EMBO/soda-nel huggingface dataset:
Nuclear Receptor Corepressor 1 Represses Cardiac Hypertrophy (2019)
Figure 2

Figure 2. Deficiency of cardiomyocyte NCoR1 exacerbates abdominal aortic constriction (AAC)-induced cardiac hypertrophy. (A) Representative images of hearts from LC or CMNKO mice subjected to sham operation or AAC for 2 weeks. Scale bar: 0.5cm. (B) VW/BW of LC or CMNKO mice 2 weeks after sham operation or AAC. n=9:9:16:15. (C) Representative H&E staining of cross sections of left ventricles. Scale bar: 25μm. (D) Representative WGA staining. Scale bar: 10μm. (E) Quantification of cardiomyocyte size. n=10:10:8:9. (F) qRT-PCR analysis of hypertrophy-related genes in left ventricles. n=8:7:15:15. (G) Representative picrosirius red staining of cross sections of left ventricles. Scale bar: 50μm. (H) Quantification of fibrotic areas. n=11:10:10:9. (I) qRT-PCR analysis of fibrosis-related genes in left ventricles. n=8:7:15:15. (J) Ejection fraction and (K) Fractional shortening measured by echocardiography. n=9:7:14:12. (L) Lung weight to body weight ratio (LW/BW) of LC or CMNKO mice 2 weeks after sham operation or AAC. n=8:7:16:15. (M) Cumulative survival rate of LC or CMNKO mice subjected to AAC. n=12:10. Two-way ANOVA followed by Bonferoni post-tests was used for statistical analysis.
Centrosomal ALIX regulates mitotic spindle orientation by modulating astral microtubule dynamics (2018)
Figure 2

Figure 2. DSpd-2/Cep192 recruits ALIX to the PCM of centrosomes

(A) Confocal images of ALIX (red), Asl (white), α-tubulin (green) and Hoechst (blue) in wild type (w1118), Sas-6c02901, DSpdG02143, cnnHK21 and γ-Tub23CPI NBs (scale bars, 5 μm). The insets show higher magnification of the boxed centrosomes. (B) The centrosomal fluorescence intensity of ALIX was measured and the graph shows the average intensity (relative to w1118 intensity) of three separate experiments (±S.E), where in total 78 w1118, 73 Sas-6c02901, 45 DSpdG02143, 61 cnnHK21 and 26 γ-Tub23CPI NBs were analyzed. Statistically significant differences are indicated (*p<0.05 and **p<0.01, Student's t-test). (C) GST or GST-ALIX were incubated with in vitro translated 35S-labelled Cnn or DSpd-2 proteins. Binding was detected by autoradiography (upper panel), and expression of GST and GST-ALIX was visualized by Ponceau S-staining (lower panel). (D) HeLa cells were transfected with control, Cep192 or Cep215 siRNA. Efficient depletion of Cep192 and Cep215 was confirmed by Western blotting analysis, which also showed unchanged expression levels of ALIX. Unspecific bands, below Cep192 and above Cep215, are indicated with *. (E) Control, Cep192 or Cep215 siRNA-transfected HeLa cells were immunostained with anti-ALIX (red), anti-α-tubulin (green) and Hoechst (blue). Confocal micrographs are presented and the insets show the indicated centrosomes (arrows) at higher magnification (scale bars, 5 μm). (F) The centrosomal ALIX fluorescence intensity was measured and the graph shows the average intensity (relative to control siRNA cells) of three independent experiments (±S.E), where in total 41 control siRNA, 40 Cep192 siRNA and 39 Cep215 siRNA-transfected cells were analyzed, **p< 0.01, Student's t-test.
Centrosomal ALIX regulates mitotic spindle orientation by modulating astral microtubule dynamics (2018)
Figure 3

Figure 3. ALIX regulates MS orientation in Drosophila NBs and asymmetrically dividing SOPs

(A) Brains were dissected from w1118, alix1, alix-l/CyO; alix1 and mud4 larvae and stained with anti-Bazooka (red), anti-Cnn (white) and Hoechst (blue). Representative confocal images are shown. Scale bars, 5 μm. The graph summarizes the average variation of the relative spindle angles in w1118 (n=74), alix1 (n= 96), alix-l/CyO; alix1 (n= 78) and mud4 (n=32) NBs (four separate experiments) (±S.E). Compared to w1118 or alix-l/CyO; alix1 NBs both alix1 and mud4 NBs showed greater variation in MS orientation (***p<0.001, ANOVA). (B) Schematic illustration of the calculation of the relative spindle angle between the polarity axis (blue line, perpendicular to the line aligning the apical Bazooka crescent (red dotted line) and the MS axis (blue dotted line) going through the two centrosomes. The basal crescent is here indicated in green. (C) Brain lysates of w1118, alix1 and alix-l/CyO; alix1 larvae were immunoblotted for ALIX, α-tubulin and GAPDH (loading control). (D) Time-lapse imaging of Asl-YFP, α-tubulin-GFP in control (left panel) and alix3 (right panel) mutant SOPs of the pupal notum. SOPs were identified by the apical meshwork of MTs (not shown). Time is indicated in min:sec. Mitotic cells are indicated with arrows. Scale bar represents 5 μm. (E) Quantification of the rotation angle (±S.E) in control (n=26) and alix3 (n=45) mutant SOPs (at least three separate experiments). **p<0.01 (Student's t-test) compared to control. (F) Schematic view of the calculation of the rotation angle. The angle α1 represents the angle made between the midline (horizontal line) and the axis of the two centrosomes immediately prior to nuclear envelope breakdown (when the two centrosomes compress the nucleus). The angle α2 represents the angle between the midline and the axis of spindle at anaphase onset. The rotation angle is equal to the difference α1-α2. (G) Western blotting analysis of lysates from control Asl-YFP, α-tubulin-GFP and Asl-YFP, α-tubulin-GFP; alix3 pupae showed absence of ALIX in the alix3 homozygous mutant and unchanged expression of α-tubulin and GAPDH (loading control).
Centrosomal ALIX regulates mitotic spindle orientation by modulating astral microtubule dynamics (2018)
Figure 4

Figure 4. ALIX regulates MS orientation in symmetrically dividing Drosophila and human epithelial cells

(A) Ovaries of adult female flies (w1118, alix1 and alix-l/CyO; alix1) were stained with rhodamine-phalloidin (red), anti-α-tubulin (green), anti-Cnn (white) and Hoechst (blue). Typical confocal micrographs of metaphase FECs are presented and the apical membrane facing the egg chamber lumen is indicated with *. Scale bar, 5 μm. The MS orientation was measured and the graph shows the average percentage of FECs (w1118, n=45, alix1, n=45, alix-l/CyO; alix1, n=39, from three independent experiments) with the indicated relative spindle angle (±S.E). Statistically significant difference is indicated as ***p<0.001 (ANOVA). (B) Schematic figure illustrating calculation of the relative spindle angle in FECs, between the polarity axis (blue line) perpendicular to the line aligning the apical membrane (red dotted line) and the MS axis (blue dotted line) spanning between the two centrosomes. (C) Lysates of ovaries from w1118, alix1 and alix-l/CyO; alix1 flies were immunoblotted for ALIX, α-tubulin and GAPDH (loading control). (D) The MS orientation was determined in metaphase cells in organoids of Caco-2 cells transfected with control (n=75), ALIX (n=88) or NuMA (n=79) siRNA (from three independent experiments). The graph shows the average percentage of Caco-2 cells with the indicated relative spindle angle (±S.E). Greater spindle angle variation was observed in ALIX- and NuMA-depleted cells compared to control cells (***p<0.001, ANOVA). Representative confocal images are shown where the cells are stained with rhodamine-phalloidin (red), α-tubulin (green) and Hoechst (blue). The apical membrane facing the lumen of the organoid is marked (*). Scale bar, 5 μm. (E) Lysates of control, ALIX or NuMA-depleted Caco-2 cells were immunoblotted for NuMA, ALIX, α-tubulin and GAPDH (loading control).
Centrosomal ALIX regulates mitotic spindle orientation by modulating astral microtubule dynamics (2018)
Figure 6

Figure 6. ALIX controls MS orientation also by γ-tubulin-independent mechanisms

(A) The MS orientation was determined in metaphase NBs from brains dissected from wild type (w1118), alix1, γ−Tub23CPI or γ−Tub23CPI; alix1 larvae. Compared to w1118, significantly greater variation in the relative spindle angles was observed in alix1 NBs and γ−Tub23CPI; alix1 NBs (*p<0.05, ANOVA). The γ−Tub23CPI;alix1 NBs displayed significantly greater variation of their spindle orientations than either alix1 or γ−Tub23CPI NBs (*p<0.05, ANOVA). The graph shows the average percentage of NBs with the indicated relative spindle angles (±S.E) from in total 57 w1118 NBs, 55 alix1 NBs, 76 γ−Tub23CPI NBs and 62 γ−Tub23CPI; alix1 NBs (three independent experiments). (B) Lysates of the respective genotypes were immunoblotted for ALIX, γ-tubulin and GAPDH (loading control). The average expression of ALIX or γ-tubulin (relative to GAPDH) from three independent experiments is given below (±S.E) and significant differences are indicated as *p<0.05, Student's t-test. (C) Dissected brains of w1118, alix1, γ−Tub23CPI and γ−Tub23CPI;alix1 larvae were subjected to MT regrowth assay and stained with anti-α-tubulin (red), anti-Cnn (white) and Hoechst (blue). Representative images of NBs after 0 and 60 seconds regrowth are presented. Scale bars, 5 μm. (D) The MT-intensities after 0 and 60 seconds regrowth were determined, here presented as the average of three separate experiments (± S.E) where in total 27 w1118-0'', 28 w1118-60'', 26 alix1-0'', 19 alix1-60'', 28 γ−Tub23CPI-0'', 26 γ−Tub23CPI-60'', 28 γ−Tub23CPI;alix1-0'' and 23 γ−Tub23CPI;alix1-60'' NBs were analyzed. Compared to w1118-NBs, significantly less MT polymerized in alix1, γ−Tub23CPI or γ−Tub23CPI;alix1 NBs (*p<0.05, ANOVA). (E) The relative spindle angle between the polarity axis perpendicular to the apical phalloidin-stained F-actin surface and the MS was determined in metaphase Caco-2 cysts cells transfected with control, ALIX and/or γ-tubulin siRNA (29, 29, 18 and 32 metaphase cells, respectively, were analyzed from three separate experiments). Compared to control cells, depletion of either ALIX or both ALIX and γ-tubulin, induced a significantly increased randomization of the relative spindle angles (**p<0.01 and ***p<0.001, ANOVA). Caco-2 cells depleted of ALIX+γ-tubulin showed a greater variation in the spindle angles compared to depletion of ALIX or γ-tubulin (*p<0.05, ANOVA). ALIX-depleted cells showed greater spindle angle-variation than γ-tubulin-depleted cells (*p<0.05, ANOVA). (F) Efficient knockdown of ALIX and γ-tubulin relative to GAPDH (loading control) was verified by Western blotting analysis, and the average expression of ALIX or γ-tubulin relative to GAPDH (±S.E) is given below (*p<0.05, Student's t-test).
Centrosomal ALIX regulates mitotic spindle orientation by modulating astral microtubule dynamics (2018)
Figure 8

Figure 8. ALIX regulates MT stability via MAP1S

(A) HeLa cells transfected with control or ALIX siRNA were fixed, permeabilized and stained with anti-MAP1S (red), anti-γ-tubulin (white) and Hoechst (blue). Interphase cells are shown and the insets show close ups of the centrosomes. Scale bars, 5 μm. (B) The fluorescence intensity of centrosomal MAP1S was determined and the graph shows the average of three independent experiments (± S.E), in which in total 52 control and 36 ALIX siRNA cells were analyzed. Significant difference is indicated as *p<0.05 (Student's t-test). (C) HeLa cells were transfected with control, ALIX, MAP1S or ALIX+MAP1S siRNA and the expression of ALIX, MAP1S and GAPDH (loading control) was determined by Western blotting analysis. Representative results are presented. (D, E) Cells treated as in C were immunostained with anti-α-tubulin (white), Hoechst (blue) (D) and anti-acetylated tubulin (green) (E). Scale bars, 5 μm. The intensities of astral MTs relative to total MTs were determined from confocal microscopy images and the graph in (D) shows the average of four separate experiments (10 cells per condition/experiment) (±S.E). *p<0.05 and **p<0.01, Student's t-test. The relative intensity of acetylated MT relative to total MT was determined in (E). The average of four separate experiments (10 cells per condition/experiment) is graphically presented (±S.E). Significant differences are indicated as *p<0.05 (Student's t-test). (F) The regrowth of MTs (i.e total tubulin intensity) was assessed in cells incubated at 37°C for 0, 60 and 150 seconds after ice-induced depolymerization (for 30 minutes). The graph represents the average of three independent experiments (calculating 10 cells per time point for each condition) (±S.E). Significant differences are noted as *p<0.05, Student's t-test. (G) HeLa cells transfected with control or ALIX siRNA grown on coverslips were transiently transfected with pEYFP-C1 (green), pEYFP-MAP1S (green), pEGFP-C1 (green) or pEGFP-CLASP2α and immunostained with anti-α-tubulin (white), anti-Pericentrin (red) and Hoechst (blue). Scale bars, 5 μm. (H) The lengths and numbers of astral MTs of HeLa cells treated as in (G) were measured (LSM software, version 3.2). The graphs show the average numbers (±S.E) based on calculating 30 control siRNA+YFP cells, 23 control siRNA+YFP-MAP1S cells, 30 ALIX siRNA+YFP cells, 30 ALIX siRNA+YFP-MAP1S cells, 28 control siRNA+GFP cells, 30 control siRNA+GFP-CLASP2α cells, 30 ALIX siRNA+GFP cells and 29 ALIX siRNA+GFP-CLASP2α cells from three separate experiments (*p<0.05, Student's t-test), respectively.
Folate levels modulate oncogene-induced replication stress and tumorigenicity. (2015)
Figure 3

The effect of folate deficiency on replication dynamics and DSB formation in cyclin E-expressing cells

Cyclin E-expressing BJ cells were grown for 7 days with and without folate.

Fork rate (Kb/min) distribution. White bars: BJ cells expressing an empty vector (n = 145); dark gray bars: BJ cells expressing the cyclin E oncogene (n = 147); light gray bars: BJ cells cultured for 7 days in a folate-free medium (n = 135); black bars: BJ cells expressing the cyclin E oncogene cultured for 7 days in a folate-free medium (n = 138).

Fork distance distribution (Kb). The color code is as in (A). Empty vector (n = 78); CycE (n = 79); empty vector −folate (n = 71); CycE −folate (n = 80).

Percent of origins with the indicated progression ratio between sister forks. Empty vector (n = 158); CycE (n = 155); empty vector −folate (n = 160); CycE −folate (n = 154). *P < 0.05.

Examples of nuclei with γH2AX and 53BP1 foci following cyclin E expression (CycE) (n = 65), empty vector (n = 65), folate-free medium for 7 days (empty vector −folate) (n = 67) or oncogene expression under folate-free conditions (CycE −folate) (n = 70). Red: γH2AX; green: 53BP1; blue: DAPI staining.

Percent of nuclei with the indicated number of γH2AX-53BP1 co-localized foci. **P < 0.01.

Data information: Bars represent average values.
Folate levels modulate oncogene-induced replication stress and tumorigenicity. (2015)
Figure 4

The effect of folate deficiency on DNA damage and signal transduction pathways

Cyclin E-expressing BJ cells grown for 7 days with and without folate.

Immunoblotting with anti-phosphorylated ATM and anti-phosphorylated CHK1 antibodies. Anti-β-catenin and anti-actin antibodies were used as loading controls.

Protein level quantification.

Examples of nuclei with RAD51 foci following cyclin E expression (CycE) (n = 67), empty vector (n = 65), folate-free medium for 7 days (−folate) (n = 71) or oncogene expression under folate-free conditions (CycE −folate) (n = 75). Green: RAD51, blue: DAPI staining.

Percent of nuclei with the indicated number of RAD51 foci (n = 66). **P < 0.01.
Folate levels modulate oncogene-induced replication stress and tumorigenicity. (2015)
Figure 5

The effect of folate deficiency on the tumorigenicity of oncogene-expressing cells <italic>in vitro</italic> and <italic>in vivo</italic>

A, B Cyclin E- and Ras (H-RasV12)-expressing 3T3 cells grown in 100 nM folate for 4 weeks and then two additional weeks in a normal medium. Control cells were grown in a normal medium for the whole period. (A) Examples of anchorage-independent growth in soft agar of 3T3 cells. (B) Average number of colonies per soft agar plate of 3T3 cells. The number of colonies per plate is expressed as the average ± SEM from three independent experiments.

C–E Ras (H-RasV12)-expressing MCF10A cells grown in 100 nM folate for 4 weeks and then two additional weeks in a normal medium. Control cells were grown in a normal medium for the whole period. (C) Examples of anchorage-independent growth in soft agar of MCF10A cells. (D) Average number of colonies per soft agar plate of MCF10A cells. The number of colonies per plate is expressed as the average ± SEM from three independent experiments. (E) Percentage of tumor-free flanks at the indicated time points after cell injection. Ten mice were injected in both sides in each group.

Data information: Cells expressing pBABE (Empty vector); cells expressing an empty vector and grown in 100 nM folate (pBABE 100 nM Folate); cells expressing the cyclin E oncogene (CycE); cells expressing the cyclin E oncogene and grown in 100 nM folate deficiency (CycE 100 nM folate); cells expressing the Ras oncogene (Ras); cells expressing the Ras oncogene and grown in a 100 nM folate-deficient medium (Ras 100 nM Folate). *P < 0.05, **P < 0.01.
A role for mitochondria in NLRP3 inflammasome activation (2010)
f1

a, b, THP1 cells were stimulated with rotenone (10 &mgr;M), TTFA (10 &mgr;M) or antimycin (40 &mgr;g ml−1) for 6 h and then stained with Mitotracker green and Mitotracker deep red (a) or MitoSOX (b) for 30 min and analysed by flow cytometry. c, THP1 cells were stimulated for 6 h with the indicated amounts of rotenone, TTFA or antimycin. Supernatants (SN) and cell extracts (input) were analysed by western blotting as indicated. d, LPS-primed bone-marrow-derived macrophages (BMDMs) from wild-type (WT), Nlrp3 or Ipaf deficient mice were stimulated for 6 h with MSU (150 &mgr;g ml−1), rotenone (10 &mgr;M for THP1 cells and 40 &mgr;M for BMDMs) or antimycin (40 &mgr;g ml−1 for THP1 cells and 10 &mgr;g ml−1 for BMDMs). The release of caspase-1 (western blot) was then determined. Data shown are representative of three independent experiments.
A role for mitochondria in NLRP3 inflammasome activation (2010)
f2

a, BMDMs expressing GFP–LC3 were stimulated with rotenone (40 &mgr;M) for 3 h and the co-localization of mitochondria and GFP–LC3 dots were analysed using confocal microscopy. b, THP1 cells stimulated with 3-methyladenine (3-MA, 10 mM) for 24 h or THP1 cells stably expressing shRNA against beclin 1 or ATG5 were stained with Mitotracker green and Mitotracker deep red for 30 min, and analysed by flow cytometry. c, LPS-primed BMDMs from wild-type, Nlrp3−/−, Asc−/− or Ipaf −/− knockout mice were stimulated with MSU (150 &mgr;g ml−1) or 3-MA (10 mM) for 6 h and the release of active caspase-1 and IL-1&bgr; was determined. d, THP1 cells stably expressing shRNA against beclin 1 or ATG5 were incubated for 24 h and media supernatants (SN) and inputs were analysed by western blotting. e, THP1 cells stably expressing shRNA against beclin 1 or ATG5 were stimulated with MSU or nigericin for 6 h. Data shown are representative of three independent experiments.
A role for mitochondria in NLRP3 inflammasome activation (2010)
f3

a–d, THP1 cells expressing Flag–NLRP3 (a, b) or Flag–ASC (c, d) were analysed for the co-localization of Flag–NLRP3 with the endoplasmic reticulum (ER) marker calreticulin or mitochondria (Mitotracker) using confocal microscopy. e, THP1 cells were stimulated with MSU or nigericin and then fractionated. The cytosolic, ER, MAM and mitochondrial fractions were analysed by western blotting as indicated in the text. f, THP1 cells stably expressing shRNA against NLRP3 were stimulated with nigericin and subcellular fractions analysed as in e. g, TXNIP associates with mitochondria after NLRP3 inflammasome activation in a ROS-dependent manner. THP1 cells were stimulated with MSU and nigericin, and TXNIP localization was investigated in the presence or absence of the antioxidant APDC. Data shown are representative of two or three independent experiments. Scale bars: a–g, 20 &mgr;m.
A role for mitochondria in NLRP3 inflammasome activation (2010)
f4

a, THP1 cells stably expressing shVDAC1 were stimulated with various NLRP inflammasome activators and then analysed for caspase-1 activation and IL-1&bgr; secretion. b, THP1 cells expressing shRNA against VDAC2 were stimulated with MSU, R837, silica, alum or nigericin and analysed for IL-1&bgr; secretion. c, THP1 cells expressing shRNA against VDAC1 were stimulated with MSU and nigericin, and NLRP3 localization was analysed by confocal microscopy. Scale bars: 20 &mgr;m.
Effector-dependent activation and oligomerization of plant NRC class helper NLRs by sensor NLR immune receptors Rpi-amr3 and Rpi-amr1 (2022)
Figure 1

Figure 1. Rpi-amr3 and AVRamr3 form protein complexes unaltered by NRC2EEE-Myc.

A. Schematic model of NRC2-dependent resistance by sensor NLRs Rpi-amr3, Rpi-amr1 and cognate effectors AVRamr3 and AVRamr1, respectively. Each domain is labelled and represented with a different color. CC, coiled-coil; NB-ARC, nucleotide binding domain shared by APAF-1, R genes, CED-4; LRR, Leucine-rich repeat; SP, signal peptide; RXLR, conserved motif found in Phytophthora effectors; WY domains, domains in RXLR effectors with conserved Trp (W) and Tyr (Y) residues; NLS, nuclear localization signal.

B. Blue native-PAGE loading of protein extracts from nrc2/3/4 knockout N. benthamiana plants after immunoprecipitation with anti-Flag antibody. Co-migration of Rpi-amr3-HF and AVRamr3-V5 are indicated (*, red). Same samples were loaded twice on one blue native-PAGE gel, transferred onto one membrane, and then the membrane was cut into two and immunoblotted separately. GUS-V5, β-glucuronidase fused with V5 tag.

C. NRC2EEE-Myc does not alter association between Rpi-amr3 and AVRamr3. Samples of Fig 1B were SDS-boiled and loaded on SDS-PAGE. Input samples were taken prior to immunoprecipitation to show expression of all proteins.

Data information: Ponceau S staining of rubisco large subunit serve as loading control for panel C. Molecular weight markers (in kilodaltons, kDa) are shown on the right. Experiments were done with at least three biological replicates with similar results. Representative images are shown.
Effector-dependent activation and oligomerization of plant NRC class helper NLRs by sensor NLR immune receptors Rpi-amr3 and Rpi-amr1 (2022)
Figure 2

Figure 2. NRC2EEE oligomerizes upon effector detection by Rpi-amr3 or Rpi-amr1.

A. NRC2EEE-Myc is oligomerized upon effector-dependent activation of Rpi-amr3. Protein lysates from Fig 1B, C were loaded on blue native-PAGE. SDS-boiled protein lysate samples serve as control for actual size of NRC2EEE-Myc. Oligomerized NRC2EEE-Myc is indicated (*, red).

B. NRC2EEE-Myc oligomerizes upon effector-dependent activation of Rpi-amr1. Protein lysates from nrc2/3/4 knockout N. benthamiana plants were loaded on blue native-PAGE. Oligomerized NRC2EEE-Myc is indicated (*, red).

C. Samples from Fig 2B were SDS-boiled and loaded on SDS-PAGE. Protein accumulation of Rpi-amr1-Flag, NRC2EEE-Myc, AVRamr1-V5 and AVRamr3-V5 are shown.

D. Semi-log plots NRC2EEE-Myc proteins loaded on blue-native PAGE gels. At least three biological replicates were used for analysis of each sample and data points are plotted on the boxplot (blue, inactive; red, active). Wilcoxon test was conducted in a pairwise manner, and statistical significance is indicated (*, p<0.05; **, p<0.01, ***, p<0.001).

Data information: Ponceau S staining serve as loading control for panels A, and C. Molecular weight markers are shown on the right. Experiments were done with at least three biological replicates with similar results. Standard curves of protein migration derived from semi-log plots of molecular weight markers on individual gels can be found in Dataset EV2.