES cells.

Silver nitrate-stained NuPAGE gel of hnRNPLL interacting proteins. Red star indicate hnRNPLL specific interacting proteins compared to IgG.

Mass spectrum identification of the hnRNPLL interactome in ES cells. RNA splicing associated proteins are well enriched by hnRNPLL IP. The enrichment score was log2-transformed before plotting.

Western blot validation the interactions between hnRNPLL and the indicated splicing associated proteins.

Cumulative distribution of PSI with exon skipping events between WT and hnRNPLL-KO ES cells. Kolmogorov-Smirnov test was used to determine the significance.

Volcano plot of differential exon skipping events between WT and hnRNPLL-KO ES cells. Indicated AS events were labeled in purple (up-regulated) and green (down-regulated) with gene names.

The regional distribution of differential exon skipping events between WT and hnRNPLL-KO ES cells.

Western blot analysis of hnRNPLL protein during EB formation of WT ES cells at indicated days.

Venn plot showed the divergent exon skipping events between EB differentiation and hnRNPLL deletion. 38 exon skipping events that were inhibited by hnRNPLL but increased during EB differentiation, and 25 events that showed the opposite profile.

Heatmap of the 38 and 25 divergent exon skipping events in Figure 5I. * indicates the different exon skipping events of the same gene.

RT-PCR validation of partial divergent exon skipping events shown in Figure 5J. Red boxes represent ESC preferred exons, blue boxes indicate EB preferred exons.
hnRNPLL controls pluripotency exit of embryonic stem cells by modulating alternative splicing of Tbx3 and Bptf (2020)
Figure 6

Figure 6. hnRNPLL promotes Bptf and Tbx3 exon skipping to facilitate ES cells exit from pluripotency.

RIP-PCR validation of the transcripts that interact with hnRNPLL. The left panel shows the results of western blots verification of hnRNPLL Immunoprecipitation efficiency. The data represent the means ± SD (n=3, from biological repeats). ***P＜0.001. T-test was used to determine the significance.

The CLIP-PCR pipeline. Specifically, hnRNPLL and its target RNA are cross-linked by UV, then enriched by immunoprecipitation. 3'linker is added to the RNA tag, then hnRNPLL-RNA complex is purified by SDS-PAGE. After protease K digestion, RNA is reversed into cDNA. qRT-PCR is performed to detect cross-linked RNA.

Purification of hnRNPLL-RNA complex and detection of the RNA targets in the complex by PCR. The left panel shows the membrane position labeled as the yellow square which was cut to extract the hnRNPLL-RNA complex. The bands around 70KD indicate hnRNPLL proteins, the band around 50KD indicate the heavy chain of antibodies used in Immunoprecipitation. The right plot indicates the enrichment of these RNA targets in the hnRNPLL-RNA complex. The data represent the means ± SD (n=3, from biological repeats). *P＜0.05; ***P＜0.001. T-test was used to determine the significance.

Bptf and Tbx3 alternative splicing model: Bptf exon24a and Tbx3 exon2a are alternatively spliced. The predicted hnRNPLL-binding motif sites were marked in purple. The red and blue lines represented include and excluded isoforms of the AS events, respectively.

Detection of Bptf and Tbx3 exon skipping events during EB formation in WT and hnRNPLL-KO ES cells (left) and in hnRNPLL-overexpression ES cells under ES cells culture conditions (right) by RT-PCR.OE, hnRNPLL overexpression.

RT-PCR validation of Bptf exon24a and Tbx3 exon2a deletion in WT ES cells.

AP staining of Bptf E24a KO or Tbx3 E2a KO ES cells maintained under normal ES cells culture conditions (upper). Scale bar, 200 μm. Colony formation assay of Bptf E24a KO or Tbx3 E2a KO ES cells under ES cells culture condition (lower left). The data represent the means ± SD (n=3, from biological repeats). ***P＜0.001. Colony diameter of Tbx3 E2a KO ES cells (lower right). The data represent the means ± SD (n＞80). ***P＜0.001. T-test was used to determine the significance.

Relative expression of Oct4 and Rex1, determined by qRT-PCR. The data represent the means ± SD (n=3, from biological repeats). ***P＜0.001. T-test was used to determine the significance.

.
hnRNPLL controls pluripotency exit of embryonic stem cells by modulating alternative splicing of Tbx3 and Bptf (2020)
Figure 7

Figure 7. Bptf and Tbx3 alternative exon loss can rescue defective differentiation in hnRNPLL-KO ES cells.

RT-PCR validation of Bptf and Tbx3 alternative exon deletion, separately in hnRNPLL-KO H8 cells (upper) and simultaneously in hnRNPLL-KO H8 cells (lower).

AP staining of H8-Bptf or H8-Tbx3 exon-KO ES cells after LIF withdrawal. hnRNPLL-H8, hnRNPLL-KO ES cells; H8-Bptf KO, alternative Bptf exon was deleted in hnRNPLL-KO H8 cells; H8-Tbx3 KO, alternative Tbx3 exon was deleted in hnRNPLL-KO H8 cells; H8-Bptf/Tbx3 KO, alternative Bptf or Tbx3 exons were simultaneously deleted in hnRNPLL-KO H8 cells. Scale bar, 200 μm.

Colony formation assay of H8-Bptf or H8-Tbx3 exon-KO ES cells after LIF withdrawal.
The data represent the means± SD (n=3, from biological repeats).

Hematoxylin and eosin labeling of teratomas derived from WT and H8-Bptf or H8-Tbx3 exon-KO ES cells. The blue triangle represent ectoderm, the dark one represent mesoderm and the green one represent endoderm. Scale bar, 100 μm.

Oct4 immunohistochemical staining of teratomas derived from WT and H8-Bptf or H8-Tbx3 exon-KO ES cells. Representative Oct4-positive regions are shown (left). Scale bar, 100 µm. The percentage Oct4-positive region (%) is shown (right). The data represent the means± SD (n=3 biological repeats). **P＜0.01; ns, not significant. T-test was used to determine the significance.

Schematic representation of hnRNPLL regulating the AS of crucial genes that have an important role in mediating exit-from-pluripotency.

Expanded View Figure legends
Huntingtin phosphorylation governs BDNF homeostasis and improves the phenotype of Mecp2 KO mice (2019)
Figure 1

Fig. 1. Huntingtin phosphorylation rescues BDNF transport in Mecp2-deficient axons.

(A) Microchamber that allows the isolation of axons within a corticostriatal network and live-cell imaging of axonal BDNF-mCherry transduced into mouse primary cortical neurons.

(B) Representative kymographs and quantification of anterograde, retrograde, mean velocity, and linear flow rate of BDNF-mCherry-containing vesicles in cortical neurons transfected with siMecp2 (n= 753 vesicles/ 81 axons) or siControl (siCtl) (n= 894 vesicles/ 94 axons), inducing significant silencing of Mecp2 (3 independent experiments; unpaired t-test).

(C) Quantification of anterograde, retrograde, mean velocity, and linear flow rate of BDNF-mCherry trafficking into cortical neurons obtained from WT (n= 753 vesicles/ 81 axons), HTTSA (n= 812 vesicles/ 81 axons), or HTTSD (n= 787 vesicles/ 82 axons) homozygous knock-in mice in which S421 of HTT was replaced by an alanine (HTTS421A/S421A or HTTSA), mimicking the absence of phosphorylation, or by an aspartic acid (HTTS421D/S421D or HTTSD), mimicking constitutive phosphorylation (3 independent experiments; one-way ANOVA test with Tukey's multiple comparisons).

(D) Cell viability in WT (n=3), HTTSA (n=1), or HTTSD (n=2) neurons measured by MTT assay. No differences were observed between the different groups (3 independent experiments; one-way ANOVA Kruskal-Wallis test, p=0.5701)

(E) Kymographs and quantification of anterograde, retrograde, mean velocity, and linear flow rate of BDNF-mCherry axonal trafficking into WT (n= 894 vesicles/ 94 axons), HTTSA (n= 812 vesicles/ 82 axons), or HTTSD (n=787 vesicles/ 94 axons) cortical neurons transfected with siCtl or siMecp2, which significantly silenced Mecp2 (3 independent experiments; one-way ANOVA with Tukey's multiple comparisons).

(F) Cell viability in WT, HTTSA, or HTTSD neurons transfected with siCtl or siMecp2 measured by MTT assay (WT siCtl: n=3; WT siMecp2: n=3; HTTSD siMecp2: n=3; HTTSA siMecp2: n=2). No differences were observed between the different groups (3 independent experiments; one-way ANOVA Kruskal-Wallis test, p=0.5701)

Data information: Scale bars = 20 µm. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns = not significant. Data are presented as the means ± SEM.
Huntingtin phosphorylation governs BDNF homeostasis and improves the phenotype of Mecp2 KO mice (2019)
Figure 2

Fig. 2. The S421D mutation improves BDNF corticostriatal transport in vivo, the motor and autonomic functions of Mecp2-deficient mice and increases their lifespan.

(A) Left: The ratio between the striatal and the cortical BDNF determined by quantifying the level of BDNF proteins at the cortex and the striatum of 55 day-old WT (n=8), Mecp2 KO (n=6), Mecp2 KO/HTTSD (mimicking the absence of phosphorylation) (n=4) and Mecp2 KO/HTTSA mice (mimicking constitutive phosphorylation) (n=5). Since striatal BDNF depends only on BDNF transport from the cortex, this ratio reflects BDNF transport through corticostriatal pathway (Mann Whitney test). Right: Quantitative analysis of phospho TrkB protein level in striatum of KO WT mice and KO HTTSD mice by immunoblotting. The relative expression levels of phospho TrkB were normalized against GAPDH and are presented as the ratio. (n = 6 mice per group) (Mann Whitney test) Middle: Quantitative analysis of PSD-95 protein level in striatum of Mecp2 KO/HTT WT mice and KO/HTTSD mice by immunoblotting. The relative expression levels of PSD-95 were normalized against GAPDH and are presented as the ratio. (n = 6 mice per group) (Mann Whitney test)

(B) Left: We investigated the behavior of Mecp2 KO mice at 35, 45, and 55 days of age and assessed their survival. Right: Mecp2 KO/HTTSD mice (n = 30) and WT mice (n = 17) had a significantly longer lifespan than KO (n=22) or KO/HTTSA mice (n = 21) (Kaplan-Meier survival test).

(C) Body weight of 10 WT, 24 KO, 32 KO/HTTSD, and 24 KO/HTTSA mice at P30 and P50 (one-way ANOVA with Tukey's comparison).

(D) Frequency of apnea of 9 WT, 10 KO, 14 KO/HTTSD, and 9 KO/HTTSA mice at P35 and P55. (Kruskal-Wallis test with Dunn's comparison).

(E) Mecp2 KO/HTTSD mice (n=19) performed as well as WT (n=17) at P35 on the accelerating rotarod test and continued to outperform Mecp2 KO (n=16) and Mecp2 KO/ HTTSA (n=13) at P55 (one-way ANOVA with Fisher's LSD test).

(F) Time before the onset of spontaneous locomotor activity of 17 WT, 20 KO, 19 KO/HTTSD, and 17 KO/HTTSA mice at P55. (Kruskal-Wallis test with Dunn's comparison).

Data information: Data are presented as the means ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns = not significant.
Huntingtin phosphorylation governs BDNF homeostasis and improves the phenotype of Mecp2 KO mice (2019)
Figure 3

Fig. 3. FK506 restores BDNF axonal transport in Mecp2-silenced axons.

(A) Western blot analysis of DIV 5 cortical neurons transfected with siMecp2 or siControl (siCtl) and treated with 1 µM FK506 or vehicle for 1 h, and quantification of pS421 HTT (n=9 per group). Data are presented as means ± SEM, *p < 0.05, **p < 0.01, ***p < 0.001, Mann-Whitney test.

(B) Representative kymographs showing axonal trafficking of BDNF-mCherry-containing vesicles in cortical neurons transfected with siMecp2 or siControl (siCtl) and treated with 1 µM FK506 or vehicle for 1 h. Scale bar = 20 µm.

(C) Quantification of anterograde, retrograde, mean velocity, linear flow rate and number of BDNF-mCherry-containing vesicles from the data in (A) (siMecp2+Vehicle: n=801 vesicles/95 axons; siMecp2+FK506: n=1020 vesicles/116 axons; siCtl+Vehicle: n=780 vesicles/83 axons; siCtl+FK506: n=1029 vesicles/102 axons). Data are presented as the mean ± SEM of at least three independent experiments (one-way ANOVA with Tukey's comparison). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns = not significant.
Huntingtin phosphorylation governs BDNF homeostasis and improves the phenotype of Mecp2 KO mice (2019)
Figure 4

Fig. 4. Calcineurin inhibition by FK506 in Mecp2-deficient mice improves motor and autonomic functions and extends lifespan through huntingtin-dependent phosphorylation.

(A) Western blot of HTT S421 phosphorylation. We treated 30-day-old Mecp2 KO mice intraperitoneally with FK506 (5 mg/kg) or vehicle and analyzed brain extracts for endogenous HTT phosphorylation by western blotting, 2 h after administration, using an anti-phospho-HTT-S421 specific antibody. The D7F7 antibody recognizes total HTT. The relative protein level of phospho HTT was normalized on total HTT protein level and are presented as the ratio (KO FK506 n = 4, KO Vehicle n = 4). Data are presented as means ± SEM, *p < 0.05, Mann Whitney test.)

(B-F) We treated 30-day-old Mecp2 KO mice (n=10) and Mecp2 KO/HTTSA mice (n=10) with 5mg/kg FK506 three times a week by intraperitoneal injection and assessed them in various behavioral tests. (B) FK506-treated KO mice lived longer than vehicle-treated KO mice and FK506-treated KO HTTSA mice (Kaplan-Meier survival test). (C) Body weight of FK506-treated KO, FK506-treated KO HTTSA and vehicle-treated KO mice at P35 and P55 (Mann-Whitney test). (D) Frequency of apnea of FK506-treated KO, FK506-treated KO HTTSA and vehicle-treated KO mice at P35 and P55 (Mann-Whitney test). (E) Motor coordination of FK506-treated KO, FK506-treated KO HTTSA and vehicle-treated KO mice on the accelerating rotarod test at P30 and P50 (Mann-Whitney test). (F) Forelimb strength of FK506-treated KO, FK506-treated KO HTTSA and vehicle-treated KO mice assessed by the grip strength test at P40 and P60 (Mann-Whitney test).

Data information: *p < 0.05, **p < 0.01, ***p < 0.001, ns = not significant. Data are means ± SEM.

Expanded View Figure Legends