EMBO/soda-nel huggingface dataset:
A tuberous sclerosis complex signalling node at the peroxisome regulates mTORC1 and autophagy in response to ROS (2013)
figf6

(a) Immunoprecipitation was performed with anti-PEX5 (left panel) and anti-PEX19 (right panel) in HEK293 cells and followed by immunoblotting for endogenous TSC1, TSC2, mTOR (negative control) and PMP70, and PEX1 (PEX19 cargo) or catalase (PEX5 cargo). (b) Schematic of the TSC2 C terminus (amino acids 1,517-1,807) showing the GAP- and PEX5-binding sequence (PxBS), naturally occurring mutations and the reintroduced SKL sequence. (c) Representative images of FAO cells transfected with DsRed–Del-ARL (deleted ARL sequence; red) and GFP–PTS1 (green; top panel). FAO cells were also transfected with DsRed–ARL (red) and stained for PMP70 or catalase (green) as indicated. Scale bars, 10 μm. (d) HEK293 cells expressing Flag–TSC2 wild type (WT) or Flag–TSC2 mutants (RQ, RW and RG) were co-immunoprecipitated using anti-Flag or anti-PEX5, and blotted for PEX5 and Flag–TSC2. (e) Representative experiment showing subcellular fractionation of HEK293 cells overexpressing Flag–TSC2 wild type and Flag–TSC2 mutants (RQ, RW and RG). Lysates from peroxisome (P) or membrane (M) fractions and whole-cell extracts (WCE) were immunoblotted with Flag or PMP70 antibodies. (f) Co-immunoprecipitation of HEK293 cells overexpressing Flag–TSC2 wild type, or Flag–TSC2 mutant (RQ) or Flag–TSC2 rescue mutant (RQ-9NT) using anti-Flag antibody or control IgG and immunoblotted with anti-Flag and anti-PEX5 antibodies. (g) Subcellular fractionation of TSC2−/− MEFs transfected with Flag–TSC2 wild type, or Flag–TSC2 mutants (RQ or RQ-9NT). β-integrin and catalase were used as subcellular markers for membrane and peroxisome fractions, respectively. WCE, whole-cell extract. The arrow indicates the position of overexpressed Flag–TSC2. (h) HEK293 cells co-transfected with Flag–TSC1 and Flag–TSC2 wild type (WT) or Flag–TSC2 G294E mutant (TSC2 mutant that cannot bind TSC1). Lysates were immunoprecipitated using anti-PEX19 and blotted for PEX19 and Flag. (i) Representative blots from subcellular fractionation of HEK293 cells overexpressing wild type (WT) or mutant (CaaX mutant) Flag–Rheb. LDH, lamin A/C and catalase were used as subcellular markers for the cytoplasmic (Cp), nuclear and peroxisome fractions, respectively. Uncropped images of western blots are shown in Supplementary Fig. S6.
A tuberous sclerosis complex signalling node at the peroxisome regulates mTORC1 and autophagy in response to ROS (2013)
figf7

(a) HEK293 cells were transfected with either control shRNA or a PEX5 targeting shRNA, and lysates were blotted using pS6K (T389), S6K, pS6 (S235/236) and S6 antibodies. (b) TSC2 functional assay was performed in HEK293 cells co-expressing myc–Rheb, Flag–TSC1 and Flag–TSC2 wild type (WT), or Flag–TSC2 mutants (RG, RQ and RW) and HA–S6K (left panel) or HA–4E-BP1 (right panel). Cells transfected with empty vector (EV) with or without myc–Rheb were used as controls. Lysates were further analysed for phospho-S6K (T389), S6K, phospho-4EB-P1 (S65), 4E-BP1 and 32P incorporation into S6. (c) Rheb GTPase activity assays were performed by co-immunoprecipitating TSC heterodimers from HEK293 cells expressing Flag–TSC1 and Flag–TSC2 wild type, Flag–TSC2 mutants or the Flag–TSC2 GAP mutant (L1624P), for the indicated times. (d) TSC2 functional assay was performed using TSC2−/− MEFs co-transfected with Flag–TSC1, HA–S6K and Flag–TSC2 wild type or Flag–TSC2 mutants (RQ or RQ-9NT), with mock transfected cells as controls. Arrows denote the positions of Flag–TSC1 and Flag–TSC2. (e) Quantification of the ratio of phospho-S6K to total HA–S6K from Fig. 7d. (±s.e.m.,
n = 3 independent experiments).
*P0.05;**P0.01. (f) In vivo guanine nucleotide loading of Rheb was measured using HEK293 cells overexpressing Flag–TSC1 and Flag–TSC2 wild type, or Flag–TSC2 mutants (RQ and RQ-9NT) with or without Rheb as indicated. (g) Graph shows quantification of the percentage of Rheb bound to GTP (indicative of Rheb GTPase activity, ±s.e.m.,
n = 4 independent experiments).
*P0.05;***P0.001. (h) Quantification of axon number in hippocampal neurons co-transfected with GFP, Flag–TSC1 and Flag–TSC2 WT or Flag–TSC2 mutants. Quantification was performed on 150–250 neurons from each of the 3 independent experiment and the results are represented as polarity (%). All error bars represent s.e.m.
*P0.05;**P0.01 compared with GFP-only transfected control. Uncropped images of western blots are shown in Supplementary Fig. S6. Source data for the statistical analysis are shown in Supplementary Table S1.
Bacterial cyclic ß-(1,2)-glucans sequester iron to protect against iron-induced toxicity (2017)
Figure 3

Fig. 3. Glucans bind Fe2+ iron. (A) Iron (Fe2+)-binding potential of different polysaccharide [used at 5.56 micromole (glucose equivalent)] using the Fe2+-ferrozine colorimetric assay. Lipopolysacchrides (LPS; E. coli 0111:B4), mannan (Saccharomyces cerevisiae), isolated and commercial (purchased from Sigma Aldrich) xanthan, extracellular glucan from Xcc. Data shown are mean ± SD (n=3). (B) Colorimetric iron-binding assay. Glucan from diverse source binds Fe2+ iron. Extracellular (ECG) or periplasmic (PPG) glucan isolated from the wild type Xcc8004 or the ndvB (pHM1J); commercial glucan from yeast, barley and Euglena purchased from Sigma Aldrich. Glucans from various sources were incubated with FeSO4 at room temperature for 15 min, amount of free iron was determined using the standard concentration of ferrozine-Fe2+ complex. (C) Iron-binding measured by Inductively Coupled Plasma-Optical Emission Spectrometry (ICP-OES). Xanthan and various glucans were mixed with 2 mM FeSO4, dialysed against deionized water before determination of Fe2+ content by ICP-OES. Data shown are mean ± SD (n≥3). ** indicates p-value < 0.01 and *** indicates p-value < 0.001 by paired student t-test. (D) Specificity of glucan-Fe2+ interaction. Purified Xcc β-(1, 2)-glucan was incubated with 100 µM FeSO4 and increasing concentrations of Ca2+, Co2+, Ni2+, Mn2+, Mg2+ or Zn2+, and free or unbound iron was measured by Fe2+-ferrozine colorimetric assay. Data shown are mean ± SD (n=3).
Bacterial cyclic ß-(1,2)-glucans sequester iron to protect against iron-induced toxicity (2017)
Figure 5

Fig. 5. Role of glucan in the iron-induced ROS generation. Xanthomonas campestris: (A) wild type Xcc 8004; (B) B5 (EPS deficient) (C) B12 EPS deficient); (D) ndvB (glucan deficient); and (E) ndvB(pHM1J)+ (complementing strain); cells were treated in the presence or absence of either FeSO4, H2O2 or FeSO4+Dp (dipyridyl) were analyzed for ROS by monitoring DCF fluorescence with flow cytometry.
Bacterial cyclic ß-(1,2)-glucans sequester iron to protect against iron-induced toxicity (2017)
Figure 7

Fig. 7. Role of glucan in the iron-induced ROS generation is conserved in E. coli. (A) Wild type E. coli (MG1655); ΔopgG (ΔmdoG; JW1035) and ΔopgH (ΔmdoH; JW1037), mutants blocked in periplasmic glucan synthesis were grown in LOS medium, and cells were treated in the presence or absence of either FeSO4, H2O2 or FeSO4+DP (dipyridyl) were analyzed for ROS by monitoring DCF fluorescence with flow cytometry. (B) The percent of ROS positive E. coli cells treated with FeSO4, H2O2 and FeSO4+DP determined by flow cytometry at 3 h after treatment. Shown are the mean percentages of ROS positive cells exceeding the fluorescence of 99% of untreated cells. Data shown are mean ± SE (n=3). *** indicates p-value < 0.001 compared with data obtained from iron treated wild type and mutant by paired student t-test. (C) E. coli cells treated with FeSO4, H2O2 and FeSO4+ Dp were analyzed for ROS by monitoring DCF fluorescence with fluorometry. Data shown are mean ± SE (n=3). *** indicates p-value < 0.001 compared with data obtained from iron treated wild type and mutant by paired student t-test.
Netrin-1/DCC-mediated PLCG1 activation is required for axon guidance and brain structure development (2018)
Figure 1

Figure 1 - Phosoholipase C gamma1 (PLCγ1)-deficient mouse embryos show a deficit in mesencephalic axon guidance (A) Mouse embryos were immunostained with anti-neurofilament antibody (blue), showing mesencephalic projection. Dispersed axon projections (arrows point to the area of the superior colliculus) and decreased axon fibers (arrowheads point to the area of the pretectal commissure) were observed in Plcg1f/f;Nes-Cre mice at E11.5 (n = 20). The areas of the superior colliculus is shown magnified in each inset (scale bar = 200 μm). (B) Representative images between the posterior and anterior borderlines of the superior colliculus (a and b). The red boxes were drawn as rostrocaudal region of interest (ROI) based on the upper part of the pretectal commissure in the embryonic mesencephalon (scale bar = 100 μm). (C and D) The axons passing through the ROI was quantified by measuring the fluorescence intensity in the ROI, and the distribution of axons was statistically analyzed (n = 5; t-test; **P < 0.005). (E) Whole-mount immunostaining images show neurofilament (red), netrin-1 (purple), and DCC (green), respectively. An E11.5 Plcg1f/f;Nes-Cre embryo showed shortened dorsal ramus and opthalmic nerves (white and yellow boxes). Scale bar: 200 μm. (F - G) Enlarged views of the opthalmic nerve (yellow arrow heads) and dorsal ramus (white arrow heads) (scale bar: 50 μm). (H) Nerve length measurements; Op: ophthalmic nerve; DR: dorsal ramus (n = 3; t-test; **P < 0.005). Data are presented as the mean ± s.e.m. (I) Double immunostaining images. The pattern of expression and colocalization of DCC (green)- and TH (red)-positive axon bundles extending toward the alar thalamus. Scale bar: 100 μm.
Netrin-1/DCC-mediated PLCG1 activation is required for axon guidance and brain structure development (2018)
Figure 2

Figure 2 - Phospholipase C gamma 1(PLCγ1) mediates netrin-1/DCC signaling. (A) In primary mesencephalic neurons, the phosphorylation of PLCγ1 on Y783 was increased proportionally to the netrin-1 concentration. The lower graph presents quantification of the band intensities in (A) (n = 3). (B) Phosphorylation of PLCγ1 on Y783 was decreased after knockdown of the DCC receptor by siRNA (siDcc) (N = 3). (C) Western blot for the NTN1 secretion test. Lane #1: transfection-free lysate, #2: transfection-free supernatant, #3: NTN1-transfected cell lyasate, #3 and #4: NTN-1- transfected supernatant. (D) Ventral mesencephalic (A9) cultures (daggers) derived from control or Plcg1f/f;Nes-Cre mice, were co-cultured with netrin-1-releasing 293T cell aggregates (asterisks; scale bar = 100 μm). The areas delineated by the white boxes between the mesencephalon tissue and 293T cell aggregates are magnified in the respective insets (n = 4). (E) Schematic diagrams of axon extensions in (D). The graphs were drawn according to the vector values between the borders of the cell aggregates and the mesencephalic tissues. (F) Mean angle of the axon extension in (E). (G) Quantification of the ventral mesencephalic axons. The bar graphs indicate the percentages of proper targeting of ventral mesencephalic axons correctly targeting the netrin-1-releasing cell aggregates (n = 200). Each experimental set was performed in quadruplicate. (H) Anti-tyrosine hydroxylase (TH) immunostaining in ventral mesencephalic (VM) tissue. (scale bar = 200 μm). (I) Primary mesencephalic neuron culture in the absence or presence of NTN1-conditioned media (n = 100; scale bar: 500μm). (J) Schematic of the method for the measurement of neurite lengths. (K) Mean length of neurites (F-value:1.82, t-test, P**<0.005).
Netrin-1/DCC-mediated PLCG1 activation is required for axon guidance and brain structure development (2018)
Figure 3

Figure 3 - Netrin-1/DCC induces specific phosphorylation of phospholipase C gamma1 (PLCγ1) on Y783 by neuronal proto-oncogene tyrosine kinase Src. (A) Co-immunoprecipitation data showed that PLCγ1 did not interact with the DCC receptor (n = 2, N = 3). (B) Immunoprecipitation assay showed that Src kinase interacted with PLCγ1. Rac1 is a positive interacting control for PLCγ1 (N = 3). (C) In vitro kinase assay confirmed that Src kinase directly phosphorylated Y783 of PLCγ1. (D) Quantification of the band intensities in (C) (N = 3). (E) Src kinase inhibitor reduced the specific phosphorylation of PLCγ1 on Y783 (arrow; Src I IC50 conc = 44 nM; N = 3). (F) Rac1 activity assay (N = 3). (G) Quantification of Rac1 activity levels in (F). (H) Representative immunoblot of cultured Plcg1f/f (n = 2, N = 3) and Plcg1f/f;Nes-Cre (n = 2, N = 3) mesencephalic neurons stimulated with conditioned media containing Netrin-1.
Netrin-1/DCC-mediated PLCG1 activation is required for axon guidance and brain structure development (2018)
Figure 4

Figure 4 - Plcg1 deletion reduces the volume of the corpus callosum and causes abnormal axon projection into the olfactory tubercle. (A) Schematic of the main phenotype (CC: corpus callosum, OT: olfactory tubercle, SI: substantia innominata) (B) Immunohistochemical staining of control and Plcg1 conditional knockout (cKO) sagittal brain sections. The deletion of Plcg1 caused structural changes in SI and OT projections in the Plcg1f/f;Nes-Cre (d) mice compared to the controls (a) (n = 4; scale bar = 1 mm). The areas delineated by the white boxes (insets) are magnified images of the OT (b and e), and the yellow boxes are magnified images of the SI (c and f). (C) Quantification of the axon bundle distribution. Green boxes in (b) and (e) were selected as tegions of interest (ROIs) using the ZEN software (n = 4) (one-way ANOVA, F-value: 118.5745, P**<0.005). (D) Representative images of tyrosine hydroxylase (TH)- positive neurons (scale bar = 1 mm). The areas delineated by the white boxes are magnified in the insets. The yellow arrow indicated misoriented TH-positive axons (magenta: TH). The red boxes were drawn as ROIs for quantitative analysis in (E) (n = 4; scale bar = 1 mm). (E) Fluorescence intensities in the ROIs were measured according to distance (n = 4) (one-way ANOVA, F-value: 271.8, **P<0.005). Cumulative values of fluorescence intensities per section are shown in the lower graph (S: section). (F) In situ hybridization of PLCγ1 in mDA neurons (scale bar = 50 μm) (green: TH, violet: DIG-labeled probe). (G) Representative images of the CC axon bundles in control (left) and a Plcg1f/f;Nes-Cre (right) mice (n = 4). The tip of the CC was triangular in the control mouse, but oval in the Plcg1f/f;Nes-Cre mouse. The areas delineated by the white boxes are magnified in the insets (red: neurofilament, green: glial fibrillary acidic protein, blue: Hoechst; scale bar = 1 mm). (H) The area of the CC in the Plcg1f/f;Nes-Cre mouse was 36% smaller than that in the control mouse (scale bar = 500 μm) (I) Quantitative comparison of the CC areas in the control and Plcg1f/f;Nes-Cre mice (n = 4; t-test; **p < 0.005). Data are presented as the mean ± s.e.m.
Netrin-1/DCC-mediated PLCG1 activation is required for axon guidance and brain structure development (2018)
Figure 5

Figure 5 - Axon tracts in the corpus callosum (CC) of the Plcg1f/f;Nes-Cre mice are more diffuse than those of control mice. (A), (C), and (E) Cumulative histograms based on fractional anisotropy (FA) values. (B), (D), and (F) show the CC volume and the number of tensors relative to FA values. As FA values increased, the structural (CC volume and the number of tensors) differences increased between the control and Plcg1f/f;Nes-Cre mice. Depending on the number of tensors, the length in the CC appeared to be shortened (E, F). (n = 4; Shapiro-Wilk and t-test; *p < 0.05). Data are presented as the mean ± s.e.m.

Expanded View Figure Legends
Consequence of the tumor-associated conversion to cyclin D1b. (2015)
Figure 1

Humanization of the Ccnd1 exon 4/5 locus results in exclusive production of cyclin D1b

Representative schematic of the targeting construct generated to humanize the Ccnd1 exon 4/5 genomic locus to produce cyclin D1b.

Top: Schematic of primer pairs designed to discriminate between wild-type and knock-in alleles. Bottom: Representative genotyping of Ccnd1+/+, Ccnd1+/KI, and Ccnd1KI/KI mice validating specificity of the primer pairs and somatic insertion of the targeting construct.

PCR analysis of transcript b expression in organs harvested from Ccnd1+/+ and Ccnd1KI/KI mice, demonstrating production of transcript b specifically in KI mice. Gapdh serves as a control (NTC, Non-template control).

Immunoblot from parallel samples in (C) utilizing antisera specific to the 33 amino acids generated by human CCND1 intron 4. Lamin B serves as a control. Arrow indicates the cyclin D1b band.
Consequence of the tumor-associated conversion to cyclin D1b. (2015)
Figure 2

Cyclin D1b selectively rescues Ccnd1−/− phenotypes

Neonates from 2 independent litters of Ccnd1+/KI × Ccnd1+/KI crosses were sacrificed at birth and genotyped. Mice were organized by genotype and total size measured.

Mass of neonates was calculated at birth and average mass quantified (n = at least 4 mice per group).

Mice were weighed weekly, and growth is plotted as the average mass of each genotype/week (left: male, right: female, n > 5 per gender and genotype).

Mice were held by the tails and analyzed for the leg clasping phenotype. Representative images of Ccnd1+/+ and Ccnd1KI/KI age-matched mice are shown (n > 10 for each group).

Top: Individual whole-cell lysates were generated from the eyes of mice of each genotype and analyzed for expression of +/+ cyclin D1 (cyclin D1a) and KI/KI cyclin D1b via immunoblot using antisera specific to each isoform. Bottom: Representative H&E staining of retinal tissue from Ccnd1+/+ and Ccnd1KI/KI mice showing individual layers of the retina. The ganglion cell layer and inner plexiform layer (G + IPL), inner nuclear layer (INL), and outer nuclear layer (ONL) were quantified (right). Images were taken at 400× magnification.

Comparison of the phenotypes described in the Ccnd1−/− mouse with those observed in the Ccnd1KI/KI mouse, previously described (Sicinski et al, 1995).

Data information: n = at least 4 for each genotype. Error bars represent the standard error of the mean (SEM), and significance was determined using a two-tailed Student's t-test.
Consequence of the tumor-associated conversion to cyclin D1b. (2015)
Figure 3

Genetic evidence for cyclin D1b as a credentialed oncogene

Murine adult fibroblasts (MAFs) were harvested from the peritoneum of Ccnd1+/+ and Ccnd1KI/KI mice, and stable cell lines were generated utilizing a 3T3 protocol.

Indicated passage-matched MAF lines were grown to 70% confluency and then harvested for RNA and protein extraction. Top: qPCR analysis of cyclin D1 transcript using primer pairs common to both transcript a and transcript b. Bottom: Immunoblot of cyclin D1 levels in Ccnd1+/+ and Ccnd1KI/KMAF lines using an antibody common to both isoforms.

Percentage of tumor-free mice post-injection of 1 million cells mixed with Matrigel (1:1 ratio) into the flanks of nude mice over a period of 10 months (n = 10 per genotype).

Indicated tumors were harvested 3 weeks after detectable tumor formation, fixed in formalin, and stained with H&E. Tumors were analyzed for features of malignancy by a board-certified veterinary pathologist. Arrows indicate the specific tumor-associated features noted. Top panels were taken at 40× magnification, and lower panels were taken at 400× magnification.

Ccnd1+/+ or Ccnd1KI/KI MAF lines were stably transfected with either cyclin D1a (+/+), cyclin D1b (KI/KI) or vector control constructs. After selection with puromycin, individual lines were assessed for the induction of cyclin D1a in the +/+ line and cyclin D1b in KI/KI lines via immunoblot.

One million cells from the indicated stable cell lines were injected subcutaneously into the flanks of nude mice and monitored for tumor formation over a period of 365 days. Indicated time points represent time of palpable tumor detection and are plotted as % of tumor-free mice over time.

Sections from KI/KI vec and KI/KI-D1b tumors were stained for the proliferative marker Ki67. Three random fields from each tumor were quantified for Ki67 positivity, and are plotted as the mean ratio of Ki67-positive cells/total cell number for each individual tumor.

Data information: Error bars represent ± SEM.
Consequence of the tumor-associated conversion to cyclin D1b. (2015)
Figure 4

Cyclin D1b cooperates with h-Ras to drive tumorigenesis

Passage-matched Ccnd1+/+ or Ccnd1KI/KI MAF lines were infected with lentivirus containing vector control or h-Ras constructs. Cells were selected with puromycin for 14 days and assayed for β-galactosidase activity. DAPI serves as a control for cell number. Images were taken at 200× magnification with insets taken at 400× magnification.

The indicated cell lines were grown in complete media and assayed for h-Ras expression via immunoblot. Lamin B serves as a loading control.

Cells were plated in soft agar and allowed to grow for a period of 3 weeks. Plates were then washed, fixed, and stained with 0.01% crystal violet. Colonies greater than 50 μm were counted and are plotted (top). Representative images of colony growth are shown for each cell line. Images were taken at 40× with insets taken at 200× magnification.

Indicated MAF lines were injected subcutaneously into the flanks of nude mice as in (A), and tumor incidence is plotted as % of tumor-free mice over time (n = 10).

Indicated tumors were harvested, fixed, and stained for the proliferative marker mKi67. Three random fields from each tumor were counted for mKi67 positivity, and are plotted as a % of total cell number, n = 5 (right: representative images). Images were taken at 400× magnification.

Data information: Boxes highlight area of magnified images. Error bars represent ± SEM, and statistical significance was determined using a Student's t-test. ***P < 0.001.
Consequence of the tumor-associated conversion to cyclin D1b. (2015)
Figure 5

Genetic switch to cyclin D1b promotes serum independence

Ccnd1+/+ and Ccnd1KI/KIMAF lines were arrested in G1, S, and G2/M phases of the cell cycle and expression of cyclin D1 isoforms determined by immunoblot. cyclin A2 and cyclin B1 serve as phase-specific cell cycle controls.

Cells were plated in serum-proficient media and protein lysates generated from each genetic line. Individual D1 cyclins were immunoblotted to verify genetic identity along with other essential cell cycle components. Lamin B serves as a control.

Indicated cells lines were immunoprecipitated for CDK4 and analyzed for their ability to incorporate 32P-ATP into full-length RB substrate. Radioactive counts were normalized to CDK4 activity of +/+ cell lines and adjusted for efficiency of CDK4 pull down as determined by densitometry. Data is representative of three independent biological replicates.

Indicated cell lines were plated in serum-proficient (10%) or serum-deficient (1%) media and total cell number counted at 24, 48, and 72 h in biological triplicate.

Ccnd1+/+ and Ccnd1KI/KI MAF lines were transfected with a validated pool of siRNA directed against the N-terminus of the murine cyclin D1 transcript or control siRNA in full serum. 48 h post-transfection cells were harvested and analyzed for biochemical markers of cell cycle kinetics via immunoblot. GAPDH and Lamin B serve as controls.

Cells were treated as (E), and incubated in the indicated serum concentration for 48 h. Cells were treated with BrdU for 1 h prior to harvesting and then stained for BrdU incorporation. Three random fields from each of three biological triplicates were counted for BrdU incorporation via immunofluorescence and data is represented as percent positive/total cell number.

Data information: Error bars represent ± SEM, and statistical significance was determined using a two-tailed Student's t-test. **P < 0.01, ***P < 0.001.