EMBO/soda-nel huggingface dataset:
Metronomic  chemotherapy  offsets  HIFα  induction  upon maximum-tolerated dose in metastatic cancers (2020)
Figure 4

Figure 4: HIFα transcriptional activation in human colon and breast cancers

A: Correlation matrix among statistically over-represented and biologically-validated HIFα targets in TCGA colon or breast cancer data. Spearman (ρ) coefficients were encoded in a pseudo-color scale wherein flattening of ellipses denotes increasing |ρ| values; blank cells indicate non-significant correlation pairs after Bonferroni post hoc comparisons. Transcripts are annotated using official gene symbols.

B: HIFα- inducible colon cancer signature (HIFi-CCS).

Left and middle/left: List of HIFα targets and overall survival in TCGA (left) or aggregated microarray data (middle/left) from colon adenocarcinomas. Cases are classified as low (blue) or high (red) HIFi-CCS according to their relationship to the median. Kaplan-Meier analysis followed by log-rank test.

Middle/right: HIFi-CCS and lymph node (LN) invasion status. Data are standardized as z-scores. t(194) = 4.399 and P< 0.0001 by Student t-test with Welch's correction.

Right: HIFi-CCS is associated with accelerated distant metastasis. Cases are classified as low (blue) or high (red) HIFi-CCS according to their relationship to the median. Kaplan-Meier analysis followed by log-rank test.

C: HIFα- inducible breast cancer signature (HIFi-BCS).

Left and middle/left: List of HIFα targets and overall survival in METABRIC (left) or recurrence- free survival in microarray (middle/left) from breast adenocarcinomas. Cases are classified as low (blue) or high (red) HIFi-BCS according to their relationship to the median. Kaplan-Meier analysis followed by log-rank test.

Middle/right: HIFi-BCS and estrogen receptor (ER) status. Data are standardized as z-scores. t(112) = 3.789 and P= 0.0002 by Student t-test with Welch's correction.

Right: HIFi-BCS is associated with worsened distant metastasis-free survival. Cases are classified as low (blue) or high (red) HIFi-BCS whenever they are below or above the distribution median. Kaplan-Meier analysis followed by log-rank test.

D: Median RNAseq expression of individual HIFα targets as split by median HIFi-CCS (left) or HIFi-BCS (right). Transcripts up/downregulated by ±1.5-fold are shown in blue or yellow, respectively. Error bars indicate 95% CI of the median. Log2 scale; n= 209 (colon, TCGA) or n= 609 (breast, METABRIC).

Data information: Gene expression omnibus accession numbers are indicated; HR, hazard ratios, brackets indicate 95% CIs; DMFS, distant metastasis- free survival; OS, overall survival; RFS, recurrence- free survival; n, number of patients. Violin plots present 50th (thick line), 25th and 75th percentiles (thin lines) Blue, low signature index (below median); red, high signature index (above median).
TERRA promotes telomerase-mediated telomere elongation in Schizosaccharomyces pombe (2016)
Figure 2

Figure 2. Polyadenylated TERRA interacts with telomerase.

A RIP experiments were performed using anti-myc antibodies and extracts from Trt1-myc expressing strains followed by qRT-PCR to detect G-rich TERRA, polyA+ TERRA, ARRET, TER1 and ACT1. Values represent fractions of input RNA detected in immunoprecipitated material expressed as fold increase over untagged (unt) wt strain. Bars and error bars are averages and SD from three independent experiments. * P < 0.05 (relative to unt; two-tailed Student’s t test).

B RIP experiments as in (A) where, prior to RNA extraction, washed beads were resuspended in DNaseI buffer (DNbuff) and incubated for 1 hour at 30° C in presence or absence of DNaseI. Values represent fractions of input polyA+ TERRA detected in immunoprecipitated material expressed as fold increase over an untagged (unt) wt strain. Bars and error bars are averages and SD from three independent experiments. * P < 0.05 (relative to unt; two-tailed Student’s t test).

C RIP experiments were performed using anti-myc antibodies and extracts from Trt1-myc expressing strains (either ter1+ or ter1Δ) cultured in YES medium for ~ 43 pds. Values represent polyA+ TERRA detected in immunoprecipitated material expressed as a fraction of the input (graph on the left) or after normalization to ACT1 RNA in the corresponding input (on the right), and expressed as fold increase over an untagged wt strain (unt ter1+). Bars and error bars are averages and SD from three independent experiments. * P < 0.05, ** P < 0.01, *** P < 0.001 (relative to unt ter1+; two-tailed Student’s t test).

D Western blot analysis of Trt1-myc in cells used in (C). Ponceau S staining serves as a loading control.
TERRA promotes telomerase-mediated telomere elongation in Schizosaccharomyces pombe (2016)
Figure 3

Figure 3. Characterization of transcriptionally-inducible telomeres (‘tiTELs’).

A Schematic representation of tiTELs. An nmt1 promoter (Pnmt1) was inserted upstream of the natural TERRA TSS. Telomeric repeats are in black and the subtelomeric tract in grey. A cassette conferring resistance to nourseothricin (natr) was used to select for positively transformed cells. The inserted cassette generates a novel HindIII restriction site, while the endogenous ApaI restriction site close to the telomeric tract is disrupted upon integration. Probes and oligonucleotides used for northern blot, Southern blot and RT-PCR analyses are indicated. The sketch is not to scale.

B Genomic DNA from wt cells (CAF13) and cells carrying two tiTELs (CAF110) maintained in YES medium was digested with HindIII and Southern blot-hybridized consecutively with the indicated probes. RET refers to telomeric sequences (tiTELs or natural telomeres, nTELs) retained in the upper part of the gel; REL refers to telomeric sequences released in the lower part of the gel. The black arrow points to the natural nmt1+ sequence on chromosome III. Marker molecular weights are on the left in kilobases.

C 5’ RACE analysis of the tiTERRA transcription start site using total RNA from cells carrying two tiTELs (CAF110) maintained in YES medium. Nucleotides boxed in blue overlap with the 5’ UTR of the nmt1+ transcript while the ones in green correspond to subtelomeric sequences. tiTERRA and TERRA transcription start sites (TSS) are indicated.

D qRT-PCR analysis of tiTERRA in cells carrying no tiTEL (CAF13), one tiTEL (CAF545) or two tiTELs (CAF110) cultured in EMM medium in presence or absence of Thiamine (THI; repressed and induced condition, respectively) for 24 h. RNA was reverse transcribed with oC and cDNA was amplified with oF3 and oR3 oligonucleotides to detect only tiTERRA (graph on the left) or with oF1 and oR1 oligonucleotides to detect simultaneously tiTERRA and natural TERRA (on the right). Values are normalized to ACT1 mRNA and expressed as fold increase over CAF545 in uninduced conditions (THI+). Bars and error bars are averages and SD from 3 independent experiments. * P < 0.05, ** P < 0.01 (relative to CAF545 THI+; two-tailed Student’s t test).

E Northern blot analysis of total RNA from wt cells and cells carrying two tiTELs (CAF110) treated with combinations of THI and Trichostatin A (TSA) for 24 h. Two identical membranes were hybridized in parallel with oligonucleotides corresponding to C-rich (oC) or G-rich (oG) telomeric repeats. To confirm similar hybridization efficiencies of the two oligonucleotides, northern blot membranes were simultaneously hybridized and exposed along with blots of digested genomic DNA (upper right insets). After signal detection, RNA membranes were stripped and re-hybridized with U6 probes to assure equal loading. Marker molecular weights are on the left in kilobases.
TERRA promotes telomerase-mediated telomere elongation in Schizosaccharomyces pombe (2016)
Figure 4

Figure 4. TiTERRA transcription induces telomerase-dependent tiTEL lengthening.

A Telomere restriction fragment analysis of genomic DNA from wt cells and cells carrying two tiTELs (CAF110) grown for 24 h in EMM medium containing THI and TSA as indicated. DNA was digested with HindIII and hybridized first with a nmt1 probe and successively with a telomeric probe. Marker molecular weights are on the left in kilobases. Numbers at the bottom indicate gel lanes.

B Genomic DNA from cells carrying two tiTELs and deleted for trt1+ (CAF113) was analyzed as in (A). Marker molecular weights are on the left in kilobases. Numbers at the bottom indicate gel lanes.

C ter1+ cells carrying two tiTELs (CAF110, left panel) or trt1Δ cells carrying one tiTEL (MKSP2104, central panel) were cultured in EMM medium in presence of THI for 24 h (THI+) or in absence of THI for 24, 48 or 72 h. In the right panel, cells carrying two tiTELs (CAF110) were treated with TSA for 24 h in the presence or absence of THI. TiTEL telomeric sequences were amplified from genomic DNA and sequenced using a PacBio platform. The statistical analysis for each set of conditions is displayed above each panel. P values for each comparison were derived from the Student’s t test between each condition and the THI+ sample using Welch’s correction when appropriate. The new population of longer tiTELs appearing in trt1Δ cells upon prolonged transcription induction is indicated in red (center panel).
1-Undecene from Pseudomonas aeruginosa is an olfactory signal for flight-or-fight response in Caenorhabditis elegans (2021)
Figure 1

Figure 1. Odor sensation controls C. elegans flight response to P. aeruginosa.

Time course of avoidance of P. aeruginosa lawn by N2, odr-3(n2150) and odr-3(n2046) worms. n ≥ 3 assays. *** P ≤ 0.001, **** P ≤ 0.0001 as determined by two-tailed unpaired t-test. Error bars indicate SEM.

Images of P. aeruginosa lawn at different time intervals showing avoidance response of N2 and odr-3(n2150) mutant worms. Scale bar = 5 mm.

Kaplan Meier survival curve of N2, odr-3(n2150) and odr-3(n2046) worms on partial lawn of P. aeruginosa at 25°C.

Kaplan Meier survival curve of N2, odr-3(n2150) and odr-3(n2046) worms on full lawn of P. aeruginosa at 25°C.

Time required for 50% of worms to die (TD50) on partial lawn and full lawn of P. aeruginosa. Individual data points indicate replicates with ~100 worms each. **** P ≤ 0.0001, ns (not significant) P > 0.05 as determined by two-tailed unpaired t-test. n ≥ 3 assays. Error bars indicate SEM.

Schematic of young (8 h) and old (24 h) lawn choice assay in a tripartite plate and choice index. n ≥ 3 assays. Individual data points indicate replicates with ~40 worms each. * P ≤ 0.05 as determined by two-tailed unpaired t-test. Error bars indicate SEM.

Representative image of intestinal lumen width, indicated by arrows, in N2 worm exposed to P. aeruginosa for 12 h. Scale bar = 50 µm.

Quantification for the width of intestinal lumen in N2 exposed to E. coli and P. aeruginosa for 12 h. ns (not significant) P > 0.05 as determined by two-tailed unpaired t-test. n = 3. Error bars indicate SEM.
1-Undecene from Pseudomonas aeruginosa is an olfactory signal for flight-or-fight response in Caenorhabditis elegans (2021)
Figure 4

Figure 4. 1-Undecene odor induces pathogen-specific immune response in C. elegans.

Real time PCR analysis of P. aeruginosa specific immune response genes in naive and 1-undecene odor exposed N2 worms. n = 3. * P ≤ 0.05, ** P ≤ 0.01 as determined by two-tailed unpaired t-test. Error bars indicate SEM. The negative values are arrived at by representing FC value less than 1 as (-1/FC).

Real time PCR analysis of irg-1, irg-2 and irg-3 genes in N2 and zip-2(tm4248) worms exposed to 1-undecene odor upon respective naive worms. n = 3. ns (not significant) P > 0.05, * P ≤ 0.05, ** P ≤ 0.01 as determined by two-tailed unpaired t-test. Error bars indicate SEM.

irg-1p::GFP induction in naive worms and worms exposed to P. aeruginosa (6 h) and 1-undecene odor (2 h). Scale bar = 500 µm.

Real time PCR analysis of irg-1, irg-2 and irg-3 genes in N2, odr-3(n2150) and odr-3(n2046) worms exposed to 1-undecene odor upon respective naive worms. n = 3. * P ≤ 0.05, ** P ≤ 0.01 as determined by two-tailed unpaired t-test. Error bars indicate SEM.

Real time PCR analysis of irg-1, irg-2 and irg-3 genes in N2, lim-4(ky403) and lim-4(yz12) worms exposed to 1-undecene odor upon respective naive worms. n = 3. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001 as determined by two-tailed unpaired t-test. Error bars indicate SEM.

Kaplan Meier survival curve on the full lawn of P. aeruginosa for N2 (naive) worms and N2 worms pre exposed to 1-undecene odor. Survival assay was performed at 20°C.

Time required for 50% of worms to die (TD50) on lawn of P. aeruginosa. Each data point indicates replicates with ~100 worms each. n = 3 assays. ** P ≤ 0.01 as determined by two-tailed unpaired t-test. Error bars indicate SEM.

Real time PCR analysis of irg-1, irg-2 and irg-3 genes in N2 worms exposed to odor of undA mutant upon PA14. n = 3. * P ≤ 0.05, ** P ≤ 0.01 as determined by two-tailed unpaired t-test. Error bars indicate SEM.
Differential regulation of glycinergic and GABAergic nanocolumns at mixed inhibitory synapses (2021)
Figure 1

Figure 1. Gephyrin SSDs and their alignment with pre-synaptic RIM1/2 in vivo.

(A-C) dSTORM imaging of gephyrin in sucrose impregnated cryosections of adult mouse spinal cord. (A) dSTORM detections of gephyrin (red dots) overlaid with the epifluorescence image (white with grey background). Synaptic clusters of gephyrin in dSTORM images were identified by the epifluorescence puncta. Scale bar: 1 µm. (B) Enlarged pointillist images of the two gephyrin clusters indicated in (A). (C) Rendered images of the two gephyrin clusters outlined with the boundaries of the epifluorescence mask. Gephyrin SSDs are indicated with red asterisks in (B) and (C). Scale bar: 200 nm.

(D) Two-color dSTORM imaging of RIM1/2 and gephyrin in cryosections. From left to right: rendered images of RIM1/2 and gephyrin clusters showing the aligned SSDs (arrows); gephyrin SSDs segmented by watershed outlined with different colors; RIM1/2 SSDs outlined with different colors; binary SSDs of gephyrin and RIM1/2. Inhibitory synapses were identified by the gephyrin clusters in the epifluorescence images. Scale bar: 200 nm. RIM1/2 was labelled with Alexa 647, gephyrin (mAb7a) with Cy3B in these experiments.
Differential regulation of glycinergic and GABAergic nanocolumns at mixed inhibitory synapses (2021)
Figure 2

Figure 2. Trans-synaptic alignment at inhibitory synapses in cultured spinal cord neurons.

(A-C) Alignment of SSDs of pre-synaptic RIM1/2 and SSDs of post-synaptic gephyrin, GlyRs, and GABAARs, respectively. (A1-C1) Distances were measured between the intensity peaks of paired SSDs in rendered dual-color dSTORM images (yellow line, values given in mean ± SD). RIM1/2 was labelled with Alexa 647 and gephyrin (mAb7a) with Cy3B in (A1). RIM1/2 was labelled with Cy3B, while GlyRs or GABAARs were labelled with Alexa 647 in (B1) and (C1). (A2-C2) Trans-synaptic alignment of SSDs of gephyrin, GlyRs, GABAARs and RIM1/2. SSDs were segmented by H-watershed. Data are plotted as mean ± SD. Number of synapses: n = 48 (A1, A2) from three independent experiments (no treatment), n = 30 (B1, B2) from two experiments with TTX treatment, n = 16 (C1, C2) from two experiments with TTX. Only synapses with side view profiles were included. All images are adjusted to the same scale. Scale bar: 200 nm.
Differential regulation of glycinergic and GABAergic nanocolumns at mixed inhibitory synapses (2021)
Figure 3

Figure 3. Differential organization of GlyRs and GABAARs at mixed inhibitory synapses.

(A-B) Representative dSTORM images of GlyRs and GABAARs at synapses in side view (A) or en face view (B). The top row of images shows the pointillist representation of the detections, the second row the rendered images and the bottom row the corresponding segmented SSDs. GlyRs were labelled with Alexa 647 and GABAARs with Cy3B in these experiments. Scale bar: 200 nm.

(C) Image correlation analysis (ICA) of GlyR and GABAAR clusters at the same PSDs. The ICQ values between GlyR and GABAAR (blue trace, 0.36 ± 0.06, n = 395 synapses from three independent experiments) are significantly lower than between GlyR clusters labelled with two secondary antibodies conjugated with different fluorophores (yellow trace, 0.38 ± 0.04, n = 218 synapses; MW test, p < 0.0001).

(D) The mean diameter of GlyR SSDs is 65 ± 29 nm (mean ± SD, n = 218 synapses from two independent experiments), that of GABAAR SSDs is 69 ± 30 nm (mean ± SD, n = 395 synapses from three independent experiments).

(E) Partial overlap between the SSDs of GlyRs and GABAARs. The fraction of overlapping SSDs was calculated based on the binarized images for each synapse (without selection for synapse orientation), dividing the number of overlapping SSDs by the total number of SSDs for each receptor. Data are shown as median, 25% and 75% quartiles of the population (only synaptic clusters with ≥ 2 SSDs in the respective channel; n = 183 for GABAAR-A647, n = 277 for GlyR-Cy3B, from three independent experiments). GlyRs labelled with both Alexa 647- and Cy3B-conjugated secondary antibodies for the same primary antibody were used as positive control (n = 158 GlyR-A647 and n = 171 GlyR-Cy3B clusters from two independent experiments). The GABAAR-GlyR overlap is significantly lower than the GlyR-GlyR match (KW test, p < 0.0001).

(F) Distance measurement between GlyR SSDs and GABAAR SSDs (blue bar; mean ± SD, n = 395 synapses from three independent experiments) and between double labelled GlyR SSDs (yellow, mean ± SD, n = 218 synapses from two independent experiments).
Differential regulation of glycinergic and GABAergic nanocolumns at mixed inhibitory synapses (2021)
Figure 4

Figure 4. Reduced GABAAR levels but not GlyRs at mixed inhibitory synapses by 4-AP treatment.

(A) Triple immuno-labelling of gephyrin, GlyRs and GABAARs at mixed inhibitory synapses (arrow) imaged with conventional fluorescence microscopy. Scale bar: 5 µm.

(B) Quantification of the apparent synaptic area (in pixels) of gephyrin clusters (KS test p < 0.0001).

(C) Lower fluorescence intensity of GABAARs but only a minor reduction of GlyRs at synaptic gephyrin clusters (KS test, p < 0.0001, both panels).

(D) Reduced GABAAR/GlyR intensity ratio at mixed inhibitory synapses (KS test, p < 0.0001). Number of synapses: n = 9416 in TTX and n = 6949 in 4-AP conditions from three independent experiments.
Differential regulation of glycinergic and GABAergic nanocolumns at mixed inhibitory synapses (2021)
Figure 5

Figure 5. Differential sub-synaptic re-organization of GABAARs and GlyRs by 4-AP treatment.

(A-E) Characterization of the nanoscale organization of GABAARs and GlyRs at mixed inhibitory synapses. (A) Synaptic receptor cluster rendered from detections of both GABAARs (labelled with Alexa 647) and GlyRs (labelled with Cy3B). (B) Detections of GABAARs (red dots) and GlyRs (blue dots) overlaid with binary synaptic mask (white). (C-E) Detections of GABAARs, but not GlyRs were strongly decreased at mixed synapses after 4-AP treatment (KS test, p < 0.0001 in C and E, p < 0.001 in D). Scale bar: 200 nm.

(F-J) Sub-synaptic characterization of GABAAR and GlyR domains. (F) Watershed segmentation of sub-synaptic domains (SSDs). Each SSD is shown in different colors. (G) Detections of GABAARs (red dots) and GlyRs (blue dots) overlaid with SSDs (white). (H-J) Detections of GABAARs but not GlyRs at the same SSDs were reduced after 4-AP treatment (KS, p < 0.0001 in H and J, p < 0.01 in I). Number of synapses: n = 439 in TTX, n = 531 in 4-AP condition, from three independent experiments.
Differential regulation of glycinergic and GABAergic nanocolumns at mixed inhibitory synapses (2021)
Figure 6

Figure 6. Reduced number of GABAAR SSDs after 4-AP treatment.

(A) The number of receptor SSDs, segmented from the combined receptor clusters of GABAARs and GlyRs, was decreased after 4-AP treatment (MW test, p < 0.0001).

(B) This was mostly due to the loss of GABAAR SSDs that were segmented from the rendered images of GABAAR detections only (MW, ****p < 0.0001).

(C) The number of GlyR SSDs (based on GlyR detections) were only marginally reduced (MW, **p < 0.01).

Data information: Number of synapses: n = 452 in TTX, n = 583 in 4-AP condition, from three independent experiments. Mean ± SEM. These analyses are based on the same dataset as in Figure 5.
Differential regulation of glycinergic and GABAergic nanocolumns at mixed inhibitory synapses (2021)
Figure 7

Figure 7. Reduction of gephyrin phosphorylation at synapses but not within SSDs by 4-AP.

(A-C) Reduced immuno-reactivity of S270 phosphorylated gephyrin but not total gephyrin levels after 4-AP treatment, revealed by conventional fluorescence microscopy (KS test, p < 0.001 in B1 and p < 0.0001 in B2, p < 0.0001 in C). Total gephyrin was probed with polyclonal rabbit primary antibody (rbGPHN), and pS270 phosphorylated gephyrin with monoclonal mouse primary antibody (m7a). Number of synapses: n = 4040 in TTX and n = 3818 in 4-AP conditions from two independent experiments. Scale bar: 2 µm.

(D-F) Reduced numbers of pS270 gephyrin (m7a) detections were recorded by dSTORM for the entire synaptic area (D, KS test, p < 0.01). The number of detections of pS270 gephyrin per SSD (E, KS, p = 0.18) and the number of SSDs (F, MW, p = 0.11) were not changed by 4-AP treatment. Gephyrin was probed with mAb7a antibody and Alexa 647 dye. Number of synapses: n = 810 in TTX and n = 727 in 4-AP conditions from three independent experiments; (F) mean ± SEM. Scale bar: 100 nm.
WASH inhibits autophagy through suppression of Beclin 1 ubiquitination (2013)
Figure 1

WASH deficiency causes embryonic lethality and extensive autophagy. (A) Strategy to generate WASH‐knockout (WASH−/−) mice. The coding exons of mWASH gene were shown as white boxes. The target vector with exon3 of mWASH gene was flanked with two loxP sites (arrow) and one neomycin resistance gene. The primers used for genotyping were KO primers for deficient genotypes, loxP primers for loxP site identification, and Southern blot probe for floxed mWASH gene. KO, knockout, m, mouse. (B) Detection of WASH gene targeting. The floxed WASH gene by gene targeting was analysed by Southern blotting (left panel) and PCR (middle panel). The genotypes of the offspring were analysed by PCR (right panel). (C) WASH deficiency is embryonic lethal. E11.5 of WASH deficiency showed a shrinked decidua. (D) Histological analysis of E7.5 WASH+/+ and WASH−/− embryos. Scale bar, 100 μm. (E) WASH‐deficient embryos do not undergo apoptosis. TUNEL assay was performed for apoptosis detection. PI, propidium iodide. FITC, fluoresceinisothiocyanate. Scale bar, 100 μm. (F) E7.5 embryos of WASH−/− mice present extensive autophagy. Ultrastructure of isolated E7.5 embryos was stained with antibody against LC3 and visualized by immuno‐electron microscopy. Black arrowhead indicates autophagosome. *, Cellular reference. Scale bar, 500 nm (upper panel). WASH+/+ or WASH−/− embryos were isolated, and LC3 conversion and p62 level were analysed by immunoblotting (lower panel). Bands were quantified and analysed by Image J and shown as means±s.d. ***P0.001. Above experiments were repeated for three independent times with similar results.Source data for this figure is available on the online supplementary information page.