EMBO/SourceData huggingface dataset:
f4a) Induction of autophagy in response to 5-FU in the GFP::LGG-1 reporter strain fed on RNAi targeting the indicated DNA repair genes (scale bars, 10 μm).(b) The fraction (as % of untreated control) of hermaphrodites having embryos with excessive GFP expression following 5-FU treatment. Autophagy induction in independent experiments (circles) and the mean (line) are presented.(c) F1 survival after control (black diamonds) or bec-1 RNAi (red squares) in N2 and msh-6(pk2504) mutants. The survival curve shows the mean±s.d. for each data point from three independent experiments.
f5(a) Expression of ATF-2::GFP in 5-FU-treated hermaphrodites fed control or RNAi targeting the indicated genes (scale bars, 20 μm). (b) The number of animals harbouring embryos with excessive ATF-2::GFP expression was scored in at least 150 animals and given as the fraction (%) of the total.(c) N2 or msh-6(pk2504) embryos were continuously monitored under differential interference contrast microscopy (scale bars, 5 μm) from the three-cell stage until completion of the four-cell stage. Magnified images of representative nuclei are shown. (d) The fraction of embryos with visible, enlarged nucleoli (arrows) after 5-FU treatment was scored. Bar graphs represent mean±s.d. from three independent experiments.
f6(a) Western blots showing autophagy induction by monitoring LC3 levels in whole-cell extracts from U2OS cells treated or not with 5-FU alone or in combination with Mx for the indicated time. Staurosporin was used as a positive control for apoptosis induction shown by the appearance of cleaved fragments of caspase-3 (17 and 19 kDa) and poly (ADP-ribose) polymerase 1 (PARP1; ΔPARP, 85 kDa). α−Actin was used as loading control. (b) Quantification of LC3-II levels in cells treated as in a. Intensities of the lower LC3 band (LC3-II) was normalized to those of actin.. (c) Autophagy induction demonstrated by accumulation of LC3-positive puncta in U2OS cells treated or not with 5-FU alone or in combination with Mx for 48 h. The cells were fixed and prepared for immunofluorescence of LC3 (green). DAPI staining is shown in blue. Scale bar, 20 μm. (d) Quantification of LC3-positive puncta in cells treated as in c. In each separate experiment, 200-1,600 cells were scored per condition.e) Western blots showing absence of LC3-II accumulation in U2OS cells transfected with siRNA against MSH-2. The same blot probed with anti-MSH-2 antibodies confirm efficient knockdown. (f) Quantification of LC3-II levels in cells transfected with control or MSH-2 siRNA with or without FU treatment for 24 h.(g) Immunofluorescence showing reduced accumulation of LC3 puncta in U2OS cells transfected with siRNA against MSH-2. (h) Quantification of LC3-positive puncta in cells treated as in g.(i) Quantification of normalized RPS3 protein level (black bars) or RPS3 mRNA level (white bars) in U2OS cells after 48 h 5-FU treatment. RPS3 mRNA levels were determined by quantitative real-time reverse transcriptase-PCR.
Figure 1 (B) When transposon insertion libraries subjected to ampicillin resistance selection, a substantial increase in transposon insertion frequency in the pstSCA genes was observed in strains containing the protein folding sensor (pBR322 bla::Im7 L53A I54A) relative to a strain containing just pBR322. Transposon insertion frequency increased 21-, 24-, and 8-fold in the pstS, pstA, and pstC genes, respectively, compared to the insertion frequency found with no ampicillin selection.
Figure 2 (A) To verify that disruption of the pst genes is sufficient to increase ampicillin resistance of strains containing the pBR322 bla::Im7 L53A I54A folding biosensor and to test which of the genes in the pst operon are sufficient for this effect, pBR322 and pBR322 bla::Im7 L53A I54A were transformed into strains containing in-frame deletion mutants of each of the pst genes (pstS, pstC, and pstA, respectively). The deletion mutants of each of these pst genes showed higher ampicillin resistance than did the WT strain MG1655 in the presence of pBR322 bla::Im7 L53A I54A, but did not shower higher ampicillin resistance in the presence of pBR322.  (B) Levels of β-lactamase (Bla) and β-lactamase-Im7 L53A I54A were detected by Western blotting using a polyclonal antibody recognizing β-lactamase in the WT strain MG1655 and in isogenic strains containing insertions in the pstSCA genes (CL248 (pstS::Tn5), CL249 (pstC::Tn5), CL250 (pstA::Tn5)). Maltose binding protein (MBP) was used as a control.  (C) β-lactamase enzymatic activities present in periplasmic extracts of the Im7 folding biosensor were monitored in pst WT and pstSCA mutant strains using a spectrophotometric assay. Data are represented as mean ± SEM from three technical replicates (unpaired t-test,** P<0.01, *** P<0.001 comparison to MG1655; ns, not significant).  (D) To test if levels of the Im7 L53A I54A protein itself, in the absence of the fusion, were also higher in the pstA mutant, expression of the Im7 L53A I54A protein from the IPTG-inducible plasmid pCDFTrc was induced using IPTG concentrations ranging from 0-5 mM. Periplasmic fractions were isolated and examined by SDS-PAGE and subsequent Coomassie staining to determine overall protein expression patterns. Im7 L53A I54A levels were determined by Western blotting using an antibody for Im7. MBP was used as a loading control. Im7 L53A I54A was increased in the pstA strain relative to the WT control strain. Coomassie staining revealed that a number of periplasmic proteins, including UgpB, were also induced in the pstA strain.
Figure 3 (A) MG1655 WT, single mutants of pstA and ugpB, and a double mutant of pstA ugpB were transformed with pBR322 and various pBR322-based folding biosensor constructs. The transformants were examined by a spot titer assay on LB agar plates containing various ampicillin concentrations. Strains containing pBR322 bla::Im7 L53A I54A gain considerable ampicillin resistance in the presence of a pstA mutation (compare uppermost left and right panels). This resistance is completely lost upon transducing in a ugpB mutation (compare next panels down), showing that ugpB is responsible for the resistance gained by the pstA mutation.  (B) Ampicillin sensitivity conferred by a ugpB deletion mutant (CL543) is complemented by UgpB expression from the arabinose inducible expression vector pBAD33 ugpB (pCL1).  (C) UgpB expression is sufficient to lead to higher levels of Im7 L53A I54A protein. Im7 L53A I54A and UgpB were expressed in a ugpB mutant strain (CL543) from pCDFTrc and pTrc99a vectors, respectively, both of which have an IPTG-inducible promoter. Periplasmic fractions were isolated and examined by Western blotting. "-" indicates strains containing empty vector and "+" indicates strains containing a recombinant plasmid encoding either Im7 L53A I54A or UgpB. MBP was used as a loading control. Data information: These experiments were repeated twice with similar results and one representative is shown.
Figure 4(A) UgpB overexpression confers bile salt resistance, and a ugpB deletion mutant is slightly CHO sensitive. (B) CHO-induced MDH protein aggregation analyzed by monitoring light scattering.  (C) CHO-induced MDH protein aggregation analyzed by a solubility assay using centrifugation (cfg) at 16,000 g for 20 min. The supernatant (Sup) represents the soluble fraction and the pellet the insoluble fraction; 12 µM MDH was used as a model aggregation-prone substrate (see Methods). Addition of increasing quantities of UgpB increasingly solubilizes MDH. The molar ratios of MDH:UgpB that were used (left to right) are as follows: 1:0, 1:0.1, 1:0.2, 1:0.5, and 1:1. 5-fold more of the pellet fraction was loaded relative to the supernatant fraction.  (D) The supernatant/pellet assay was performed using a 1:1 MDH:chaperone ratio for Spy, SurA, and OsmY. The pellet fraction was concentrated 5-fold.  (E) 12 µM of MDH alone and a 1:1 ratio of either MDH:UgpB or MDH:MBP were incubated at 37°C for 60 min in the presence of CHO and then fractionated via centrifugation for use in a solubility assay as described above (C) . The pellet fraction was concentrated 5-fold. In some samples (+G3P), 120 µM of G3P was added prior to the addition of any other protein. Addition of G3P inactivates UgpB's ability to inhibit protein aggregation.  (F) 800 μg of E. coli total lysate and 60 µM of UgpB/MBP were used for the supernatant/pellet assay. 600 µM of G3P was added before adding proteins. The pellet fraction was concentrated 10-fold. As before, addition of UgpB inhibited aggregation, but only in the absence of G3P.  (G) The anti-aggregation activity of tryptophan single mutant (W169S) and double mutant (W169S/W172S) was examined by CHO-induced MDH aggregation assay. Light scattering values were monitored.  (I) The anti-aggregation activity of the UgpB variants (E66A and R374V) was examined by CHO-induced MDH aggregation assay. CHO-induced MDH protein aggregation analyzed by monitoring light scattering.  (J) Solubility of MDH is also monitored by supernatant/pellet assay. 12 µM of MDH alone and a 1:1 ratio of MDH:UgpB proteins, 120 µM of G3P were used. 15 mM CHO were used for all the aggregation and supernatant/pellet assays.
Figure 6(A) Acid denatures UgpB. The secondary structure of 2 µM UgpB was monitored by circular dichroism (CD) spectroscopy in 10 mM sodium phosphate buffer at pH 2 and pH 7. (B) G3P binds to acid-stripped UgpB as observed by monitoring the tryptophan fluorescence spectrum, either at pH7 or after transition from pH 2 to 7 (see Methods). Transitions from pH 2 to 7 in the presence of G3P are accompanied by a blue shift, indicating G3P binding.  (C) The anti-aggregation activity of UgpB is monitored by CHO-induced MDH aggregation assay. Light scattering values were monitored. The anti-aggregation activity of UgpB is activated after transition from pH 2 to 7 and the addition of G3P (120 µM) can again inactivate its chaperone activity.  (D) Addition of G3P to UgpB inhibits its anti-aggregation chaperone activity . 12 µM of MDH and a 1:1 ratio of UgpB:MDH were used in a CHO-induced aggregation assay. At 30 min, the indicated concentrations of G3P were added to solutions containing MDH, and MDH aggregation was monitored by light scattering. Note that G3P binding to UgpB still occurs in the presence of 15 mM CHO as monitored by tryptophan fluorescence
Figure 1B Growth curves of the indicated strains shown as population doublings (pds) on the y axis and hours in liquid culture on the x axis. Before inoculation in liquid YES medium, single cells underwent approximately 25 pds to produce visible colonies on solid medium. For each strain, averages and SDs (error bars) of three biological replicates are shown. Arrows indicate times of harvesting and corresponding pds for experiments shown in (C) and (D).C Telo-PCR analysis of telomere length in wt (ter1+ and est1+) and two independent clones (a and b) deleted for ter1+ or est1+ grown for ~ 25+10 (~ 35), ~ 25+14 (~ 39) and ~ 25+18 (~ 43) pds in YES medium. PCRs were performed with the subtelomeric oligonucleotide oF5, located 620 bp upstream of the first telomeric repeat, and the oligo(dG)-containing oligonucleotide odG18. Marker (m) molecular weights are on the left in kilobases. tels: telomere-containing Telo-PCR products.D qRT-PCR analysis of G-rich TERRA, polyA+ TERRA and ARRET/αARRET levels in cells as in (B). Values are normalized to ACT1 mRNA and expressed as fold increase over control wt strains (ter1+ and est1+) grown in parallel. Bars and error bars are averages and SD from 4 (ter1Δ clones) or 3 (est1Δ clones) independent experiments. * P < 0.05, ** P < 0.01, *** P < 0.001 (relative to wt; two-tailed Student's t test).E ChIP experiments with anti-RNA polymerase II (RPII) antibodies and extracts from ter1+ and ter1Δ cells grown for ~ 43 pds. Quantitative PCRs were performed using oligonucleotides oF1 + oR1 (subtel) or oligonucleotides amplifying a region from the actively transcribed act1+ locus. Values are expressed as fold increase over ter1+. Bars and error bars are averages and SD from three independent experiments. * P < 0.05 (relative to ter1+; two-tailed Student's t test). The western blot to the right shows no difference in RNPII levels between ter1+ and ter1Δ cells used in ChIP experiments. Ponceau Sstaining serves as loading control.G Western blot analysis of cellular fractions from ter1+ and ter1Δ cells grown for ~ 43 pds. Total proteins (tot) and equivalents of soluble (sol) and insoluble (ins) proteins were loaded. Actin serves as a control for soluble proteins, H3K9ac for chromatin-bound proteins.H qRT-PCR analysis of total TERRA and polyA+ TERRA in cellular fractions as in (G). Values are expressed after normalization to total cellular extracts. Bars and error bars are averages and SD from 3 independent experiments.
Figure 2A 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.
Figure 3B 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.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, RNAmembranes were stripped and re-hybridized with U6 probes to assure equal loading. Marker molecular weights are on the left in kilobases.
Figure 4A 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).
Figure 5A qRT-PCR analysis of G-rich and polyA+ tiTERRA in cells carrying one tiTEL and expressing Trt1-myc (CAF610) cultured in EMM medium with the indicated combinations of THI and TSA for 24 h. Values are normalized to ACT1 mRNA and expressed as fold increase over THI+ TSA- samples. Bars and error bars are averages and SD from 4 independent experiments. * P < 0.05, ** P < 0.01, *** P < 0.001, (relative to THI+ TSA-; two-tailed Student's t test).B Telo-PCR analysis of tiTELs using genomic DNA extracted from cells as in (A). Marker (m) molecular weights are on the right in base pairs.C RIP experiments performed using anti-myc antibodies and extracts from cells as in (A) followed by qRT-PCR analysis of G-rich and polyA+ tiTERRA. Values correspond to fraction of input RNA expressed as fold increase over an untagged (unt) control tiTEL strain (CAF545). Bars and error bars are averages and SD from three independent experiments. * P < 0.05, ** P < 0.01 (relative to unt; two-tailed Student's t test).D ChIP experiments performed using anti-myc antibodies and extracts from cells as in (A) followed by qPCR using tiTEL specific oligonucleotides. Values correspond to fraction of input DNA expressed as fold increase over THI+ TSA-. Unt: untagged strain (CAF545). Bars and error bars are averages and SD from at least three independent experiments. * P < 0.05, ** P < 0.01 (relative to THI+ TSA-; two-tailed Student's t test).
Figure 1A) MALDI imaging was performed on tumor-containing brain sections at a lateral resolution of 100µm and (M-H) ions were analysed in negative mode with a FTICR Mass Spectrometer (analytical range 100-1000Da). Hematoxylin-eosin (HE) staining of the same sections was performed after MALDI analysis. Patient-derived glioma xenografts (PDXs) with IDH wildtype (IDHwt), IDH mutant (IDHm), and control brain tissue (CB) were analyzed in a non-targeted and targeted approach using regions of interest (ROI) of different sizes to compare tumor (T, yellow line) and contralateral control brain (CB, blue line). The non-targeted approach was used for statistical pair-wise comparisons between samples, while the targeted approach on a selected set of metabolites allowed distribution and quantification analyses.B) Histological sections of 3 IDHwt PDXs (P3, T434, P8) and 3 IDHm PDXs ((T394, E478, T186) showing a large ROI (boxed rectangle, >500 pixels) within the tumor area applied for targeted quantification of selected metabolites. Left rectangle in P3 PDX was used as control contralateral brain (CB) for quantifications. Middle panel shows tissue distribution of D-2-hydroxyglutarate (D2HG) by MSI, which is exclusively detected in IDHm PDXs (D2HG in IDHwt tumours is below detection limit). Right panel shows tissue distribution of a key metabolite (m/z 778.51) presenting strong accumulation in IDHm lower grade gliomas (LGG) versus glioblastomas (GBM) independent of IDH status. An intensity-dependent color code indicates the relative amount of a specific compound (defined by m/z value) throughout the tissue section.C) Quantification of several high mass metabolites differentially present in IDHm PDXs (IDHwt glioblastomas (GBM) in blue, IDHm GBM in orange, IDHm lower grade gliomas (LGG) in red, contralateral control brain (CB) in grey). Box plots represent log values of metabolite intensities measured within a large ROI (>500 pixels). Many metabolites in this mass range (m/z 700-900) have been putatively assigned to phospholipids (Tables EV2-3). m/z represents the mass over charge ratio of ionized metabolites, as measured by the mass spectrometer. Except where otherwise indicated, statistical significance was calculated for IDHm (n=3) versus IDHwt (n=3) PDXs using T test. pv: p-values.
Figure 2A) MALDI imaging of indicated metabolites in IDHwt PDX (P3), IDHm PDX (E478) and control brain (CB). Hex-P: hexose-phosphate; AMP, ADP, ATP: adenosine mono-, di- and triphosphate respectively.B) LC-MS quantification of indicated metabolites in 6 PDXs. Relative quantities of metabolites are expressed in log scale and normalized to tissue quantity used for extract preparation. IDHwt glioblastomas (GBM) in blue, IDHm GBM in orange, IDHm lower grade gliomas (LGG) in red, contralateral control brain (CB) in grey. Sdh7-P: sedoheptulose-7-phosphate.C) Energetic charge defined as the ratio of (ATP+1/2ADP)/(ATP+ADP+AMP) was calculated at each pixel to present its distribution throughout the tumor.(D) The energetic charge ratio was confirmed by the LC-MS data performed on separate tumor extracts (n=3/ sample/ group). Indicated statistical significant group differences are based on T test, pv: p-values.
Figure 3A) LC-MS quantification of steady-state level of glycolysis and TCA cycle metabolites in three IDH1wt and three IDH1m PDXs and contralateral control brain (CB) (n=3/ sample/ group). Metabolites showing statistically significant group differences between IDH1wt and IDH1m are indicated (pvalue: * <0.05). Color code as in Fig. 1-2.C) In vivo flux analysis of 13C-labelled glucose injected 20 minutes prior to sacrifice in one IDH1wt PDX (P3) and one IDH1m PDX (E478) (n=3/ sample). The percentage of glucose-derived 13C label is shown in selected metabolites containing 0 to 6 13C atoms (13C0 - 13C6 as indicated by color code). Note the absence of 13C2 label of αKG and D2HG in IDHm tumours, while the label was detectable in IDHwt PDX and in contralateral brain of both tumors (lower panel).D) Quantities of 13C3 and 13C2 labeled metabolites from glucose tracer experiment in (C). D2HG in IDH1m PDX shows a detectable 13C2 signature here, but represents a minor fraction of the total cellular D2HG pool in this tumor. Hex-P: hexose phosphate, PEP: phosphoenolpyruvate, Pyr: pyruvate, Cit: citrate, Mal: malate, Asp: aspartate, Glu: glutamate, Gln: glutamine, α-KG: α-Ketoglutarate, D2HG: D2-Hydroxyglutarate. Indicated statistical significant group differences are based on T test, pv: p-values.
Figure 4A) MALDI imaging of key metabolites in IDHwt (P3) and IDHm (E478) glioma PDX, compared to control brain (CB) sections.B) Quantification of metabolite distribution by MSI in 6 PDX with IDH1wt and IDH1m status (n=3 / group) and in contralateral control brain (CB). No significant differences between IDH groups were detected, although significant differences are found between tumors and control brain (glutamate, apsartate). Color code as in Fig. 1, NAA: N-acetylaspartic acid; NAAG: N-acetylaspartylglutamic acid.