EMBO/soda-nel huggingface dataset:
High density of REC8 constrains sister-chromatid axes and prevents illegitimate synaptonemal complex formation (2016)
Figure 6

Figure 6. Analysis of REC8 distribution along the sister-chromatid axes of wild-type autosomes.

A) Representative yet-unsynapsed autosome from wild-type zygotene spermatocytes. Nuclear spreads were immunostained for SYCP3 and SYCE2. Magnified autosome is indicated by dashed area. Filled arrowheads indicate yet-unsynapsed axes regions. Bars, 10 μm in spreads and 1 μm on insets

B) Representative unsynapsed autosome from wild-type zygotene spermatocytes. Nuclear spreads were immunostained for SYCP3, REC8 and ACA. Magnified autosome is indicated by dashed area. Bars, 10 μm in spreads and 1 μm on insets.

C) Scatterplot comparing the average number of REC8 foci along zygotene autosomes and X-chromosomes. Number of REC8 foci were quantified along 15 zygotene autosomes and 14 X-chromosomes. Each dot represents the number of REC8 foci on each chromosome. Total number of foci identified: zygotene autosomes (343), X-chromosomes (225). Horizontal lines indicate the mean, error bars indicate s.d.: 7.42 and 3.02 for zygotene autosomes and X-chromosomes, respectively. p = 0.0060, obtained with two-tailed Mann-Whitney test.

D) Histogram showing the relative frequency of inter-REC8 distances along yet-unsynapsed zygotene autosomes. 294 inter-REC8 distances analyzed in 14 chromosomes. Grey area represents 15% of the chromosome axis length. The respective cumulative distribution function is shown.

E) Scatterplot comparing the measured inter-REC8 distances on yet-unsynapsed zygotene autosomes, X-chromosomes and sites of LSAEs in Stag3 mutant. Number of inter-REC8 distances for yet-unsynapsed zygotene autosomes, X-chromosomes and sites of local separation of axial elements in Stag3 mutant, n=294, n=211 and n=10, respectively. Horizontal lines indicate the median. p < 0.0001, obtained with two-tailed Mann-Whitney test.
MIG-6 is essential for promoting glucose metabolic reprogramming and tumor growth in TNBC (2021)
Figure 1

Figure 1. MIG-6 upregulation is positively correlated with disease progression and worse survival in TNBC patients.

(A) cBioPortal was used to access and visualize TCGA data for MIG-6 (ERRFI1) gene expression in 81 cases of invasive carcinoma of basal-like breast cancer Cancer Genome Atlas, 2012(). Each column represents an individual sample.

(B) Box plots show the MIG-6 (ERRFI1) gene expression in different breast tumor subtypes in the Servant dataset of 343 primary breast cancer carcinomas, analyzed using Illumina HumanWG-6_v3 Arrays. The gene expression levels are determined using the R2 Genomics Analysis and Visualization Platform (http://r2.amc.nl). In the box plot, error bars are the 95% confidence interval, the bottom and top of the box are the 25th and 75th percentiles, the line inside the box is the 50th percentile (median), and any outliers are shown as open circles. **p < 0.01, by Student's t-test.

(C) Immunoblotting analysis for MIG-6 protein expression in various human breast cancer cell lines.

(D) Representative images of histological analyses of MIG-6 in TNBC patients in low-grade (stage I) and high-grade (stage III) tumors. Scale bar, 200 µm.

(E) Quantification analysis of MIG-6 expression in 85 cases of resected TNBCs. In the box plot, error bars are the 95% confidence interval, the bottom and top of the box are the 25th and 75th percentiles, the line inside the box is the 50th percentile (median). The p-value 0.011 is used for the comparison of all three groups, determined by the Kruskal-Wallis H test.

(F) Kaplan-Meier plot analysis of the disease-specific survival of 85 TNBC breast cancer patients with low or high expression of MIG-6.

(G) Kaplan-Meier plot analysis of the metastasis-specific survival of 85 TNBC patients with low or high expression of MIG-6. MIG-6 protein expression in the 85 TNBC specimens was determined by H-score (please refer to "Immunohistochemistry and scoring" for details) and widely distributed. MIG-6 expression greater than or equal to the median is classified as "high," while expression less than the median is classified as "low."
MIG-6 is essential for promoting glucose metabolic reprogramming and tumor growth in TNBC (2021)
Figure 2

Figure 2. MIG-6 deficiency suppresses the growth and colony formation of TNBC cells.

(A) Cell proliferation and immunoblotting assays in BT549 cells with Luciferase or MIG-6 knockdown.

(B) Cell proliferation and immunoblotting assays in MDA-MB-231 cells with Luciferase or MIG-6 knockdown.

(C) Representative images and quantification results for a colony formation assay in BT549 cells with GFP or MIG-6 knockdown.

(D and E) Immunoblotting analysis for EGFR signaling in BT549 (D) and MDA-MB-231 (E) cells with GFP or MIG-6 knockdown.

(F) Cell growth assay in BT549 cells with GFP or MIG-6 knockdown in the absence and presence of gefitinib for 72 hours.

Data information: Two different MIG-6-targeting shRNAs were used in (A)-(E).The quantified results are presented as mean ± SD (n=3; biological replicates). *p < 0.05, **p < 0.01, by Student's t-test.
MIG-6 is essential for promoting glucose metabolic reprogramming and tumor growth in TNBC (2021)
Figure 3

Figure 3. MIG-6 deficiency attenuates glucose metabolism and lactate production in TNBC cells.

(A) Immunoblotting analysis for MIG-6 expression in BT549 cells under normoxia and hypoxia.

(B) Extracellular acidification rate (ECAR) in BT549 cells with GFP or MIG-6 knockdown. The ECAR flux profile of GFP and MIG-6 knockdown BT549 cells upon stimulation of glucose, oligomycin, and 2-DG is shown on the left. Quantitative results for the basal glycolysis and maximal glycolysis capacities in GFP and MIG-6 knockdown BT549 cells are indicated by orange and green boxes, graphed on the right. Results are presented as mean ± SEM (n=15; biological replicates).

(C) A heatmap diagram shows the expression profile of genes involved in glycolysis, the TCA cycle, the pentose phosphate pathway, pyruvate oxidation, gluconeogenesis, and glycogen metabolism obtained from two sets of GFP and MIG-6 knockdown BT549 cells.

(D to F) Histograms show the percentage of gene dysregulation in glycolysis, TCA cycle, and pyruvate oxidation pathways upon MIG-6 knockdown in BT549 cells. For each gene, the change was calculated based on the equation: (shGFP-shMIG6)/shMIG-6. Results are presented as mean ± SEM (n=3; biological replicates).

(G) Schematic illustration depicts the metabolic changes in the glycolytic pathway and oxidative phosphorylation (OXPHOS) regulated by MIG-6. MIG-6-upregulated genes are indicated in red; MIG-6-downregulated genes are marked in green.

(H) Lactate production assay in BT549 cells with GFP or MIG-6 knockdown (n=3). The medium was collected to measure lactate concentration using lactate test strips and an Accutrend Lactate analyzer. The rate of lactate production was determined (lactate production rate = lactate concentration/cells/time) and normalized to the rate detected in the control group. The quantified results are presented as mean ± SEM (n=3; biological replicates).

Data information: In B and H, **p < 0.01, by Student's t-test
MIG-6 is essential for promoting glucose metabolic reprogramming and tumor growth in TNBC (2021)
Figure 4

Figure 4. MIG-6 deficiency inhibits GLUT1 expression and glucose uptake in TNBC cells. (A) Box plots show the GLUT1 (SLC2A1) gene expression in different breast tumor subtypes in the Servant dataset of 343 primary breast cancer carcinomas, analyzed using Illumina HumanWG-6_v3 Arrays. The gene expression levels are determined using the R2 platform. In the box plot, error bars are the 95% confidence interval, the bottom and top of the box are the 25th and 75th percentiles, the line inside the box is the 50th percentile (median), and any outliers are shown as open circles. **p < 0.01, by Student's t-test..

(B) Real-Time PCR analysis for GLUT1 and MIG-6 mRNA expression in GFP and MIG-6 knockdown BT549 cells (n=3).

(C) Immunoblotting analysis for GLUT1 protein expression in BT549 cells with GFP or MIG-6 knockdown.

(D) Confocal image analysis results for membrane-bound GLUT1 in GFP and MIG-6 knockdown BT549 cells are shown in the top panel. The arrow indicates membrane-localized GLUT1. Scale bar, 25 μm. Quantitative analysis results of the percentage of GFP and MIG-6 knockdown BT549 cells expressing membrane-localized GLUT1 are shown in the bottom panel (n=3).

(E) Representative flow cytometry images (upper) and quantitative results (lower) of measuring glucose uptake in GFP and MIG-6 knockdown BT549 cells (n=3). Cells were grown in the presence of the fluorescent analog 2-NBDG for various time periods, and glucose uptake was quantified using flow cytometric analysis. The red box indicates the gating for 2-NBDG+ cells and the number indicates the percentage of 2-NBDG+ cells.

(F) Immunoblotting analysis for GLUT1 protein expression in GFP and MIG-6 knockdown BT549 cells with or without MG132 treatment.

Data information: In B, D, and F, the quantified results are presented as mean ± SD (n=3). **p < 0.01, by Student's t-test.
MIG-6 is essential for promoting glucose metabolic reprogramming and tumor growth in TNBC (2021)
Figure 5

Figure 5. MIG-6 regulates GLUT1 gene expression by stabilizing HIF1α protein expression. (A) Schematic illustration of the potential mechanisms by which MIG-6 regulates GLUT1 gene expression.

(B) Immunoblotting analysis for HIF1α protein expression in BT549 cells with GFP or MIG-6 knockdown.

(C) Immunoblotting analysis for HIF1α protein expression in MDA-MB-231 cells with GFP or MIG-6 knockdown.

(D) Real-Time PCR analysis for HIF1α mRNA expression in BT549 cells with GFP or MIG-6 knockdown. The quantified results are presented as mean ± SD (n=4). ***p < 0.001, by Student's t-test.

(E) Immunoblotting analysis for HIF1α protein expression in GFP and MIG-6 knockdown BT549 cells in the absence or presence of the proteasome inhibitor MG132.

(F) GFP and MIG-6 knockdown BT549 cells transfected with the indicated plasmids were treated with MG132, subjected to immunoprecipitation (IP) with Flag-tag or HIF1α antibody, followed by immunoblotting analysis. MG132 was used to rescue HIF1α protein degradation mediated by MIG-6 knockdown, leading to similar HIF1α protein levels in control and MIG-6 knockdown BT549 cells, which were used as the input to examine the role of MIG-6 in the interaction between HIF1α and HAUSP.

(G) Luciferase and MIG-6 knockdown 293 cells were transfected with the indicated plasmids and subjected to MG132 treatment. Afterward, cells were harvested for IP with HA antibody, followed by immunoblotting analysis to determine the level of K48-linked ubiquitination of HIF1α. MG132 was used to preserve the degradative K48-linked ubiquitination signals.

(H) Immunoblotting analysis for MIG-6 and HIF1α expression in Luciferase and MIG-6 knockdown 293 without MG132 treatment.

(I) Immunoblotting analysis for GLUT1 protein expression upon MIG-6 overexpression in GFP and HIF1α knockdown BT549 cells.

(J) Immunoblotting analysis for GLUT1 protein expression upon MIG-6 overexpression in GFP and HAUSP knockdown BT549 cells.

(K) Immunoblotting analysis for HIF1α protein expression upon HAUSP overexpression in GFP and MIG-6 knockdown BT549 cells.
MIG-6 is essential for promoting glucose metabolic reprogramming and tumor growth in TNBC (2021)
Figure 6

Figure 6. MIG-6 regulates aerobic glycolysis and cell growth through GLUT1.

(A and B) Lactate production assay in BT549 (A) and MDA-MB-231 (B) cells with Luciferase or GLUT1 knockdown.

(C) Cell proliferation assay in BT549 cells with Luciferase or GLUT1 knockdown.

(D) Cell proliferation assay in MDA-MB-231 cells with GFP or GLUT1 knockdown.

(E and F) Immunoblotting analysis for GLUT1 protein expression in control and MIG-6 knockdown BT549 (E) and MDA-MB-231 (F) cells with or without GLUT1 overexpression.

(G and H) Lactate production assay in control and MIG-6 knockdown BT549 (G) and MDA-MB-231 (H) cells with or without GLUT1 overexpression.

(I and J) Cell proliferation assay in BT549 (I) and MDA-MB-231 (J) cells with control knockdown, MIG-6 knockdown, or MIG-6 knockdown plus GLUT1 overexpression.

Data information: The quantified results are presented as mean ± SD (n=3; biological replicates). *p < 0.05, **p < 0.01, by Student's t-test.
MIG-6 is essential for promoting glucose metabolic reprogramming and tumor growth in TNBC (2021)
Figure 7

Figure 7. MIG-6 deficiency inhibits tumor growth in TNBC.

(A and B) In vivo primary tumor growth derived from BT549 cells with Luciferase or MIG-6 knockdown (six mice per group). Cells were injected into the mammary fat pads of nude mice, and tumor sizes were measured weekly by caliper. Kaplan-Meier plot analysis is used to determine the incidence of Luciferase or MIG-6 knockdown BT549-xenograft tumors (A). Volumes of Luciferase or MIG-6 knockdown BT549 tumors at week 10 are presented as mean ± SEM (B).

(C and D) In vivo primary tumor growth derived from MDA-MB-231 cells with Luciferase or MIG-6 knockdown (nine mice per group). Volumes of Luciferase or MIG-6 knockdown MDA-MB-231 tumors were measured weekly by caliper are presented as mean ± SEM (C). Tumor weights of MDA-MB-231-derived xenografts were measured at the endpoint (day 33) and are presented as mean ± SEM (D).

(E) Immunoblotting analysis for MIG-6 expression in BT549 cells with MIG-6 inducible knockdown (iMIG-6-shRNA) and the non-targeting shRNA control (iNT-shRNA) upon doxycycline treatment.

(F) In vivo primary tumor growth derived from BT549 cells containing iNT-shRNA and iMIG-6-shRNA (n=7 per group). The arrow indicates the time point when the diet was given to induce MIG-6 knockdown in vivo. Volumes of iNT-shRNA and iMIG-6-shRNA BT549 tumors are presented as mean ± SEM.

(G) Representative images for tumor growth and quantitative results for tumor weight at the experimental endpoint (seven mice per group). Results are presented as mean ± SEM.

Data information: *p < 0.05, **p < 0.01, ***p < 0.001, by Student's t-test.
MIG-6 is essential for promoting glucose metabolic reprogramming and tumor growth in TNBC (2021)
Figure 8

Figure 8. MIG-6 deficiency inhibits GLUT1 protein expression in vivo.

(A) Representative images of histological analysis for GLUT1 protein expression in BT549-derived xenograft tumors. Scale bar, 100 μm.

(B) Representative images of histological analysis for GLUT1 protein expression in xenograft tumors derived from BT549 cells containing iNT-shRNA and iMIG-6-shRNA. Scale bar, 100 μm.

(C) Representative images of histological analyses for GLUT1 and MIG-6 expressions in TNBC patients in low-grade (stage I) and high-grade (stage III) tumors Scale bar, 200 µm.

(D) Scatterplot analysis for the correlation between GLUT1 and MIG-6 expression levels (n=85). The r value was calculated by Spearman's rank correlation coefficient analysis.

(E) Representative images of the immunofluorescence multiplex assay for colocalization of MIG-6 and HIF1α protein expression in TNBC tumor specimens. The triangle indicates nuclear colocalization, and the star indicates cytoplasmic colocalization.

(F) A working model depicts the mechanism by which MIG-6 exerts its tumor-promoting function in TNBC. MIG-6 overexpression promotes HIF1α stabilization and the subsequent upregulation of GLUT1 gene transcription, metabolic reprogramming, and tumorigenesis.

Expanded View Figure Legends
Histamine H3 receptors aggravate cerebral ischaemic injury by histamine-independent mechanisms (2014)
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(a) H3R expression in cultured rat cortical neurons at DIV7 after OGD/R injury (2 h OGD and 24 h reperfusion) shown by western blot (n=6 per condition; **P0.01 versus control with Student’s t-test). Full-size blots are shown in Supplementary Fig. 9. (b) When the H3R antagonist thioperamide (THIO, 10−6 mol l−1 at reperfusion) and the agonist immepip (IMME, 10−6 mol l−1 at reperfusion) were administered, viability was tested in cultured neurons by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay after OGD/R (n=7 per condition; ***P0.001 with analysis of variances (ANOVAs) followed by the Bonferroni/Dunn post hoc test). (c) 2,3,5-triphenyltetrazolium chloride-stained brain sections showing the infarct area in WT and H3R−/− mice receiving saline, the H3R antagonists A331440, clobenpropit (CLOB) and thioperamide, (THIO; 5 mg kg−1, i.p., immediately at reperfusion and 6 h later), and the H3R agonist immepip (IMME, 1 μg, i.c.v., at reperfusion) after I/R (1 h MCAO followed by 24 h reperfusion; scale bar, 5 mm). The bar graphs show infarct volume (d) and neurological score (e) (n=6–8 mice per condition, *P0.05, **P0.01, ***P0.001 versus WT saline group with ANOVAs followed by the Bonferroni/Dunn post hoc test). Data are presented as mean±s.e.m.
Histamine H3 receptors aggravate cerebral ischaemic injury by histamine-independent mechanisms (2014)
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(a) TUNEL staining of apoptotic cells in penumbral brain slices in vivo (scale bar, 100 μm) and (c) in cultured neurons when H3R was inhibited with thioperamide (scale bar, 50 μm) after tMCAO and OGD/R. TUNEL-positive cells are green, and all cells are stained with 4',6-diamidino-2-phenylindole (blue). (b,d) H3R was inhibited by thioperamide, and cleaved caspase-3 protein expression is shown by western blot in WT and H3R−/−mice after tMCAO (b) (n=6–7 per condition; **P0.01, ***P0.001 with analysis of variances (ANOVAs) followed by Bonferroni/Dunn post hoc test) and in cultured neurons subjected to OGD/R (d) (n=8 per condition; *P0.05, ***P0.001 with ANOVAs followed by the Bonferroni/Dunn post hoc test). Data are presented as mean±s.e.m. Full-size blots are shown in Supplementary Fig. 10.
Histamine H3 receptors aggravate cerebral ischaemic injury by histamine-independent mechanisms (2014)
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(a) 2,3,5-Triphenyltetrazolium chloride (TTC)-stained brain sections from WT and HDC−/− mice showing the infarct area in those receiving saline, the H3R antagonist thioperamide (THIO), the H1R antagonist pyrilamine (PYRI; 10 mg kg−1, i.p., at reperfusion), the H2R antagonist cimetidine (CIME; 10 mg kg−1, i.p., at reperfusion) and α-FMH (50 mg kg−1, i.p., 2 h before ischemia) after 24 h reperfusion (n=6 per condition; scale bar, 5 mm). (b) Infarct volumes and (c) neurological deficit scores (*P0.05, **P0.01, ***P0.001 with analysis of variances followed by the Bonferroni/Dunn post hoc test). Data are presented as mean±s.e.m.
Histamine H3 receptors aggravate cerebral ischaemic injury by histamine-independent mechanisms (2014)
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(a) Akt/GSK-3β/mTOR/P70S6K phosphorylation assessed by western blots after thioperamide administration and OGD/R. LiCl (5 mmol l−1 immediately at reperfusion) was used to rephosphorylate GSK-3β/mTOR/P70S6K. The digits below represent the semiquantified optidensity of the bands, the control bands were defined as 1.00. (b) Representative western blots showing the phosphorylation of Akt/GSK-3β/mTOR/P70S6K signaling in H3R−/− mice after OGD/R injury. The digits below represent the semiquantified optidensity of the bands, the control bands were defined as 1.00. (c) Representative images showing LC3-positive autophagic vacuoles (red) with 4',6-diamidino-2-phenylindole staining (blue) after administration of thioperamide and LiCl with OGD/R (scale bar, 10 μm). (d) Cell viability assessed by MTT when thioperamide and LiCl were administered after OGD/R (n=7, **P0.01, ***P0.001 with analysis of variances (ANOVAs) followed by the Bonferroni/Dunn post hoc test). (e) Representative western blots and bar graph showing the expression of LC3-I and LC3-II in steady-state autophagy and autophagic flux under OGD/R injury when thioperamide (THIO) and immepip (IMME) were administered (n=6; **P0.01, ***P0.001 with ANOVAs followed by the Bonferroni/Dunn post hoc test). Data are presented as mean±s.e.m. Full-size blots are shown in Supplementary Fig. 11.
Histamine H3 receptors aggravate cerebral ischaemic injury by histamine-independent mechanisms (2014)
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(a) Representative immunohistochemical staining of LC3 (green) along with NeuN staining (red) in WT and H3R−/− mice (scale bar, 25 μm). (b,c) When thioperamide was administered in vivo, LC3 expression was assessed by western blot after I/R in WT mice (b) (*P0.05, ***P0.001; n=6 mice per condition, with analysis of variances (ANOVAs) followed by the Bonferroni/Dunn post hoc test), and in H3R−/− mice (c) (***P0.001 versus H3R−/− sham group; n=5–6 mice per condition, with ANOVAs followed by the Bonferroni/Dunn post hoc test.) Data are presented as mean±s.e.m. Full-size blots are shown in Supplementary Fig. 12.
Histamine H3 receptors aggravate cerebral ischaemic injury by histamine-independent mechanisms (2014)
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(a) Western blots showing the inhibitory effect of 3-MA on autophagy in I/R. Full-size blots are shown in Supplementary Fig. 13. (b) TTC-stained sections showing the infarct area after administration of 3-MA (100 nmol, i.c.v., at reperfusion) in WT and H3R−/− mice (scale bar, 5 mm), and (c) bar graph showing infarct volume (n=6–8 mice per condition; *P0.05, ***P0.001, with analysis of variances (ANOVAs) followed by the Bonferroni/Dunn post hoc test). (d) TTC-stained sections (scale bar, 5 mm) and bar graph showing the infarct area after administration of thioperamide in Atg5+/−mice (n=4–6 mice per condition). (e) In cultured neurons, viability was tested by MTT assay after 3-MA (2.5 mmol l−1 at reperfusion) and thioperamide was administered in OGD/R (n=7 per condition; ***P0.001, with ANOVAs followed by the Bonferroni/Dunn post hoc test). (f) In cultured neurons, viability was tested by MTT after siRNA for Atg7 and OGD/R (n=7 per condition; ***P0.001, with ANOVAs followed by the Bonferroni/Dunn post hoc test). (g) Viability was tested by MTT assay on Atg5+/+and Atg5−/− MEFs after thioperamide administration after OGD/R (n=7 per condition; **P0.01, ***P0.001, with ANOVAs followed by the Bonferroni/Dunn post hoc test). Data are presented as mean±s.e.m.
Histamine H3 receptors aggravate cerebral ischaemic injury by histamine-independent mechanisms (2014)
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(a) Representative co-immunoprecipitation results showing the effect of thioperamide on the interaction of H3R with CLIC4 during OGD/R. Full-size blots are shown in Supplementary Fig. 14. (b) Cell viability was tested by MTT assay after administration of Tat-H3RCT414–436 (10−6 mol l−1 at reperfusion) under OGD/R (data are presented as mean±s.e.m.; n=7 per condition; *P0.05, **P0.01, with analysis of variances (ANOVAs) followed by the Bonferroni/Dunn post hoc test). (c) Representative western blots showing the effect of Tat-H3RCT414-436 on phosphorylation of Akt/GSK-3β/mTOR/P70S6K signalling and LC3 expression in OGD/R. Full-size blots are shown in Supplementary Fig. 14. (d) Representative images of the effect of Tat-H3RCT414-436 on LC3-positive autophagic puncta (red) in 4',6-diamidino-2-phenylindole-stained (blue) neurons after OGD/R (scale bar,10 μm). (e) Western blots showing the effect of siRNA for CLIC4 on phosphorylation of Akt/GSK-3β/mTOR/P70S6K signalling and LC3 expression in OGD/R. Full-size blots are shown in Supplementary Fig. 14. (f) Representative western blots and bar graph showing the expression of LC3-I and LC3-II on steady-state autophagy and autophagic flux under OGD/R when CLIC4 was knocked down (data are presented as mean±s.e.m.; n=6; *P0.05, **P0.01; , with ANOVAs followed by the Bonferroni/Dunn post hoc test). Full-size blots are shown in Supplementary Fig. 14. (g) Cell viability was assessed by MTT assay after siRNA for CLIC4 under OGD/R (data are presented as mean±s.e.m.; n=8 per condition; ***P0.001, with ANOVAs followed by the Bonferroni/Dunn post hoc test).