EMBO/soda-nel huggingface dataset:
Reprogrammed mRNA translation drives resistance to therapeutic targeting of ribosome biogenesis (2020)
Figure 5

Figure 5: Metformin inhibits cAMP-EPAC1/2 signaling and re-sensitizes combination therapy-resistant early passage Eµ-Myc lymphoma cells to CX-5461-everolimus co-treatment in vivo

(A) Western analysis demonstrating the effects of metformin treatment for 48 hours on the levels of active GTP-bound RAP1 in CX and CMB cells (n=3) and its quantitation.

(B, C) Proportion of green fluorescent protein (GFP)-positive CMB (clone #8) cells in (B) lymph node and (C) spleen of transplanted C57BL/6 mice treated as indicated for 6 hours on day 12 post-transplant. Graphs represent mean ± SEM of 6 mice per group.

(D) Kaplan-Meier curve of C57BL/6 mice transplanted with CX-5461-everolimus-resistant (CMB #8) early-passage Eµ-Myc lymphoma cells treated with vehicles (everolimus vehicle: 1% methylcellulose; CX-5461/metformin vehicle: 25 mM NaH2PO­­­4) (n=6); CX-5461 (35 mg/kg every twice weekly) and everolimus (5 mg/kg daily) (n=8), metformin (400 mg/kg twice daily) (n=6), or CX-5461, everolimus and metformin (35 mg/kg twice weekly, 5 mg/kg daily and 400 mg/kg twice daily, respectively) (n=8). Light grey: 5-day metformin pre-treatment period, dark grey: treatment period. Data were analyzed by a log-rank (Mantel-Cox) test. Vehicle vs. CX-5461-everolimus: P = 0.0006, Vehicle vs. CX-5461-everolimus-metformin: P = 0.0001. CX-5461-everolimus vs. CX-5461-everolimus-metformin: P = 0.0003.

Data information: (A) Graphs represent mean ± SEM of n=3. Data were analyzed by Student's t test (A) or one-way ANOVA (B, C). ns, not significant, P≥ 0.05; *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001.

Expanded View Figure Legends
A Neuropathy-Associated Kinesin KIF1A Mutation Hyper-stabilizes the Motor-Neck Interaction during the ATPase Cycle (2021)
Figure 2

Figure 2 - Axonal transport of KIF1A cargoes by the WT and the β7 mutant E239K.

(A-F) Immunocytochemistry of DRG neurons and the corresponding quantifications of the indicated genotypes for surface TrkA (A), surface TRPV1 (B), Rab3A (C), synaptotagmin (D), DENN/MADD (E) and α-tubulin (F). TrkA and TRPV1 were visualized without permeabilization. ns, p > 0.05; *, p < 0.05; **, p < 0.01; ***, p < 0.001; Welch's t test from at least three independent cultures. Error bars show SEM. Scale bars, 2 µm.

(G-J) Anterograde (red) and Retrograde (blue) velocities of ECFP-TrkA (G), TagRFP-Rab3QL (H), TagRFP-synaptotagmin (I), and mitochondria labelled with MitoBright (J). ns, p > 0.05; **, p < 0.01; ****, p < 0.0001. ECFP-TrkA, n = 25­-72; TagRFP-Rab3QL, n = 31-71; TagRFP-synaptotagmin, n = 33-62; Mitochondria, n = 21-49 moving particles were quantified from at least three independent cultures and statistical analysis performed using unpaired t test. Error bars show SEM.
A Neuropathy-Associated Kinesin KIF1A Mutation Hyper-stabilizes the Motor-Neck Interaction during the ATPase Cycle (2021)
Figure 3

Figure 3 - Live-cell imaging of the WT KIF1A and the β7 mutant E239K.

(A) Fluorescence images of dissociated mouse DRG neurons at DIV5 transduced with adenoviral vectors encoding KIF1A-EGFP and KIF1A(E239K)-EGFP. The boxes show the axonal area for the time-lapse images. Scale bar, 50 µm.

(B, C) Time-lapse fluorescence images of DRG neurons expressing KIF1A-EGFP and KIF1A(E239K)-EGFP (B) and representative kymographs of signal movements (C). Arrowheads in (B) show the anterogradely moving particles. Scale bar in (B), 2 μm. See Movies EV1 and 2.

(D) Velocities of the moving particles calculated from the slopes of the kymographs. Data are shown as mean and SD from n = 49 moving particles of three independent cultures. ****, p < 0.0001; Unpaired t test.

(E) Direction of the moving particles at the start of observation. ns, p = 0.1484; chi-square test.

(F) Movements of KIF1A-EGFP and KIF1A(E239K)-EGFP expressed in KIF1A knockdown DRG and the scrambled control. White scale bar, 1 μm.

(G) Anterograde velocities of the indicated genotypes calculated from the kymograph slopes. Data are shown as mean and SD. KD+KIF1A-EGFP, n = 29; KD+KIF1A(E239K)-EGFP, n = 32; scrambled+KIF1A-EGFP, n = 44; scrambled+KIF1A(E239K)-EGFP, n = 42 from three independent cultures. ns, p = 0.3329; **, p < 0.01; ****, p < 0.0001; Unpaired t test.
A Neuropathy-Associated Kinesin KIF1A Mutation Hyper-stabilizes the Motor-Neck Interaction during the ATPase Cycle (2021)
Figure 4

Figure 4 - Effect of the β7 mutant E239K on KIF1A motility and kinetics in vitro.

(A) Representative kymographs of tetramethylrhodamine-labelled microtubule movement driven by the WT and the E239K mutant. The arrowheads represent the end positions of the WT and E239K.

(B) Movement of microtubules on glass driven by the WT and E239K. ****, p < 0.0001, chi-square test.

(C) Microtubule gliding velocity of the WT (green) and E239K mutant (magenta) determined by kymograph slope. Data and error bars in the inserted graph are presented as the mean ± SD. n = 269 microtubules for WT. n = 263 microtubules for E239K. ****, p < 0.0001, unpaired t test­.

(D) ATPase activity of WT (green) and E239K mutant (magenta) activated by microtubules. Data are represented as the mean ± SD. The data from at least three independent experiments were analysed. ****, p < 0.0001, two-way ANOVA.

(E) Measurements of the association and dissociation of mant-ATP. Nucleotide-free KIF1A(WT) and KIF1A(E239K) (final concentrations 2 µM) were rapidly mixed with varying concentrations of mant-ATP (2.5-25 µM) in a stopped-flow fluorometer. Representative transients at 25 µM, 12.5 µM and 2.5µM mant-ATP and fitted single-exponential function curves are shown. Data are represented as the mean ± SD. The data from three independent measurements were analysed.

(F) Rate constants (kobs) determined for the reaction of mant-ATP with nucleotide-free KIF1A(WT) and KIF1A(E239K) were plotted against concentration of mant-ATP. Data are represented as the mean ± SD. The data from three independent measurements were analysed.

(G) Measurements of the dissociation of mant-ADP for 80 s (left panel) and the enlarged view for the first 8 s (right panel). KIF1A(WT) and KIF1A(E239K) (final concentrations 50 µM) preloaded with mant-ADP were mixed into an excess of unlabeled ATP (5 mM) with or without taxol-microtubule. Fluorescence decreases were observed upon the dissociation of mant-ADP.

(H) Summary of the experimentally determined kinetics parameters of KIF1A(WT) and KIF1A(E239K). Data are represented as the mean value ±SD for mant-ATP measurements.
Loss of enteric neuronal Ndrg4 promotes colorectal cancer via increased release of Nid1 and Fbln2 (2021)
Figure 1

Figure 1. Loss of Ndrg4 does not alter intestinal morphology or physiology.

A Representative microscopic views of Ndrg4+/+ and Ndrg4-/- murine intestinal sections (n=4, 12 months of age) reveal an even distribution of alkaline phosphatase along the enterocyte brush border, and a similar number and distribution of Paneth cells (Lysozyme) in the small intestine; and neuroendocrine (Chromogranin A), Goblet (PAS+), and proliferating (Ki67) cells in the colon. Scale bars, 50 µm.

B, C Representative microscopic views of the myenteric plexus of Ndrg4+/+ and Ndrg4-/- murine colonic sections labeled with either TuJ1 and S100 (B, n=3) or HuC/D (C, n=6) did not reveal structural or organizational differences in the ganglionic network of Ndrg4+/+ and Ndrg4-/- mice. Scale bars, 100 µm.

D Quantification of the enteric neuronal cell number (HuC/D) shows a similar number of enteric neurons in the proximal and distal colon of Ndrg4+/+ and Ndrg4-/- mice (n=6).

E-I Gastrointestinal motility assays reveal a similar whole-gut transit time (E; n=12 vs. 12), with a similar weight per stool and stool water content of the fecal pellets (F, G; n=11 vs. 10), a comparable small intestinal transit (H; n=10 vs. 12) and colonic propulsion time (I; n=7 vs. 6) in Ndrg4-/- compared to Ndrg4+/+ mice.

Data information: All data are presented as mean ±SEM, with p-values determined using a two-tailed, unpaired t-test.
Loss of enteric neuronal Ndrg4 promotes colorectal cancer via increased release of Nid1 and Fbln2 (2021)
Figure 2

Figure 2. Enhanced intestinal adenoma progression in Ndrg4-/- mice crossed with APCmin/+ mice (A,C,E,G) or treated with azoxymethane (AOM; B,D,F,H), but not after epithelial loss of Ndrg4 (I-L).

A, B Ndrg4+/+ and Ndrg4-/- mice have a similar bodyweight (n=17 vs. 14 in A and n=25 vs. 19 in B).

C, D The total number of small intestinal (SI) and colonic adenomas that develops remains the same after deletion of Ndrg4 (n=18 vs. 15 in C and n=25 vs. 19 in D).

E-H Ndrg4-/- mice develop significantly enlarged small intestinal (SI) polyps (E, G) and colon adenomas (F, H). N=10 vs. 13 in E and n=20 vs. 17 in F)

I-L In contrast, Ndrg4fl/fl and Ndrg4fl/fl-VillinCre mice have a similar bodyweight (I) and develop an equal number of adenomas (J) with an equivalent diameter (K,L). N=13 vs. 12 in I, n=13 vs. 11 in J and n=10 vs. 8 in K.

Data information: All data are presented as mean ± SEM with p-values determined using a two-tailed, unpaired t-test. Scale bars in (G, H, L), 50 µm. Intestinal adenomas are delineated with a blue dotted line in (G, H, L).
Loss of enteric neuronal Ndrg4 promotes colorectal cancer via increased release of Nid1 and Fbln2 (2021)
Figure 3

Figure 3. Medium derived from Ndrg4-/- ENS cultures boosts organoid development.

A-C Relative Ndrg4 mRNA (A) and Ndrg4 protein expression by Western blotting (B) and immunofluorescence (C) confirms absence of Ndrg4 in enteric neurons of Ndrg4-/- mice (n=3). EIF4E is used as a loading control in (B).

D qRT-PCR analysis of the Ndrg4+/+ and Ndrg4-/- ENS cells used in the co-culture model showed that Ndrg4-/- ENS cultures have reduced levels of vesicle trafficking-related genes, with the expression of Rabac1 (78.3%), Nsf (12.8%), Vamp2 (49.2%), Snap25 (13.3%), Stx1a (8.0%), Rab3a (48.1%), Stx1bp (18.3%), Caly (82.1%) and Cadps (60.4%) being down-regulated in Ndrg4-/- ENS cultures compared to Ndrg4+/+ ENS cultures. Data are derived from three independent experiments and log10 transformed ± SEM, with p-values determined using a Mann-Whitney U test.

E, F Compared to Ndrg4+/+ ENS-derived medium, addition of medium derived from Ndrg4-/- ENS cultures enhances the relative growth of intestinal epithelial organoids (E, IEOs) and accelerates the formation of new crypt buds per IEO in the course of five days (F).

G Medium from both Ndrg4+/+ and Ndrg4-/- ENS cultures ensures IEOs progression from crypt to (entero-) sphere, budding enterosphere, enteroid and a microadenoma-like structure.

H After five days of culture, significantly more nonviable structures are formed upon influence of Ndrg4-/- ENS-derived medium.

Data information: The data represented in panel E-H are derived from three independent co-culture experiments with ± 35 IEOs per experimental condition. Data are shown as mean ± SEM (A, E, F). P-values are calculated using a two-tailed, unpaired t-test (A, E ,F) or logistic regression (H). Scale bars in (C and G), 50 µm.
Loss of enteric neuronal Ndrg4 promotes colorectal cancer via increased release of Nid1 and Fbln2 (2021)
Figure 4

Figure 4. Nidogen-1 (Nid1) and Fibulin-2 (Fbln2) are highly present in the Ndrg4-/- ENS cell secretome and expressed within the ENS.

A Box plot showing the quantitative protein expression results of nanoLC-MS/MS analysis reveals the significantly higher presence of Nid1 and Fbln2 in the Ndrg4-/- compared to the Ndrg4+/+ ENS cell secretome (n=4; NSAF, normalized spectral abundance factor). Data are analyzed with R version 3.5.2 and the ibb R package. Each dot within the box plot represents the NSAF of an individual sample; the inside band reflects the median, and the bottom and top of the box the first and third quartile, respectively. The whiskers reflect the minimum and maximal values within 1.5× the interquartile range. NSAF values were compared using the Mann-Whitney U test.

B Data derived from the Linnarsson mouse database (Linnarsson) displays the expression of Nid1 and Fbln2 within cells of the central, peripheral and enteric nervous system. Unit color scale: Expression (log(UMI)); UMI, unique molecular identifier. Abbreviations: ENS, enteric nervous system (i.e. enteric neurons and glial cells); OL, oligodendrocytes; Exc N, excitatory neurons in CNS; Hippo, hippocampus; MB, midbrain; HB, hindbrain; Str, striatum; Hyp, hypothalamus; BG, basal ganglia; OB, olfactory bulb; CB, cerebellum; SC, spinal cord; PNS, peripheral nervous system; Glia, glial cells; Vasc, vasculature.

C-E Representative immunohistochemistry (C) and more detailed immunofluorescence (D, E) labeling indicate that Nid1 (C, D) and Fbln2 (C, E) are expressed within the myenteric plexus and by primary ENS-cells ((C), black arrowheads, scale bars, 50 µm; (D, E), n=3, scale bars, 10 µm).
Loss of enteric neuronal Ndrg4 promotes colorectal cancer via increased release of Nid1 and Fbln2 (2021)
Figure 5

Figure 5. NID1 and FBLN2 enhance HIO proliferation and CRC cell migration in vitro and are significantly up-regulated in the cancer tissue secretome.

A Representative microscopic views show that NID1 and FBLN2 are highly expressed in the human colonic ganglia (Black arrowheads) and interconnecting nerve fibers, and in a limited level throughout the epithelium (n=3). Scale bar, 50 µm.

B Addition of NID1&FBLN2 to the Matrigel® dome significantly enhances the relative growth rate of HIOs after five days of culture as compared to the PBS control condition.

C Compared to the PBS-control condition (white), stimulation with NID1&FBLN2 (black) enhanced the proliferation rate of HCT116 cells after 72 hours, but did not affect Caco-2 cell proliferation.

D The migration rate of HCT116 cells was already significantly increased 24 hours after addition of NID1&FBLN2 (black), whereas the migration of Caco-2 cells only significantly increased after 48 hours of culture with NID1&FBLN2 (black).

E Box plot reflecting the quantitative nanoLC-MS/MS analysis results shows the up-regulation of NID1 and FBLN2 in the colorectal cancer tissue secretome (n=17) compared to the normal colon tissue secretome (n=17). Data are analyzed with R version 3.5.2 and the ibb R package. Within the box plots, each dot represents the log10 transformed raw count of an individual sample; the inside band is the median value; the bottom and top of the box the first and third quartile, respectively; and the whiskers reflect the minimum and maximal values within 1.5× the interquartile range.

F Representative images of human colonic cancer epithelium (n=3) shows a high scattered expression of NID1 and FBLN2 within the cancerous epithelium. Scale bar, 50 μm.

Data information: Data in B are represented as relative mean percentage ± SEM versus day 0, with the p-value determined using a two-tailed, unpaired t-test. Data in C and D are derived from three independent experiments and are represented as mean ± SEM, with p-values determined using a two-tailed, unpaired t-test. To compare the paired normal colon and CRC secretome data in E (n=17), a paired Beta-Binominal test, taking into account the sample origin (i.e. comparing protein signatures between tissues derived from the same patient) was performed (de Wit et al., 2014; Pham & Jimenez, 2012).

Expanded View Figure legends
Upfront admixing antibodies and EGFR inhibitors preempts sequential treatments in lung cancer models (2021)
Figure 1

Figure 1. Combinations of mAbs and EGFR-specific TKIs downregulate in vitro several receptors for survival factors and effectively block the ERK and AKT pathways.

(A) NSCLC expressing single site mutants of EGFR, PC9 (3X106) or H3255 (8X106), were seeded in 10-cm dishes. On the next day, complete media were replaced with media containing serum (1%) and the cells were treated for 24 hours with different EGFR-specific TKIs (erlotinib, 50 nM; osimertinib, 50 nM, or afatinib, 10 nM), either alone or in combination with 2XmAbs (cetuximab and trastuzumab, 5 μg/ml each). Thereafter, cells were washed with cold saline and extracted. Proteins were separated using gel electrophoresis and transferred onto nitrocellulose membranes. After blocking, membranes were incubated overnight with the indicated primary antibodies, followed by incubation with horseradish peroxidase-conjugated secondary antibodies (60 minutes), and treatment with Clarity™ Western ECL Blotting Substrates (Bio-Rad). ECL signals were detected using the ChemiDoc™ Imaging System (Bio-Rad) and images were acquired using the ImageLab software. Signals (relative to Control) were quantified and normalized to the signals of GAPDH (numbers shown below each lane).

(B) PC9 cells (1X106) were seeded in 6-well plates and treated as in (A). After washing with acidic buffer (glycine 100 mM, pH 3.0), cells were incubated with fluorescently-labelled antibodies against EGFR, HER2 and HER3, and surface expression levels of each receptor were analyzed using flow cytometry. The normalized fluorescence intensity is shown as averages + SEM of four experiments. Significance was assessed using two-way ANOVA followed by Dunnett's multiple comparisons test. Note that non-significant comparisons are not shown. See p-values in Appendix Table S2.

(C) PC9 cells were seeded on coverslips and treated for 24 hours as in (A). Cells were washed in acidic buffer, fixed in paraformaldehyde (4%) and incubated with specific primary antibodies, followed by an Alexa Fluor 555-conjugated secondary antibody (pseudo colored in green). DAPI (blue) was used to stain nuclei. Images were captured using a confocal microscope (40X magnification). Scale bars, 40 μm.
De novo mutations in the autophagy gene WDR45 cause static encephalopathy of childhood with neurodegeneration in adulthood (2013)
figf3

(a) Immunoblot analysis of the WIPI4 protein (apparent mobility at ∼35 kDa) in LCLs (top). Truncated forms were not detected (middle). HSP90 was used as a loading control (bottom). An asterisk indicates nonspecific immunoreactive bands. (b) Cells were treated with 250 nM Torin1 in the presence or absence of 20 μM chloroquine for 2 h. Cell lysates were analyzed by SDS-PAGE and immunoblotting using antibodies to LC3, WIPI4 and HSP90. The positions of LC3-I (cytosolic) and LC3-II (membrane bound) are indicated. (c–e) Cells were cultured in the presence of Torin1 for 2 h. (c) Cytospun cells were fixed and analyzed by immunofluorescence microscopy using antibodies to LC3 and ATG9A. Abnormal colocalization of LC3 with ATG9A was observed in the LCLs of affected subjects. Scale bars, 10 μm and 1 μm in the inset. (d,e) The numbers of LC3+ (d) and LC3+ATG9A+ (e) foci were quantified from more than 20 images from 3 independent samples (Online Methods). Data are presented as mean ± s.e.m. *P 0.05, ANOVA followed by Bonferroni-Dunn post-hoc test.
PrimPol-mediated repriming facilitates replication traverse of DNA interstrand crosslinks (2021)
Figure 1

Figure 1. PrimPol interacts with factors involved in ICL traverse.

A. Experimental design for the proteomic identification of PrimPol-interacting factors in S phase-synchronized cells. Immunoblots showing PrimPol levels in WT and KO cells. MEK2 is shown as loading control.

B. Volcano plot showing statistical significance (-log p-value) vs enrichment ratio (log2) of identified proteins after PrimPol immunoprecipitation in WT and KO cells. The positions of PrimPol, RPA1-3, BLM, RMI1, RMI2, MHF1, SSBP1, RUNX1, HERC2 and BCCIP proteins are indicated.

C. Volcano plot showing statistical significance (-log p-value) vs enrichment ratio (log2) of identified proteins after PrimPol immunoprecipitation in WT cells with or without MMC (2 µg/mL; 5 h). BLM, RMI1, RMI2, MHF1, SSBP1, RUNX1, HERC2 and BCCIP proteins are indicated.

D. Immunoblot detection of the indicated proteins in whole cell extracts (WCE), PrimPol immunoprecipitates (IP), and non-precipitated flow-through fractions (FT). IgG shows the presence of anti-PrimPol antibody in the IP fractions (lanes 4-6).
PrimPol-mediated repriming facilitates replication traverse of DNA interstrand crosslinks (2021)
Figure 3

Figure 3. PrimPol mediates the ICL traverse reaction through its primase activity.

A. Experimental design and examples of stretched DNA fibers with different patterns of DNA synthesis around the ICL lesion. Fibers were stained with anti-CldU (red), anti-IdU (green) and anti-Dig (magenta). Schematics over the fiber images highlight the position of the ICL (circle) in each replicative structure. Scale bar, 10 µm.

B. PrimPol mutants used in the experiments. AxA is a PrimPol catalytic mutant. ∆Zn and CH are primase-null, polymerase-proficient PrimPol mutants. RBDm is a PrimPol mutant defective for RPA-binding.

C. Histograms show the percentage of the different replication patterns observed in each experimental condition (average and SD of three assays). Circle dots in each column represent the values of individual replicates. Statistical analysis: two-way ANOVA followed by Bonferroni post-test comparing each condition to the WT. p-values of individual comparisons are indicated. Immunoblots showing the levels of V5-PrimPol proteins (WT and mutant derivatives). Ponceau-S staining is shown as loading control.

D. Experimental design of ICL-localized DNA fibers followed by S1 nuclease incubation. Hypothetical fiber containing ssDNA downstream of the ICL (circle) turns into a DSB after S1 nuclease action. This would lead to an increase in apparent stalled forks or shortening of IdU length downstream of the lesion.

E. Histograms show the percentage of the different replication patterns observed in each experimental condition (average and SD of two assays). Circle dots in each column represent the values of individual replicates. Statistical analysis: two-way ANOVA followed by Bonferroni post-test. p-values of individual comparisons are indicated.

F. Dot plot shows the distribution and median (horizontal red line) of IdU track length after an event of ICL traverse in each condition. Data pooled from two replicates are represented (n ≥100 cells per condition). Statistical analysis was conducted with Kruskal-Wallis test and Dunns post-test. p-values of individual comparisons are indicated.

G. Histograms show the percentage of the different replication patterns observed in each experimental condition (average and SD of two assays). Circle dots in each column represent the values of individual replicates. Statistical analysis: two-way ANOVA followed by Bonferroni post-test. p-values of individual comparisons are indicated.

H. Immunoblots showing the levels of BLM, FANCD2 and PrimPol after BLM or FANCD2 downregulation in WT and PRIMPOL KO cells for experiments in (G). Ponceau-S is shown as loading control.