EMBO/soda-nel huggingface dataset:
Arabidopsis MAKR5 is a positive effector of BAM3-dependent CLE45 4 signaling (2016)
Figure 2

Figure 2 - Second site mutation in MAKR5 partially suppresses brx root phenotypes. (A) Root phenotypes of 7-day-old seedlings of indicated genotypes. (B) Primary root length of 7-day-old seedlings. (C) Root meristem size in 5-day-old seedlings. (D) Root meristem phenotype of 5-day-old seedlings, confocal microscopy, propidium iodide-stained (red). (E) Auxin activity as visualized by the constitutively expressed inverse DII-NLS-VENUS marker (green) in root meristems of 5-day-old seedlings. (F) Gap cell frequency in brx single or brx makr5 double mutants (note: gap cells were absent from Col-0 wild type or makr5 single mutants). (G) Illustration of gap cells (arrowheads) in developing protophloem sieve element strands (asterisks) of brx single or brx makr5 double mutants. Differences are not statistically significant unless indicated. (B, C): student’s t-test; (F): Fisher’s exact test; (B, C): a: versus Col-0; b: versus brx; ** = p < 0.01; *** = p < 0.001; error bars indicate standard error of the mean;
Arabidopsis MAKR5 is a positive effector of BAM3-dependent CLE45 4 signaling (2016)
Figure 3

Figure 3 - MAKR5 loss-of-function confers CLE45 resistance. (A) Primary root length of 7-day-old seedlings of indicated genotypes in response to continuous CLE45 treatments. (B) Root meristem phenotypes of (CLE45-treated) 5-day-old seedlings (asterisks: protophloem sieve element strands). Note absence of protophloem in CLE45-treated Col-0 wild type roots. (C) Transverse sections of (CLE45-treated) 5-day-old roots, just before the onset of protoxylem (asterisks) differentiation. Note failure of protophloem sieve elements (arrowheads) to differentiate in CLE45-treated Col-0 (indicated by dense cytoplasm). (D) Expression pattern of GUS reporter gene driven by the MAKR5 promoter in roots of 5-day-old Col-0 seedlings. (E) Corresponding transverse sections from different positions along the root meristem. (F) Expression of a MAKR5-GFP fusion protein under control of the MAKR5 promoter in roots of 5-day-old seedlings. (G) Corresponding optical sections illustrating MAKR5-GFP expression in developing protophloem sieve elements (asterisk) and adjacent cell files. (H) Complementation of makr5 CLE45 insensitivity by MAKR5 expression under control of a promoter specific for developing sieve elements (CVP2), but not by expression under control of a companion cell-specific promoter (SUC2) (7-day-old seedlings; 3 independent transgenic lines per construct are shown). (I) Subcellular localization of MAKR5-GFP (meristematic zone). Note plasma membrane association (white, green arrowheads) and dissociation from the cell wall (red arrowhead) upon osmosis. Differences are not statistically significant unless indicated (student’s t-test); * = p < 0.05; *** = p < 0.001; (A): versus Col-0; (H): a: versus CLE45-treated Col-0; b: versus CLE45-treated makr5; error bars indicate standard error of the mean;
Arabidopsis MAKR5 is a positive effector of BAM3-dependent CLE45 4 signaling (2016)
Figure 4

Figure 4 - Specificity for CLE45 signal transduction resides in the MAKR5 C-terminus. (A) Schematic presentation of the MAKR5 protein, indicating the K/R-rich membrane hook motif, the S-rich conserved C-terminus, the position of the makr5 mutation, and the truncated fragments tested. (B) Expression of truncated MAKR5 fusion proteins under control of the MAKR5 promoter in 5-day-old roots (asterisks: protophloem sieve element strands). (C) Close up of protophloem sieve element strands expressing truncated MAKR5-CITRINE fusion proteins under control of the UBQ10 promoter. (D) Failure of truncated MAKR5 fusion proteins to complement CLE45 insensitivity of the makr5 mutant (7-day-old seedlings; 1-2 independent transgenic lines per construct are shown). (E) Sensitivity of makr5 mutant roots to continuous brassinolide (BL) treatment (7-day-old seedlings). (F) Expression of MAKR5-BKI1 fusion proteins under control of the MAKR5 promoter in roots of 5-day-old makr5 seedlings. (G) Complementation of CLE45 insensitivity of makr5 roots by BKI1-MAKR5, but not MAKR5-BKI1 fusion protein (7-day-old seedlings; 3 independent transgenic lines per construct are shown). Differences are not statistically significant unless indicated (student’s t-test); * = p < 0.05; ** = p < 0.01; *** = p < 0.001; (D, G): a: versus mock; b: versus makr5; (E): versus Col-0; error bars indicate standard error of the mean;
Arabidopsis MAKR5 is a positive effector of BAM3-dependent CLE45 4 signaling (2016)
Figure 5

Figure 5 - Post-transcriptional regulation of MAKR5 by CLE45 treatment. (A) Increased expression of BAM3-CITRINE fusion protein under control of the BAM3 promoter in developing protophloem sieve elements (asterisks) of 5-day-old bam3 roots upon CLE45 treatment. (B) Expression of BAM3-CITRINE fusion protein under control of the BAM3 promoter in 5-day-old Col-0 roots or makr5 roots. (C) Expression of MAKR5-GFP fusion protein under control of the MAKR5 promoter in 5-day-old Col-0 or bam3 roots. (D) GUS reporter gene expression driven by the MAKR5 promoter in roots of 5-day-old Col-0 seedlings upon CLE45 treatment. (E) Expression of MAKR5-GFP fusion protein under control of the MAKR5 promoter in 5-day-old roots upon CLE45 treatment (3 h). Note accumulation of MAKR5-GFP in protophloem sieve elements (magnified) and its dependence on functional BAM3 and CLE45. The frequency of representative observations per total observations is indicated. (F) Close up view of MAKR5-GFP accumulation in the protophloem cell file upon CLE45 treatment. (G-H) Accumulation of MAKR5-BKI1 fusion proteins in protophloem cells upon CLE45 treatment. The frequency of representative observations per total observations is indicated. (I-J) Quantification of fusion protein abundance in developing protophloem cells upon expression under control of the MAKR5 promoter, showing mean total fluorescence (I) and plasma membrane to cytosol signal ratio (J). MM: MAKR5-GFP; BM: BKI11-252-MAKR5207-326-CITRINE; MB: MAKR51-206-BKI1253-337-CITRINE; Differences versus mock are not statistically significant unless indicated (student’s t-test); (I, J): a: versus mock; b: versus MAKR5-GFP; * = p < 0.05; ** = p < 0.01; *** = p < 0.001; error bars indicate standard error of the mean;
Defective glutamate and K+ 1 clearance by cortical astrocytes in familial hemiplegic 2 migraine type 2. (2016)
Figure 3

Figure 3. The size of GLT-1a immunoreactive (ir) puncta overlapping with VGLUT1 ir puncta is reduced in the cortex of W887R/+ FHM2 KI mice, suggesting a reduced expression of the glutamate transporter GLT-1a in the vicinity of cortical glutamatergic synapses.

(A) Simultaneous visualization of GLT-1a+ (green) and VGLUT1+ puncta (red) in first somatic sensory cortex (SI) of a WT and a KI mouse (P35). Arrows point to some GLT-1a+ puncta overlaying with VGLUT1+ puncta (i.e., GLT-1a/VGLUT1 related puncta); framed regions (enlarged below) are examples of GLT-1a/VGLUT1 related puncta (arrowheads). All microscopic fields are from layers II/III. Scale bars: 3.5 µm for left and right panels and 1 µm for enlarged framed areas.

(B) Percentage and size of GLT-1a+ puncta overlaying with VGLUT1 in P35 WT and KI mice. Left, percentage of GLT-1a+ puncta overlaying with VGLUT1 is comparable in WT and KI mice (data were obtained from 14 and 18 fields of 20 µm x 20 µm from 2 WT and 2 KI mice (4 sections/animal), respectively) (unpaired t-test: P = 0.52). Right, size of GLT-1a/VGLUT1 related puncta is 19 % reduced in KI mice (169 GLT-1a+ puncta analyzed from 2 mice) compared to WT (174 GLT-1a+ puncta analyzed from 2 mice) (Mann-Withney U test: P = 0.016).

Data are mean ± SEM.
Defective glutamate and K+ 1 clearance by cortical astrocytes in familial hemiplegic 2 migraine type 2. (2016)
Figure 4

Figure 4. The density of GLT-1a in the membrane of cortical perisynaptic astrocytic processes is reduced in W887R/+ FHM2 KI mice, while the density of GLT-1a in axon terminals is unaltered.

(A) Distribution of GLT-1a gold particles in astrocytic processes contacting asymmetric synapses (perisynaptic astrocytic processes: PAP) in SI of 5 WT and 5 KI (2 male, 3 female) mice (P34-35). In cortical PAP of KI mice the density (particles/µm2) of total and membrane-associated gold particles (arrowheads) are reduced compared to WT mice, whereas the density of cytoplasmic gold particles (arrows) is comparable to that in WT (cf Table I for numerical values and statistics).

(B) Distribution of GLT-1a gold particles in excitatory axon terminals forming asymmetric synaptic contact (AxT) in SI of P34-35 WT and KI mice. In cortical AxT of KI and WT mice, GLT-1a density is comparable (arrowheads: membrane-associated gold particles; arrows: cytoplasmic gold particles) (cf Table I).

All microscopic fields in (A) and (B) are from layers II/III. t, total density; cyt, cytoplasmic density; mem, membrane density. Scale bar: 100 nm.

Data are mean ± SEM.
Defective glutamate and K+ 1 clearance by cortical astrocytes in familial hemiplegic 2 migraine type 2. (2016)
Figure 7

Figure 7. The CSD threshold is increased in cortical slices of FHM2 KI mice after Cef treatment that increases the GLT-1a expression in WT mice.

(A) Western blottings of GLT-1a in cortical crude synaptic membranes of P39 WT mice following Cef treatment for 8 days. GLT-1a levels are significantly increased in mice treated with Cef (Cef WT, N = 4) compared to control saline-injected mice (Ctr WT, N = 4) (Mann-Withney U test: P = 0.028).

(B) Visualization of GLT-1a+ puncta (green) and VGLUT1+ puncta (red) in sections from SI of P45-46 WT mice that were treated with saline (Ctr WT) or Cef (Cef WT). Arrows point to some GLT-1a/VGLUT1 related puncta. Framed regions (enlarged below) are examples of GLT-1a/VGLUT1 related puncta (arrowheads). Right, Cef treatment increased significantly the size of GLT-1a+ puncta overlaying VGLUT1 (142 and 186 GLT-1a+ puncta analyzed from 4 Ctr WT and 4 Cef WT mice, respectively; 3 sections/animal) (Mann-Withney U test: P < 0.0001). All microscopic fields are from layers II/III. Scale bars: 3.5 µm for left and right panels and 1 µm for enlarged framed areas.

(C) CSD threshold and CSD velocity in cortical slices from P30-33 KI mice that were injected with saline (Ctr KI, n = 38; N=7) or Cef (Cef KI, n = 31; N=6). CSD threshold is 12% higher (Mann-Withney U test: P = 0.02) in Cef-treated compared to saline-treated KI mice. CSD velocity is not altered by Cef treatment (P = 0.90).

Data are mean ± SEM.
Defective glutamate and K+ 1 clearance by cortical astrocytes in familial hemiplegic 2 migraine type 2. (2016)
Figure 8

Figure 8. The density of GLT-1a in the membrane of cortical perisynaptic astrocytic processes is not altered by ceftriaxone treatment in W887R/+ FHM2 KI mice, while the density of GLT-1a in the axon terminals is increased.

(A) Distribution of GLT-1a gold particles in PAP of saline-injected (Ctr KI, N=4) and Cef-treated KI mice (Cef KI, N=4) (P45-46). Cef treatment does not modify the density of total, cytoplasmic (arrows) and membrane-associated (arrowheads) gold particles in PAP of KI mice (cf Table II).

(B) Distribution of GLT-1a gold particles in AxT in SI of Ctr KI and Cef KI KI mice. The density of the membrane-associated gold particles (arrowheads) is increased in Cef-treated KI mice (cf Table II).

All microscopic fields in (A) and (B) are from layers II/III. t, total density; cyt, cytoplasmic density; mem, membrane density. Scale bar: 100 nm.

(C) Western blottings of GLT-1a in cortical crude synaptic membranes of P39 KI mice following Cef treatment. GLT-1a levels are similar in Cef-treated (Cef KI, N=4) and and saline-injected (Ctr KI, N=4) (Mann-Withney U test: P = 0.83).

(D) Visualization of GLT-1a+ puncta (green) and VGLUT1+ puncta (red) in KI mice that received saline (Ctr KI) and in Cef-treated KI mice (Cef KI) (P45-46). Arrows point to some GLT-1a/VGLUT1 related puncta; framed regions are examples of GLT-1a/VGLUT1 related puncta (arrowheads). Right, Cef treatment does not increase the size of GLT-1a+ puncta overlaying with VGLUT1 (160 and 175 GLT-1a+ puncta analyzed from the same 4 Ctr KI and 4 Cef KI used for post-embedding electron microscopy analysis; 3 sections/animal) (Mann-Withney U test: P = 0.31). Scale bar: 3.5 µm for left and right panels and 1 µm for enlarged framed areas. All microscopic fields are from layers II/III.

Data are mean ± SEM.
Impaired telomere integrity and rRNA biogenesis in  PARN-deficient patients and knock-out models (2018)
Figure 1

Figure 1. Identification of PARN mutations in two unrelated HH patients. (A) Pedigree of Patients 1 and 2 and their families. (B) Direct sequencing of PARN in a control, P1 and her parents. (C) Alignment of the two first conserved blocks of sequences from the DEDDh subfamily of nucleases (Uniprot accession numbers). Conserved residues are coloured and the conserved glutamine is highlighted with a red arrowhead. Secondary structures are reported on top, as observed in the experimental 3D structure of human PARN (pdb 2A1R)(Wu et al, 2005). (D) Direct sequencing of PARN in a control, P2 and her parents. (E) Aberrant splicing products detected in P2 and her mother but not in P2's father nor in a healthy control. GAPDH RT-PCR was performed as control. (F) Detection of the exon6/7 deletion by PCR with specific primers (1F/2R). (G) PARN detection in cell lysates from P1 and P2 and a healthy control. Actin was used as loading control. (H) In vitro deadenylation activity using protein extracts from control and P1 and P2 SV40T-transformed fibroblasts. Mixing protein extracts from control together with P1 or P2 eliminated the deadenylation defect whereas pairwise mixing of P1 and P2 failed to complement the deadenylation defect.
Impaired telomere integrity and rRNA biogenesis in  PARN-deficient patients and knock-out models (2018)
Figure 2

Figure 2. Short and dysfunctional telomeres in cells from HH patients. (A) Mean telomere length (kb) of whole blood cells from patients and their parents estimated with the TRF method. (B) Representative picture of telomeric signals used for Q-FISH analysis on metaphase spreads from SV40T-transfomed fibroblasts and Muntjac cells used to normalize the signals. (C) Individual and mean values of Q-FISH analyses for control and patients. Mean fluorescence intensity of Muntjac cells was set to 1. Error bars indicate s.d.. (D) Graphical representation of (C) showing the average of fluorescence ratios (relative to Muntjac). Error bars indicate s.e.m.. The non-parametric Kruskal-Wallis test was applied to compare relative fluorescence values between Ctl and P1, and Ctl and P2. (E) Representative pictures of nuclei from two controls and patient's primary fibroblasts showing 53BP1 foci (green) and telomeres (red, detected by Telo-FISH). Yellow arrows indicate telomere dysfunction-induced foci (TIF). The scale bar corresponds to 5µm. (F) Quantification of (E) in primary fibroblasts from two healthy controls (passages > 4) and from patient 1 (passages < 4). Control 1: n = 224; Control 2: n = 263; Patient 1: n = 231. Averages are shown and error bars indicate s.e.m.. Unpaired Student's t tests were applied when indicated. (G) Representative pictures of SA-β-galactosidase staining in control (passage 15) and P1's primary fibroblasts (passage 6). Results are expressed as the percentage of SA-β-galactosidase-positive cells (averages, lower panel). Control: n = 436; P1: n = 436. A test to compare two population proportions was applied. (H) Representative pictures of chromosomes with normal or aberrant telomeres detected by Telo-FISH. The scale bar corresponds to 1µm. (I) Quantification of terminal deletions and sister telomere losses (J) from three independent experiments (counted chromatids: Ctl1: n=2224; Ctl2: n= 5696; P1: n=4764; P2: n=7908). Averages are shown and χ2-tests were applied to compare Ctl1/2 with either P1 or P2.
Impaired telomere integrity and rRNA biogenesis in  PARN-deficient patients and knock-out models (2018)
Figure 3

Figure 3. PARN KO cells display reduced telomerase activity and down-regulation of shelterin and telomere-related gene expression. (A) Validation of PARN KO in HT1080 cells by Western blot. GAPDH is used as loading control. (B) In vitro deadenylation activity assay using protein extracts from HT1080 (WT) and HT1080PARN KO (PARN KO) cells. (C) TRAP assay using successive dilutions (500 ng, 250 ng, 125 ng and 62.5 ng) of cell extracts from WT or PARN KO cells. An internal control (IC) for PCR was used. (D) qRT-PCR analysis of hTR expression in WT and PARN KO cells. Expression levels were normalized first to ACTB and then to WT. Three independent RNA extractions were performed for each cell line. Error bars indicate s.e.m.. (E) Representative pictures of FISH against hTR in WT and PARN KO cells. Arrows indicate hTR foci. Scale bar 5 μm. (F) PARN detection by Western blot in the indicated conditions. Expression was induced by incubating cells with 10 ng/ml doxycycline for 72 h. Actin is used as loading control. PARN/actin ratios, normalized to doxycycline-treated WT/PARN cells, are shown below. (G) In vitro deadenylation activity assay using protein extracts from doxycycline-treated WT/empty, WT/PARN, KO/empty and KO/PARN cells. (H) qRT-PCR analysis of the indicated gene transcripts in doxycycline-treated WT/empty, WT/PARN, KO/empty and KO/PARN cells. Expression levels were normalized first to ACTB and then to WT/empty. Three independent doxycycline inductions were performed for each cell line. Averages are shown and error bars indicate s.e.m.. Unpaired Student's t tests were applied. (I) Representative western blot analysis of PARN, TRF2 and p53. Actin was used as loading control. Mean TRF2/Actin levels from two independent experiments are indicated below. (J) Same as (H) for p53 and p21 transcripts. Averages are shown and error bars indicate s.e.m.. Unpaired Student's t tests were applied. (K) qRT-PCR analysis of TERRA from the indicated chromosome ends in doxycycline-treated KO/empty and KO/PARN cells. Expression levels were normalized first to ACTB and then to KO/empty. Three independent doxycycline inductions were performed for each cell line. Error bars indicate s.e.m..
Impaired telomere integrity and rRNA biogenesis in  PARN-deficient patients and knock-out models (2018)
Figure 4

Figure 4. PARN KO cells display telomere instability independently of telomere length. (A) Analysis of telomeric aberrations by Telo-FISH in doxycycline-treated WT/empty, WT/PARN, KO/empty and KO/PARN cells. Two independent experiments were performed. Counted chromatids: WT/empty: n=1824; WT/PARN: n= 1744; KO/empty: n=1452; KO/PARN: n=1920. Averages are shown and χ2-tests were applied to compare KO/empty with either WT/empty or KO/PARN. (B) TRF analysis of telomere length (kb) in KO/empty and KO/PARN cells treated with 10 ng/ml Dox for 72h. A control Western blot with PARN and Actin is shown below for the corresponding samples.
Impaired telomere integrity and rRNA biogenesis in  PARN-deficient patients and knock-out models (2018)
Figure 5

Figure 5. Down-regulation of DKC1 mRNA in PARN KO cells is dependent on p53. (A) HT1080 (WT) and HT1080PARN KO (KO) cells stably transfected with GFP plasmid (empty) were transfected with siRNAs against p53 for 72h before RNA extraction and qRT-PCR. siRNAs against luciferase (siLuci) were used as control. p53 expression levels were normalized first to ACTB and then to WT/empty/siLuci cells. Experiment was performed in triplicate. Error bars indicate s.e.m. (B) Cells were treated as in (A) and transcript levels of the indicated genes were measured by qRT-PCR. cDNA expression levels were normalized first to ACTB and then to WT/empty/siLuci cells. Experiment was performed in triplicate. Error bars indicate s.e.m. (C) qRT-PCR analyses of the indicated genes were performed on RNA extracted from P1's primary or SV40T-transformed fibroblasts. cDNA expression levels were normalized to primary fibroblasts.
Impaired telomere integrity and rRNA biogenesis in  PARN-deficient patients and knock-out models (2018)
Figure 6

Figure 6. PARN dysfunction impacts human pre-ribosomal RNA processing. (A) Schematic representation of human ribosomal precursors with rRNAs sequences displayed as grey (18S; small ribosomal subunit) or white boxes (5.8S and 28S rRNAs; large ribosomal subunit). These three rRNAs are flanked by external (5'ETS, 3'ETS) and internal transcribed spacers (ITS1, ITS2). The position of endonucleolytic cleavages is represented by vertical lines along these transcribed spacers, or corresponds to 5' and 3' ends of rRNA sequences. (B) Graphical representation of DNA probes used for Northen blot hybridization displayed along the 47S primary rRNA transcript. (C) Northern blot analysis of pre-rRNAs from control and patient B-LCLs. (D) Log2 values of 18S-E/21S and 18S-EFL/21S for P1 and P2 were normalized to the values of the control and presented in a graphical format, as Log averages + standard deviation for two independent experiments. (E) Northern blot analysis of pre-rRNAs from HT1080PARN WT and HT1080PARN KO cells, transduced with an empty vector. (F) Ectopic expression of PARN was induced in HT1080PARN KO cells and compared to cells transduced with an empty vector. Western blot relative to actin assessed PARN levels. (G) Quantitative analyses of 18S-E/21S and 18S-E FL/21S ratios were as described in (D) for HT1080PARN KO cells relative to HT1080PARN WT (left panel; see (E)), and for HT1080PARN KO cells rescued by ectopic expression of PARN relative to HT1080PARN KO cells transduced with an empty expression vector (right panel; see (F)), with Log averages + standard deviation for four independent experiments.
Impaired telomere integrity and rRNA biogenesis in  PARN-deficient patients and knock-out models (2018)
Figure 7

Figure 7. The early embryonic lethality of Parn KO is not rescued in p53-/- mice. (A) Western blot analysis of Parn expression in control and Parn+/- MEFs. (B) Northern blot analysis of total RNAs from WT and Parn+/- MEFs. (C) Schematic representation of mouse rRNA precursors and position of DNA probes used in northern blot. (D) Frequencies of expected and observed newborns of the indicated genotype obtained from Parn+/- intercrosses. n indicates the number of animal analyzed. Statistical significance (P = 1.5417E-08) of the differences between the observed and expected genotype distributions was assessed by χ2-test. (E) Frequencies of expected and observed E11.5 embryos of the indicated genotype obtained from Parn+/- intercrosses. n indicates the number of animal analysed. Statistical significance (P = 0.02535) of the differences between the observed and expected genotype distributions was assessed by χ2-test. (F), (G) p53 knock-out does not rescue Parn-/-. No Parn-/- p53-/- mouse was born from various combinations of Parn+/- and either p53+/- or p53-/- crosses (49 and 56 pups, respectively). Statistical significance (P = 0.000886) of the differences between the observed and expected genotype distributions was assessed by χ2-test.