EMBO/soda-nel huggingface dataset:
TRAF6 directs FOXP3 localization and facilitates regulatory T cell function through K63-linked ubiquitination (2019)
Figure 5

Figure 5. Ablation of the K63-type ubiquitination site in Foxp3 disrupts its cellular distribution, expression, and regulatory function.

A Impact of K63-ubiquitination loss on FOXP3's gene silencing capacity. Jurkat T cells transfected with a wild type Foxp3 expression vector, one encoding the K262R mutant, or an empty (control) vector also received a duel luciferase reporter construct where luciferase expression was under the control of the Il2 promoter. After activation with PMA and ionomycin for 8 hours, luciferase activity was assayed.

B IL-2 production in the presence of wild type and K63-ubiquitination resistant FOXP3. Naïve CD4+ T cells isolated from Traf6fl/flCD4Cre+ or WT mice were activated overnight by anti-CD3/CD28 antibodies, then transduced by retroviruses carrying normal Foxp3, the K262R mutant, or an empty vector. Ires-GFP in the retroviral vector serves as an internal control. GFP+ cells were sorted out 48 hours post-transduction. The sorted cells were cultured for one additional day. Culture supernatants were collected for measuring the levels of IL-2 by ELISA.

C Cellular distribution of wild type and K262R FOXP3. Hela cells were transfected with expression vectors encoding either wild type FOXP3 or the K262R mutant. The relative overlap of FOXP3 protein signal and DAPI-stained nuclei was observed by fluorescence microscopy. (blue: DAPI, green: FOXP3, Scale bars: 50 μm).

D Immunostaining of FOXP3 in Traf6fl/flCD4Cre+ and wild type (Traf6fl/flCD4Cre-) derived suspensions of lymph node and spleen cells. (blue: DAPI, green: FOXP3, Scale bars: 50 μm).

E Quantification of FOXP3 distribution in murine Tregs. The degree of FOXP3-nuclear coIocalization in CD4+/FOXP3+ cells isolated from the lymph nodes and spleens of Traf6fl/flCD4Cre+ and wild type (Traf6fl/flCD4Cre-) mice was determined by Imagestream analysis, and the frequencies of cells with either a low or high probability of nuclear FOXP3 distribution are shown (red: nuclear stain, green:FOXP3, Scale bars: 10 um).

Data information: A, B and E (right panels) represents mean results from 3 experiments +/- SEM. *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001; ns, no significance (unpaired t test). C and D show representative staining results, while E (left panels) depict exemplars for high and low nuclear overlap from Imagestream analysis.
TRAF6 directs FOXP3 localization and facilitates regulatory T cell function through K63-linked ubiquitination (2019)
Figure 6

Figure 6. Genetic prevention of ubiquitination at Foxp3 residue K262 disrupts immune suppression in vitro and in vivo.

A, B The in vitro suppressive potency of wild type- and K262R Foxp3-expressing T cells. Naïve CD4+ T cells were purified from Thy1.1+ BALB/c mice and subjected to retroviral transduction to express either wild type FOXP3 or a K262R mutant resistant to ubiquitination that lysine residue 262. These engineered "Tregs" were then co-cultured with naïve responder CD4+ T cells stained with CFSE at the indicated ratios and activated with anti-CD3/CD28 antibodies. Dilution of CFSE signal by the responder populations (i.e. proliferation) was assessed by flow cytometry.

C In vivo suppression of colitis by wild type- and K262R mutant-Foxp3 expressing T cells. 2x105 naïve CD4+ transductants from A were mixed with 1x106 colitogenic naïve CD4+ T cells from wild type mice and transferred i.v. into Rag2-/- mice. Rag2-/- mice receiving no cell transfers, naïve CD4+ T cells alone, or purified wild type Tregs served as controls (n= 6/group). Recipient mouse body weights were monitored weekly for each group.

D, E After 8 weeks, colons were excised, fixed in buffered formalin, and processed for H and E staining (D) and histopathology scoring (E). Scale bars shown in 1 mm).

Data information: Panels B, C, and E represent mean values +/-SEM from 2 experiments. **P<0.01; ***P<0.001; ns, no significance (unpaired t test). Panel A and the micrographs depicted in D are representative findings.

Expanded View Figure Legends:
Therapeutic Correction of ApoER2 Splicing in Alzheimer’s Disease Mice Using Antisense Oligonucleotides (2016)
Figure 2

Figure 2. Antisense oligonucleotides improve human ApoER2 exon 19 inclusion.

A. Gene structure of ApoER2 exons 18-20 and the resulting RNA and protein products. Boxes are exons and lines represent introns. Diagonal lines represent splicing.

B Top, diagram of ASOs tested below, mapped to their position of complementarity on ApoER2. Bottom, RT-PCR of RNA isolated from HeLa cells transfected with the indicated ASO at a final concentration of 80 nM. RNA spliced forms are labeled. Quantification of the percentage inclusion is shown below the image and calculated as: Inclusion (%)=[exon 19 included/(exon 19 included+exon 19 skipped)] x 100.

C Sequence and ApoER2 target region of most active ASOs from (B). Exonic and intronic sequences are in capital and lower-case letters, respectively. Conserved nucleotides between human and mouse ApoER2 are shown in black, non-conserved nucleotides are red. Predicted, conserved binding sites for splicing factors are labeled as intronic splicing silencers (ISS1 and ISS2).

D Top, RT-PCR analysis of ApoER2 exon 19 splicing in HeLa cells depleted of SRSF1 using an SRSF1-specific siRNA. Control is a non-specific siRNA. Bottom, Immunoblot analysis of SRSF1 from Hela lysates depleted of SRSF1. β-actin is included as a control.

E Quantification of exon 19 splicing in (D). Error bars show s.e.m. P-value was determined using Student’s two-tailed t-test.

F Top, RT-PCR analysis of exon 19 splicing in a primary mouse kidney cell line depleted of SRSF1 by RNAi. Control is a non-specific siRNA. Bottom, Immunoblot analysis of SRSF1 from Hela lysates depleted of SRSF1. β-actin is included as a control.

G Quantification of exon 19 splicing in E. Error bars show s.e.m. P-value was calculated using Student’s two-tailed t-test.
Therapeutic Correction of ApoER2 Splicing in Alzheimer’s Disease Mice Using Antisense Oligonucleotides (2016)
Figure 3

Figure 3. Antisense oligonucleotides improve mouse ApoER2 exon 19 inclusion.

A Top, ASOs mapped onto the mouse ApoER2 exon 19 gene region. Bottom, RT-PCR of RNA isolated from mouse cells transfected with different ASOs. Quantification of percent exon 19 inclusion is shown below the gel image.

B ASO improves exon 19 inclusion in a dose-responsive manner. RT-PCR analysis of RNA from mouse cells transfected with increasing doses of ASO-15. The percent inclusion of exon 19 is shown below the gel image. The half-maximal effective concentration (EC50) was calculated using GraphPad Prism version 6.0 (GraphPad Software, San Diego, CA) after fitting the data using nonlinear regression with normalized response and variable slope.

C Sequence and ApoER2 target region of most active mouse ASOs from A. Exonic and intronic sequences are in capital and lower-case letters, respectively.

D RT-PCR analysis of RNA isolated from the hippocampus of mice treated as adults by ICV injection of indicated ASOs. C refers to a non-specific control ASO.

E Quantitation of ApoER2 exon 19 inclusion from D (mean s.e.m., one-way ANOVA with Bonferroni multiple comparison test.
Therapeutic Correction of ApoER2 Splicing in Alzheimer’s Disease Mice Using Antisense Oligonucleotides (2016)
Figure 4

Figure 4. ASO-21 improves ApoER2 exon 19 inclusion in vivo four-months post-treatment.

A RT-PCR analysis of ApoER2 exon 19 splicing in RNA isolated from hippocampus of 4 month old AD and WT mice treated with ASO-21 or ASO-C at P1-2.

B Quantitation of exon 19 splicing. (mean s.e.m.; Kruskal-Wallis test with Dunn’s multiple comparison test.

C Immunoblot for exon 19 containing ApoER2 (top) and total ApoER2 (bottom). β-actin was used as loading control.

D Quantification of ApoER2 protein isoforms shown in C (mean s.e.m., student’s t-test).
Cellular and molecular determinants of all-trans retinoic acid sensitivity in breast cancer: Luminal phenotype and RARα expression. (2015)
Figure 3

ATRA-dependent anti-tumor activity in short-term cultures of primary breast tumors

Tissue slices deriving from surgical specimens were challenged with vehicle (DMSO) or ATRA (0.1 μM) for 48 h.

The panel illustrates examples of the immuno-histochemical data obtained in four representative cases: (i) Luminal-A (Lum A); (ii) Luminal-B (Lum B); (iii) Her2+ with RARA coamplification (Her2+/RARA+) and (iv) triple negative (TN).

The percentage of Ki67-positive tumor cells in the 45 samples considered are illustrated by the bar graphs. Each value represents the mean ± SE of at least five separate fields for each experimental sample. *Significantly lower than the corresponding vehicle-treated control (P-value < 0.05, Student's t-test). **Significantly lower than the corresponding vehicle-treated control (P-value < 0.01, Student's t-test).
Cellular and molecular determinants of all-trans retinoic acid sensitivity in breast cancer: Luminal phenotype and RARα expression. (2015)
Figure 4

Associations between components of the retinoid signaling pathway and the phenotype in breast cancer cell lines

The figure illustrates the associations between the indicated retinoid receptors/binding proteins and the Luminal versus Basal phenotype (left panels), ER positivity versus ER negativity (middle panels) as well as HER2 positivity versus HER2 negativity (right panels). The gene-expression microarray and RNA-seq data refer to 42 and 40 breast cancer cell lines, respectively. The P-values of the indicated comparisons after Student's t-test are shown in red. L (red) = Luminal cell lines; B (blue) = Basal cell lines; ER+ (red) = ER-positive cell lines; ER− (red) = ER-negative cell lines; ER− (blue) = ER-negative Luminal cell lines; H+ (red) = HER2-positive cell lines; H− (red) = HER2-negative cell lines; H+ (blue) = HER2-positive Luminal cell lines; H− (blue) = HER2-negative Luminal cell lines. fpkm = fragments per kilobase of exon per million fragments mapped.
Cellular and molecular determinants of all-trans retinoic acid sensitivity in breast cancer: Luminal phenotype and RARα expression. (2015)
Figure 6

Associations between components of the retinoid signaling pathway and ATRA sensitivity

The gene-expression microarray and RNA-seq data associated with 40 of the breast cancer cell lines were used for the analyses. The panels illustrate the associations of RARα and PPARβ/δ with ATRA sensitivity. The left panels show the basal average levels of the indicated transcript in the cell lines belonging to the T1 and T3 groups (13 cell lines in each of the T1 and T3 groups) defined by ascending ATRA scores. The intermediate and right panels indicate the same results after stratification for the Luminal (microarray and RNA-seq data = 7 cell lines in each of the T1 and T3 groups) and the Basal (microarray and RNA-seq data = 7 cell lines in each of the T1 and T3 groups) phenotype, respectively. fpkm = fragments per kilobase of exon per million fragments mapped.

The basal expression levels of the indicated RAR-isoform variants were determined with the use of specific Taqman assays. The results are associated with ATRA sensitivity before (TOTAL) and after stratification of the cell lines for the Luminal and Basal phenotype as in (A).

Data information: Significant P-values (Student's t-test) are indicated in red.
Cellular and molecular determinants of all-trans retinoic acid sensitivity in breast cancer: Luminal phenotype and RARα expression. (2015)
Figure 7

Basal levels of RARα, RARβ, and RARγ mRNA splicing variants in mammary tumors

Total RNA was extracted from the tissue slices deriving from the surgical specimens of breast cancer patients used in Fig3 before any treatment with DMSO or ATRA. RNA was subjected to RT–PCR analysis to determine the basal expression of the indicated RAR splicing variants.

Each value represents the mean ± SD of two replicate measurements. The table shows the statistical significance of the indicated comparisons. *Significantly different (P-value < 0.05, Student's t-test). **Significantly different (P-value < 0.01, Student's t-test).

The plots illustrate the average expression levels of the indicated mRNAs (mean ± SD of two replicates) in tumor samples classified as ATRA-sensitive (Sens) and ATRA-resistant (Res) according to the response of Ki67. *Significantly different (P-value < 0.05, Student's t-test).
Cellular and molecular determinants of all-trans retinoic acid sensitivity in breast cancer: Luminal phenotype and RARα expression. (2015)
Figure 8

RARα protein and ATRA sensitivity in breast cancer cell lines

Total proteins were extracted from logarithmically growing cell lines and subjected to Western blot analysis.

The panel illustrates representative Western blots for a number of cell lines. The blots were sequentially developed with RARα-specific and control tubulin antibodies. The positions of the RARα and tubulin bands (right) along with the position of a relevant molecular weight marker (left) are indicated. To normalize the Western blot signals in different gels, the same preparation of RARα-transfected COS-7 cell extracts (COS-7) and MCF7 extracts was loaded in each gel. The blots are representative of at least two independent experiments providing similar results.

The quantitative results obtained after densitometric analysis of the RARα bands are plotted against the ATRA scores. Cell lines are grouped according to the ATRA score (A–D groups and T1–T3 tertiles).

Left: The upper graph indicates the averages expression levels of the RARα protein in T1–T3 cell lines. The middle and lower graphs indicate the average expression levels after stratification of the cell lines for the Luminal (six cell lines in each of the T1 and T3 groups) and the Basal (six cell lines in each of the T1 and T3 groups) phenotype, respectively. Right: The levels of the RARα protein in Basal and Luminal cell lines (upper graph), in ER+, ER−, and Luminal ER− cell lines (middle graph) and the indicated classes of HER2+ and HER2− cell lines (lower graph) are shown. *Significantly different (P-value < 0.05, Student's t-test). **Significantly different (P-value < 0.01, Student's t-test).

The plots show the correlation curves between the levels of the indicated RARα-variant transcripts and the RARα protein.
Cellular and molecular determinants of all-trans retinoic acid sensitivity in breast cancer: Luminal phenotype and RARα expression. (2015)
Figure 9

Effects of RAR agonists on the growth of Luminal and Basal breast cancer cell lines

The indicated Luminal and Basal cell lines were challenged with increasing concentrations of ATRA, the RARα agonist, AM580, the RARβ agonist, UVI2003, and the RARγ agonist, BMS961, for 6 days. The complement of RAR-variant transcripts expressed in each cell line is shown in the left bar graphs (mean ± SD of two replicate measurements). The growth curves (sulforhodamine assay) of the cell lines are illustrated by the right linear plots. The results are expressed in % values relative to the corresponding control dishes treated with vehicle alone (right graphs). Each result is the mean ± SD of five replicate wells. ATRA sc = ATRA score.
Cellular and molecular determinants of all-trans retinoic acid sensitivity in breast cancer: Luminal phenotype and RARα expression. (2015)
Figure 10

Over-expression of RARα1/3 in ATRA-resistant MDA-MB453 cells and silencing of RARα1/3 in ATRA-sensitive SKBR3 cells

A RARα1/3 plasmid construct and the corresponding control void vector were stably transfected into ATRA-resistant MDA-MB453 cells. Two cell clones over-expressing RARα (RARA-C5 and RARA-C7) and two appropriate control clones (Vect-C1 and Vect-C2) were isolated. A RARα3 shRNA plasmid construct and the corresponding void vector were stably transfected into ATRA-sensitive SKBR3 cells. After selection, two cell clones silenced for RARα3 (RARA-sh18 and RARA-sh19) and two appropriate control clones (Vect-C6 and Vect-C8) were isolated.

A, D The indicated clones and the parental cell line (WT) were transiently transfected with the RARE-DR5-Luc retinoid reporter construct and the level of luciferase activity was measured 24 h after treatment with vehicle (DMSO) and ATRA (100 nM), as illustrated in the upper bar graph. Each value is the mean ± SD of three replicate cultures. The levels of the RARα protein measured in the indicated clones by Western blot analysis is shown under the bar graph. To demonstrate that similar levels of total proteins were loaded in each lane, the β-actin band signal obtained after re-blotting of the gel is shown. FI = Fluorescence intensity.

B, E The panels illustrate the growth curves of the indicated MDA-MB453 and SKBR3 clones and the SKBR3 parental cell lines (WT) measured with the sulforhodamine assay.

C The bar graphs illustrate the effect of increasing concentrations of ATRA on the growth of the indicated MDA-MB453 clones and the parental cell line. The cell lines were challenged with vehicle (DMSO) or ATRA for 3, 6, and 9 days prior to the sulforhodamine assay. OD = optical density at 540 nm. Each value is the mean ± SD of five replicate culture wells.

F The graphs illustrate the effect of increasing concentrations of ATRA on the growth of the indicated SKBR3 clones and the parental cell line. The cell lines were challenged with vehicle (DMSO) or ATRA for 3 and 6 days prior to the sulforhodamine assay. Each value is the mean ± SD of five replicate culture wells.

G The panel illustrates the effects exerted by ATRA (0.1 μM) on random cell motility of the indicated MDA-MB453 and SKBR3 clones. The results are representative of two independent experiments. Each value is the mean ± SE of the motility of at least 60 cells.

Data information: * and **, significantly different from the corresponding vehicle-treated control (*P-value < 0.05, **P-value < 0.01, Student's t-test).
Exercise-induced α-ketoglutaric acid stimulates muscle hypertrophy and fat loss
through OXGR1-dependent adrenal activation (2020)
Figure 3

Figure 3. AKG increases fat thermogenesis and lipolysis

(A-B). Oxygen consumption in male C57BL/6 mice after 11 weeks of AKG supplementation (n = 8 per group).

(C). Body temperature of male mice after 11 weeks of AKG supplementation (n = 9 per group).

(D-E). Representative images (D) and quantification (E) of BAT thermogenesis induced by 6 hr cold exposure at 4℃ in male mice supplemented with AKG for 11 weeks (n = 9 per group).

(F-G). The mRNA expression of thermogenic genes (F) and Immunoblots and quantification (G) of UCP1 protein in BAT of male mice after 11 weeks of AKG supplementation (n = 3-6 per group).

(H-I). DAB staining (H) and quantification (I) of UCP1 in BAT of male mice supplemented with AKG for 11 weeks (n = 9 per group).

(J). Serum levels of NEFA in male mice supplemented with AKG for 11 weeks (n = 9 per group).

(K). The mRNA expression of CD137, CD40, TBX1, TMEM26, CITED1 and slc27a1 in iWAT of male mice supplemented with AKG for 11 weeks (n = 8 per group).

(L-M). Respiratory exchange ratio (RER) in male C57BL/6 mice after 11 weeks of AKG supplementation (n = 8 per group).

(N-O). Immunoblots (N) and quantification (O) of p-HSL and ATGL protein in gWAT of male mice after 11 weeks of AKG supplementation (n = 3 per group).

(P-Q). Representative images (P) and quantification (Q) of p-HSL DAB staining in gWAT and iWAT of male mice after 11 weeks of AKG supplementation (n = 9 per group).

(R). The mRNA expression of PPARγ, FASN and ACC in the gWAT and iWAT from male mice supplemented with AKG for 11 weeks (n = 6 per group).

(S-V). Serum levels of E (S), NE (T), T3 (U), and T4 (V) in male mice supplemented with AKG for 11 weeks (n = 8-9 per group).

Data information: Results are presented as mean ± SEM. In (B-C), (E-G), (I-K), (M), (O), and (Q-V), *p≤0.05, **p≤0.01, and ***p≤0.001 by non-paired Student's t test.
Exercise-induced α-ketoglutaric acid stimulates muscle hypertrophy and fat loss
through OXGR1-dependent adrenal activation (2020)
Figure 4

Figure 4. Metabolic effects of AKG is mediated by adrenergic stimulation of thermogenesis and lipolysis

(A). Serum AKG concentration-time profile obtained from male C57BL/6 mice (10 weeks) fed with normal chow before or after i.p AKG (10 mg/kg body weight). The serum AKG level were tested at 0, 1, 2, 4 and 6 hrs after injection (n = 8 per group).

(B-C). Representative images (B) and quantification (C) of BAT thermogenesis after 6 hrs cold exposure at 4℃. Male C57BL/6 mice (10 weeks) were i.p. injected with 10 mg/kg AKG or saline and immediately exposed to cold stress at 4℃ (n = 8 per group).

(D). Immunoblots and quantification of p-HSL and ATGL in the gWAT of male C57BL/6 mice (10 weeks) 3 hrs after i.p. injection of 10 mg/kg AKG or saline (n = 3 per group).

(E). Serum E level in AKG treated male mice 3 hrs after i.p. injection (n = 8 per group).

(F-I). Oxygen consumption (F-G) and RER (H-I) in male C57BL/6 mice (10 weeks) i.p. injected with saline, 10 mg/kg AKG, 1 mg/kg SR59230A (ADRB3 inhibitor) or AKG + SR59230A (n = 8 per group). All injections were performed at 7:00 am of second day. Data was summarized in bar graph (G and I) by light or dark cycle of second day.

(J-N). Body weight gain (J), cumulative food intake (K), body composition (L), fat weight (M) and serum NEFA (N) of sham or adrenalectomized male C57BL/6 mice. Male mice were adrenalectomized at 8 weeks of age. Two weeks after surgeries, male mice were switched to HFD and given free access to tap water or 2% AKG for 9 weeks (n = 8 per group).

(O-P). Representative images (O) and quantification (P) of BAT thermogenesis after 6 hrs cold exposure at 4℃ in sham or adrenalectomized male mice treated with AKG for 9 weeks (n = 8 per group).

(Q). The mRNA expression of themogenic genes in the BAT of sham or adrenalectomized male mice treated with AKG for 9 weeks (n = 6 per group).

(R-S). Immunoblots (R) and quantification (S) of p-HSL and ATGL protein in the gWAT of sham or adrenalectomized male mice treated with AKG for 9 weeks (n = 4 per group).

(T-U). Immunoblots (T) and quantification (U) of UCP1 protein in the BAT of sham or adrenalectomized male mice treated with AKG for 9 weeks (n = 4 per group).

Data information: Results are presented as mean ± SEM. In (A), *p≤0.05 by non-paired Student's t test compared with before injection. In (C-E), *p≤0.05, **p≤ 0.01 by non-paired Student's t test. In (J-K), *p≤0.05 by two-way ANOVA followed by post hoc Bonferroni tests. In (G), (I), (L-N), (P-Q), (S) and (U), different letters between bars indicate p≤0.05 by one-way ANOVA followed by post hoc Tukey's tests.