EMBO/SourceData huggingface dataset:
Figure 4qRT-PCR analysis of SRF target genes (MYH6, MYH7 and ACTC1) in CTR and DCM single cardiomyocytes under basal condition (no infection, NI) and after infection with control U7snRNA-ScrAONs-IRES-GFP (Scr-AON) and the U7snRNA-TTNAONs-IRES-GFP (TTN-AON) lentiviruses. Statistical difference was tested using the two-sided Student's t-test (**P = 0.009, CTR Scr-AON versus DCM Scr-AON; *P = 0.04, DCM Scr-AON versus DCM TTN-AON for MYH6; **P = 0.002, CTR Scr-AON versus DCM Scr-AON; **P = 0.002, DCM Scr-AON versus DCM TTN-AON for MYH7; **P = 0.004, CTR Scr-AON versus DCM Scr-AON; *P = 0.02, DCM Scr-AON versus DCM TTN-AON for ACTC1). No significant differences were observed comparing the CTR NI, CTR Scr-AON and CTR TTN-AON groups and comparing the DCM NI and DCM Scr-AON groups. Expression values were relative to CTR Scr-AON, normalized to GAPDH, and presented as mean ± SEM, n = 3.Immunofluorescence images showing normal (a, b, e, f, i, l, o, p) and altered (c, d, g, h, m, n, q, r) intracellular distribution of SRF (a and b, nuclear; c and d, cytoplasmic), MURF2 (e and f, sarcomeric; g and h, nuclear), Nbr1 (i and l, sarcomeric; m and n, diffused), and SQSTM1/p62 (o and p, sarcomeric; q and r, diffused) in representative single cardiomyocytes (left). Sarcomeres are marked by α-actinin. On the right, percentage of CTR and DCM cardiomyocytes showing cytoplasmic expression of SRF, nuclear accumulation of MURF2, and diffused expression of Nbr1 and of SQSTM1/p62 after infection with control U7snRNA-ScrambleAONs-IRES-GFP (Scr-AON) and the U7snRNA-TTNAONs-IRES-GFP (TTN-AON) lentiviruses (right). Data represent mean values ± SEM from two control and two DCM clones. Statistical difference was tested using the two-sided chi-squared test (CTR Scr-AON: n = 874, n = 874, n = 882 and n = 890, CTR TTN-AON: n = 880, n = 990, n = 878 and n = 890, DCM Scr-AON: n = 890, n = 887, n = 884 and n = 886, DCM TTN-AON: n = 900, n = 875, n = 899 and n = 891 for SRF, MURF2, Nbr1 and SQSTM1/p62, respectively; *P = 0.01, CTR Scr-AON versus DCM Scr-AON; *P = 0.04, DCM Scr-AON versus DCM TTN-AON for SRF; *P = 0.02, CTR Scr-AON versus DCM Scr-AON; *P = 0.04, DCM Scr-AON versus DCM TTN-AON for MURF2; *P = 0.03, CTR Scr-AON versus DCM Scr-AON; *P = 0.03, DCM Scr-AON versus DCM TTN-AON for Nbr1; **P = 0.009, CTR Scr-AON versus DCM Scr-AON; *P = 0.01, DCM Scr-AON versus DCM TTN-AON for SQSTM1/p62). No significant differences were observed comparing CTR Scr-AON and CTR TTN-AON groups. Scale bars, 50 μm.
Figure 5B, C Immunofluorescence images of sarcomeric α-actinin and myosin (B) and assessment of cardiac function (C) in homozygous Ttn knock-in embryos cultured for 24 h after mScrAON and mTtnAON transient transfection (n = 3 per group) or lentivirus delivery (n = 7 per group). Scale bars, 10 μm.D Electron microscopy analysis in Ttn knock-in embryos cultured for 24 h after mScrAON and mTtnAON transient transfection. mTtnAON treatment of homozygous embryos rescued myofibril formation (left, black arrows), resulting in thicker filaments (right).
Figure 6Echocardiographic analysis of adult knock-in mice. Statistical difference was tested using the two-sided Student's t-test (WT: n = 6, HET: n = 6; HET + vPMO-mScrAON: n = 10 and HET + vPMO-mTtnAON: n = 14; *P = 0.013, HET versus HET + vPMO-mTtnAON and *P = 0.02, HET + vPMO-mScrAON versus HET + vPMO-mTtnAON for LVEDD differences; **P = 0.006, HET versus HET + vPMO-mTtnAON and *P = 0.02, HET + vPMO-mScrAON versus HET + vPMO-mTtnAON for LV-EF differences; *P = 0.03, HET versus HET + vPMO-mTtnAON and *P = 0.04, HET + vPMO-mScrAON versus HET + vPMO-mTtnAON for IVSd differences; **P = 0.001, HET versus HET + vPMO-mTtnAON and *P = 0.03, HET + vPMO-mScrAON versus HET + vPMO-mTtnAON and for PWd differences). No significant differences were observed among groups at day 0 and day 7. Data represent mean values ± SEM.Masson's trichrome staining of heart sections from HET animals injected with vPMO-mScrAONs and vPMO-mTtnAON. Scale bars, 1 mm.
Figure 7Fluorescence in situ hybridization (FISH) of heart muscle tissue from adult knock-in mice with a probe complementary to vPMO-mTtnAON. Scale bars, 250 and 50 μm (magnification).RT-PCR analysis of Ttn exon 326 transcripts from heart tissue of untreated WT and HET animals and vPMO-mScrAON- and vPMO-mTtnAON-treated mice (left). Representative direct sequencing of Ttn exon 326 transcripts from vPMO-mTtnAON-treated HET heart tissue (right).Mass spectrometry-based analysis of titin peptides in adult knock-in mice injected with vPMO-mScrAONs and vPMO-mTtnAONs. Unsupervised hierarchical clustering identified a cluster enriched in exon 326 peptides that was down-regulated in vPMO-mTtnAON-treated animals compared to vPMO-mScrAON-treated littermates. Another cluster enriched in C-terminal peptides was up-regulated in the vPMO-mTtnAON group compared to the vPMO-mScrAON group (n = 3, P = 0.02, Fisher's exact test, FDR = 0.04).
Figure 1A-B) Western-blot analysis for GAPDH and LC3 of protein lysates obtained from HeLa cells treated with DMSO or 1 µM rapamycin (Rapa) for the indicated time periods (A) (n = 7) or for 5 h with the indicated concentrations (B) (n = 6). One hour before harvesting, 100 nM bafilomycin A1 was added. Upper panels: representative Western blots; lower panels: quantification of the LC3-II/GAPDH ratio.C) GFP-LC3-punctae quantification in HeLa cells treated for 5 h with different concentrations of rapamycin. Left: representative pictures. The scale bar represents 10 µm. Concentrations are mentioned in the right lower corner. Right: Quantification of the number of punctae per cell (n = 3). * p<0.05, repeated measurements ANOVA.
Figure 2A) Representative measurements (n = 4) of cytosolic Ca2+ signals, displayed as Fura2 ratio (F340/F380), showing the effect of 0.3 µM and 100 µM ATP, 1 µM thapsigargin (Tg) or 10 µM ionomycin (Iono) in intact HeLa cells treated with different concentrations of rapamycin (Rapa) for 5 h. 45 s prior to the addition of ATP, Tg or Iono, EGTA (3 mM) was given to buffer extracellular Ca2+ as indicated.B) Quantification of the average amplitude of the response (F−F0) (n = 4). * p<0.05; ** p<0.01; *** p<0.001, repeated measurements ANOVA.C) Mean resting cytosolic [Ca2+], measured in Fura2-loaded HeLa cells treated with the indicated concentrations of rapamycin for 5 h, as well as in the absence (Ctrl) or presence of DMSO (n = 3). * p<0.05, repeated measurements ANOVA.D) Unidirectional 45Ca2+-flux experiments in permeabilized cells pretreated with 1 µM rapamycin for 5 h or with DMSO (Ctrl). Mean fractional 45Ca2+ release (%/2 min) is shown as a function of time with the effect of 0.7 µM IP3 (circles) or no addition (squares). The horizontal bar indicates the presence of IP3.E) Quantitative analysis of the IP3-induced 45Ca2+ release in cells pretreated for 5 h with 1 µM rapamycin or DMSO (Ctrl) (n = 8). *** p<0.001, paired Student's t-test.
Figure 3A-B) Western-blot analysis for luminal Ca2+-binding proteins in HeLa cells treated with the indicated concentrations of rapamycin (Rapa) for 5 h: calreticulin (CRT) (A) and BiP/Grp78 (BiP) (B). Upper panels: representative Western blots; lower panels: quantification of the protein/GAPDH ratio (n = 4).C) Western-blot analysis for SERCA2 in HeLa cells treated with the indicated concentrations of rapamycin for 5 h. Upper panels: representative Western blots; lower panel: quantification of the SERCA2/GAPDH ratio (n = 4).D) Representative plot showing the decrease in ER 45Ca2+ content (logarithmic scale) in a Ca2+-free efflux medium without ATP as a function of time in permeabilized HeLa cells pretreated for 5 h with 1 µM rapamycin or with DMSO. The passively bound Ca2+ was determined by loading the cells with 45Ca2+ in the presence of 10 µM of the Ca2+ ionophore A23187 and then subtracted from the stored 45Ca2+. The ER Ca2+-leak rate can be estimated as the rate of decline of the ER 45Ca2+-store content as a function of time.E) Quantification of the mean 45Ca2+-store content at the beginning of the measurement (t0) (n = 5).F) Quantification of the mean slope of the curve in D after transformation to a linear scale, which is a measure of the 45Ca2+-leak rate (n = 5). * p<0.05; ** p<0.01, paired Student's t-test.
Figure 4A) Representative Western-blot analysis for Atg12 (showing the autophagic Atg12-Atg5 complex), GAPDH and LC3 of protein lysates obtained from MEF cells pretreated with (+Dox) or without (-Dox) doxycycline and treated with DMSO or 0.1, 1 or 5 µM rapamycin (Rapa) for 5 h (n = 3).B-C) Representative measurements of cytosolic Ca2+ signals, displayed as Fura2 ratio (F340/F380), showing the effect of 1 mM ATP (B) or 10 µM ionomycin (Iono) in intact MEF cells pretreated with or without doxycycline and treated with different concentrations of rapamycin for 5 h. Prior to the addition of ATP or Iono, EGTA (3 mM) was added to chelate the extracellular Ca2+ as indicated.B-C) Representative measurements of cytosolic Ca2+ signals, displayed as Fura2 ratio (F340/F380), showing the effect of 1 mM ATP (B) or 10 µM ionomycin (Iono) in intact MEF cells pretreated with or without doxycycline and treated with different concentrations of rapamycin for 5 h. Prior to the addition of ATP or Iono, EGTA (3 mM) was added to chelate the extracellular Ca2+ as indicated.D) Quantification of the average amplitude of the response (F−F0) (n = 3, 4, 5 and 6 for ATP-Dox, ATP+Dox, Iono-Dox and Iono+Dox, resp.) * p<0.05; ** p<0.01; *** p<0.001, repeated measurements ANOVA.
Figure 52+-dependent. Western-blot analysis for GAPDH and LC3 of protein lysates obtained from HeLa cells treated for 5 h with DMSO, 1 µM rapamycin (Rapa), 10 µM BAPTA-AM or both. One hour before harvesting, 100 nM bafilomycin A1 was added. Left: representative Western blots; right: quantification of the LC3-II/GAPDH ratio (n = 6). * p<0.05, repeated measurements ANOVA.
Figure 1(b) Temperature-sensitive growth assay. Yeast strains (wild-type or deletion mutants, indicated on the left) were transformed with either an empty vector or a plasmid carrying an artificial guide RNA gene targeting U2 pseudouridylation at position 42 and 44 (indicated on the top), and grown at various temperatures (indicated at the bottom).(c) Blockage of pseudouridylation by deletion of specific pseudouridylase genes. RNAs isolated from wild-type and pseudouridylase-deletion strains (indicated on the top) were used for U2 pseudouridylation assay (CMC modification followed by primer-extension). Primer-extension pauses/stops correspond to ψ sites (indicated on the left and right). Note: The above-background signal of ψ42 in the snr81Δ lane (and to some extent, in the snr81Δ pus7Δ lane) is likely caused by the presence of strong ψ44 band. This is a primer-extension artifact, which often occurs when there is a strong ψ signal in the neighboring position. Alternatively, it is possible that having ψ at position 44 allows for an unidentified enzyme other than snR81 to modify U42 (albeit inefficiently) (Perhaps Pus1, which is responsible for ψ44 formation, can be a bit "processive"). Lanes 1-8 and lanes 9-16 are from two separate gels.(d) Restoration of U2 pseudouridylation by an artificial box H/ACA RNA. RNAs isolated from various yeast strains (indicated on the top), including the snr81Δ pusΔ strain that was transformed with an artificial guide RNA targeting positions 42 and 44 (lanes 9 and 10), were assayed for pseudouridylation [see legend to (c)].
Figure 2(a) Growth assay using the ACT1-CUP1 reporter system. Yeast cells carrying the ACT1-CUP1 reporter were deleted of pseudouridylase genes (indicated on the left), and were then assayed for growth at 30°C on media containing various concentrations of [Cu2+] (indicated at the bottom).(b) Growth rescue by restoration of pseudouridylation. The wild-type strain [from (a)] or the snr81Δ pusΔ strain [also from (a)], which exhibited the most severe growth-deficiency phenotype, was transformed with an empty vector or a plasmid carrying a pseudouridylation guide RNA gene targeting either position 42, position 44, or both (indicated). The resulting cells were assayed for growth at 30°C on media containing various concentrations of [Cu2+] (indicated at the bottom).(d) Splicing assay using the wild-type and mutant ACT1-CUP1 reporters. RNAs isolated from the wild-type and the snr81Δ pusΔ strains (indicated on the top), which carry the wild-type ACT1-CUP1 reporter pre-mRNA or any of the mutant ACT1-CUP1 reporter pre-mRNAs [indicated, also see (c)], were used for splicing assay (primer-extension analysis). The un-spliced pre-mRNA, lariat intermediate, and spliced mRNA are indicated. A primer complementary to U6 was also used (as an internal control) in the assay, and the U6 band is indicated as well. In addition, the growth phenotype of each strain (in various concentrations of [Cu2+]) is also shown. Lanes 1-12, lanes 13 and 14, and lanes 15 and 16 are from separate gels.(e) Quantification of ACT1-CUP1 mRNA levels. Spliced ACT1-CUP1 mRNA levels were calculated relative to U6 in each lane. The quantification was based on three independent experiments.(f) Relative improvement/reduction for the mutant reporters. Relative improvement or reduction in splicing was calculated by normalizing the splicing efficiency (mRNA/U6) of each mutant reporter to that of WT reporter (set at 1).
Figure 5(a) Prp5's ATPase activity assay using synthetic U2. The 5' sequence of S. cerevisiae U2 is shown (top). The three ψs (35, 42 and 44) as well as the branch site recognition sequence (underlined) are indicated. In the ATPase assay (bottom), no RNA (lane 3), or an equal amount of LiCl-precipitated RNA (lane 1) or tRNA (lane 2), a short U2 fragment (nts 1-76) (lane 4), a fully pseudouridylated short U2 fragment (nts 1-76) (lane 5), a long U2 fragment (nts 1-120) (lane 6), or a fully pseudouridylated long U2 fragment (nts 1-120) (lane 7), was used. The un-hydrolyzed ATP and hydrolyzed product ADP are indicated. Lanes 1 and 2, lanes 3-5, and lanes 6 and 7 are from separate gels.(b) Quantification of Prp5's ATPase activity activated by in vitro transcribed U2. Prp5's ATPase activity shown in (a) was calculated using the formula (ADP) / (ADT+ATP). The numbers at the bottom of (b) correspond to the lane numbers of (a). Quantification was based on three independent experiments.(c) U2-Prp5 binding assay. FLAG-Prp5 and a radiolabeled short (nts 1-76) (lanes 1 and 2) or long (nts 1-120) (lanes 5 and 6) U2 fragment, or FLAG-Prp5 and a radiolabeled fully pseudouridylated short (nts 1-76) (lanes 3 and 4) or long (nts 1-120) (lanes 7 and 8) U2 fragment, were incubated under the conditions used for ATPase activity assay. Anti-FLAG IP was then carried out. Co-precipitated (lanes 2, 4, 6 and 8) and un-precipitated (lanes 1, 3, 5 and 7) U2 RNAs were analyzed by electrophoresis. The U2 band is indicated.(d) Quantification of Prp5-U2 binding. Relative intensities of U2 bands shown in (c) were calculated [setting the un-precipitated uridine-containing U2 (lane 1 and 5) to 1]. The numbers at the bottom of (d) correspond to the lane numbers of (c). Quantification was based on three independent experiments.(e) Prp5's ATPase activity assay using cellular U2. U2 RNA isolated from the wild-type strain (lanes 2 and 4) or from the snR81Δ pus1Δ strain (lanes 6 and 8) was used for the ATPase activity assay. In odd-numbered lanes, no RNA was added. Both the wild-type Prp5 (lanes 1-4) and the mutant Prp5 (GAR) (lanes 5-8) were tested.(f) Quantification of Prp5's ATPase activity activated by U2 from cells. Prp5's ATPase activity shown in (e) was calculated using the formula (ADP) / (ADT+ATP). The numbers at the bottom of (f) correspond to the lane numbers of (e). Quantification was based on four independent experiments.(g) U2 level in the ATPase activity reactions. RNAs from lanes 2 and 4 in (c) were recovered, and U2 level was measured using primer-extension. Lanes 1-5, a titration of U2 snRNA. Lane 6, U2 from the wild-type strain [lane 2 of (c)]. Lane 7, U2 from the snR81Δ pus1Δ strain [lane 4 of (c)].
Figure 6(a) Native gel analysis of pre-splicing complexes. Pre-splicing complex assembly was carried out in the test tube with Prp5-reconstituted cell extracts and labeled pre-mRNA. Lane 1, Prp5-depleted wild-type cell extract. Lane 2, Prp5-depleted wild-type cell extract, plus wild-type Prp5. Lane 3, Prp5-depleted snR81Δ pus1Δ cell extract, plus wild-type Prp5. Lane 4, Prp5-depleted wild-type cell extract, plus mutant Prp5 (GAR). Lane 5, Prp5-depleted snR81Δ pus1Δ cell extract, plus mutant Prp5 (GAR). The pre-splicing complexes A and B are indicated.(b) DMS in vivo probing. After being exposed to DMS, yeast cells were lysed, total RNA collected, and primer-extension analysis carried out (left panel). Lane 1, cells that were not exposed to DMS. Lane 2, wild-type cells exposed to DMS. Lane 3, snR81Δ pus1Δ cells exposed to DMS. Lane 4, snR81Δ pus1Δ cells, transformed with a plasmid carrying an artificial guide RNA gene targeting positions 42 and 44, and exposed to DMS. Lane 5, snR81Δ pus1Δ cells, transformed with a plasmid carrying a control guide RNA gene with random guide sequences, and exposed to DMS. The nucleotides in parentheses are DMS-modified C and A residues that are one nucleotide after the actual primer-extension stops (indicated by the arrows). A partial U2 sequence (along with the BSL structure) is shown (right panel). DMS-modified adenosine and cytosine in the BSL are also indicated.(c) Residue accessibility quantification. Relative accessibility (relative band intensity) was quantified based on three independent experiments. The intensity of every band in each lane was normalized against the C46 band (the least changed band; compare lanes 2-5 in (b)) in its respective lane [(intensity of a band) / (intensity of C46 in the same lane)]. Each C46-normailzed band in lanes 3-5 (refer to (b)) was then further normalized against its respective counterpart in lane 2 [(the value of a C46-normalized band in lanes 3-5) / (the value of its counterpart in lane 2)]. The error bars represent the standard deviations of the measurements.
Figure 1 Mice were infected with sub-lethal dose (25 pfu) of influenza virus and assessed as follows (A) Percentage body weight loss Data information: Data are representative of three independent experiments with 3-5 mice per group and represented as mean ± S.E.M  Mice were infected with sub-lethal dose (25 pfu) of influenza virus and assessed as follows (B) Relative amount of PR8 viral NP mRNA, by qPCR Data information: Data are representative of three independent experiments with 3-5 mice per group and represented as mean ± S.E.M  Mice were infected with sub-lethal dose (25 pfu) of influenza virus and assessed as follows (C) Histology of lungs by H&E staining with pathology scores. Scale bars = 200 μm Data information: Data are representative of three independent experiments with 3-5 mice per group and represented as mean ± S.E.M  Mice were infected with sub-lethal dose (25 pfu) of influenza virus and assessed as follows (D) Absolute number of live CD45+ cells in whole lungs and BAL, by flow cytometry Data information: Data are representative of three independent experiments with 3-5 mice per group and represented as mean ± S.E.M Statistical significanc was determined with unpaired student's T-test Mice were infected with sub-lethal dose (25 pfu) of influenza virus and assessed as follows (E) Level of LDH in BAL, by LDH cytotoxicity assay Data information: Data are representative of three independent experiments with 3-5 mice per group and represented as mean ± S.E.M  Mice were infected with sub-lethal dose (25 pfu) of influenza virus and assessed as follows (F) Expression of PDPN in lungs, by western blot Data information: Data are representative of three independent experiments with 3-5 mice per group and represented as mean ± S.E.M  Mice were infected with sub-lethal dose (25 pfu) of influenza virus and assessed as follows (G) Total protein in BAL, by Bradford assay. (H) Concentration of albumin in BAL, by ELISA Data information: Data are representative of three independent experiments with 3-5 mice per group and represented as mean ± S.E.M. Statistical significanc was determined with unpaired student's T-test
Figure 3Mice were superinfected as described in (A) (B) Survival of mice following bacterial superinfection Data information: Data from (B) are combined from two independent experiments, n=13 mice per experimental group All data are represented as mean ± S.E.M. Statistical significance was determined by logrank test in (BMice were superinfected as described in (A) (C) Pneumococcal cfu in BAL of mice Data information Dat are representative of at least two independent experiments with 3- mice per experimental group All data are represented as mean ± S.E.M Statistical significance wa determined b Mann-Whitney U test in (CMice were superinfected as described in (A) (D) Absolute immune cell counts in lungs, assessed by flow cytometry Data information Dat are representative of at least two independent experiments with 3- mice per experimental group. All data are represented as mean ± S.E.M. Statistical significanc determined b unpaired student's T-test in (DMice were superinfected as described in (A) (E) Pneumococcal cfu in BAL of mice 12 h after infection with 2x105 cfu of S. pneumoniae serotype 19F alone (-Flu) or following influenza virus infection (+Flu) Data information Dat are representative of at least two independent experiments wit 5- mice per experimental group. All data are represented as mean ± S.E.M. Statistical significance was determined b Mann-Whitney U tes n.s. = not significantMice were superinfected as described in (A) (F) Pneumococcal cfu in BAL 36 h after infection of naïve mice with 2x105 cfu of S. pneumoniae serotype 19F alone Data information Dat are representative of at least two independent experiments wit 5- mice per experimental group. All data are represented as mean ± S.E.MMice were superinfected as described in (A) (G) Absolute neutrophil and eosinophil cell count in lungs infected as described in (E), assessed by flow cytometry Data information Dat are representative of at least two independent experiments with 3- mice per experimental group All data are represented as mean ± S.E.M. Statistical significance wa determined b ), unpaired student's T-test i (G (E). n.s. = not significant
Figure 4 (A) Concentrations of cytokines or chemokines in the BAL of days 5 and 9 post sub-lethal influenza virus infection as determined by protein bead array Data information: Data in (A) are obtained from one independent experiment with 4 mice per group, and results of selected analytes IFNγ, CXCL10, IL-6, IL-13 and IL-17 are reconfirmed in a separate independent experiment by a customized protein bead array Significance i was determined by unpaired student's T-test. n.s. = not significantMice were superinfected as described in Fig 3A. (B) Concentrations of IL-17 and IL-22 in BAL of mice, as determined by ELISA Data informatio Data in (B are representative of two independent experiments with 3-4 mice per group. All data are represented as mean ± S.E.M. Significance i was determined by unpaired student's T-test. n.s. = not significantMice were superinfected as described in Fig 3A (C) Concentrations of IL-17 and IL-22 in BAL of mice 12 h after with 2x105 cfu of S. pneumoniae serotype 19F alone (-Flu), or following superinfection (+Flu), assessed by ELISA Data information Dat i (C) are representative of two independent experiments with 3-4 mice per group. All data are represented as mean ± S.E.M. Significanc was determined by unpaired student's T-test. n.s. = not significant