EMBO/SourceData huggingface dataset:
Figure 5(H) Representative Iba1+ immunohistochemistry in the leptomeninges of SPF, GF, ABX-treated, ASF, recolonized ASF, sDMDMm2, TLR2,3,4,7,9-deficient, SCFA-treated GF mice or FFAR2-deficient mice. Arrows indicate Iba1+ meningeal macrophages. Scale bar = 50 μm. (I-O) Quantification thereof. Each symbol represents one mouse. Three to four sections per mouse were examined. Means ± s.e.m. are indicated. No significant differences were determined by unpaired t-test or one-way ANOVA followed by Tukey's post-hoc comparison test. Data are representative of at least two independent experiments.
Figure 6(A) Iba1 immunohistochemistry depicting cortical microglia, meningeal macrophages, perivascular macrophages and choroid plexus macrophages 4 days after i.c. challenge with LCMV or vehicle controls. Arrows indicate Iba1+ meningeal or perivascular macrophages, respectively. (B) Quantification of Iba1+ microglia, choroid plexus macrophages, meningeal macrophages and perivascular macrophages. Each symbol represents one mouse. Three to four sections per mouse were examined. Significant differences were evaluated by two-way ANOVA followed by Tukey's post-hoc comparison test and marked with asterisks (*P < 0.05, **P < 0.01, ***P < 0.001). Data are representative of two independent experiments. (C, D) Representative immunofluorescence images (left) of (C) Iba1 (green) and KI67 (red) or (D) CD74 (red) on cortical sections from SPF and GF mice. Nuclei were stained with DAPI (blue). Scale bars: 25 μm. White arrows indicate KI67+/Iba1+ or CD74+/Iba1+ macrophages and arrowheads show KI67-/Iba1+ or CD74-/Iba1+ macrophages. Quantification thereof (right). Each symbol represents one mouse. At least three slides were examined per individual mouse. Data are presented as mean ± s.e.m. Significant differences were evaluated by two-way ANOVA followed by Tukey's post-hoc comparison test and marked with asterisks (*P < 0.05, **P < 0.01, ***P < 0.001). Data are representative of two independent experiments.
Figure 7(A-C) Representative immunofluorescence images visualizing (A) cortical mMΦ, (B) cpMΦ located in the lateral ventricle and (C) hippocampal pvMΦ from SPF (left), GF (middle) and ABX-treated 5xFAD mice (right). Iba1 (green) and Aβ (6E10, red). Nuclei were stained with DAPI (blue). White arrowheads indicate Iba1+Aβ+ MΦ and white arrows show Iba1+Aβ- MΦ. Scale bar: 50 μm.(D) Quantification of the number of 6E10+ Aβ-plaques per mm2. Each symbol represents one mouse. Means ± s.e.m. are indicated. Significant differences were evaluated by one-way ANOVA followed by Tukey's post-hoc comparison test and marked with asterisks (***P < 0.001). Data are representative of four independent experiments.(E-G) Quantification of Iba1+ (E) mMΦ, (F) cpMΦ and (G) pvMΦ per mm2. Each symbol represents one mouse. At least three slides were examined per individual mouse. Data are presented as mean ± s.e.m. Significant differences were determined by two-way ANOVA followed by Tukey's post-hoc comparison test and marked with asterisks (*P < 0.05, **P < 0.01, ***P < 0.001). Data are representative of two independent experiments.(H-J) Percentage of Iba1+Aβ+ (H) cortical mMΦ, (I) cpMΦ and (J) hippocampal pvMΦ of SPF, GF and ABX-treated 5xFAD mice. Means ± s.e.m. are indicated. Significant differences were evaluated by one-way ANOVA followed by Tukey's post-hoc comparison test and marked with asterisks (*P < 0.05). Data are representative of two independent experiments.
Figure 1B RT-PCR on cytoplasmic (C) and nuclear (N) extracts showing the subcellular localization of lnc-SMaRT. RNA was isolated from C2C12 cells after two days of differentiation. GAPDH mRNA and pre-mRNA (pre-GAPDH) were used respectively as cytoplasmic and nuclear controls. Representative results from three independent experiments are shown.C qPCR analysis of lnc-SMaRT RNA expression in C2C12 cells undergoing differentiation at the indicated time points. The RNA expression levels in qPCR analyses were normalized against GAPDH mRNA and expressed as relative quantities with respect to GM samples set to a value of 1. Data are presented as the mean ± s.e.m. of three biological replicates (dots).D qPCR on RNA extracts from C2C12 myoblasts treated with either control siRNAs (si-SCR) or with two different siRNAs against lnc-SMaRT (si-SMaRT-1, si-SMaRT-2) and induced to differentiate for two days. The RNA expression levels in qPCR analyses were normalized against GAPDH mRNA and expressed as relative quantities with respect to GM samples set to a value of 1. Data are presented as the mean ± s.e.m. of three biological replicates (dots). Statistical analysis was performed with ordinary analysis of variance (ANOVA) followed by Dunnett's multiple comparison test. ***P < 0.001.E Representative immunofluorescence for Myosin Heavy Chain (MHC in red) in combination with DAPI staining (in blue) C2C12 murine myoblasts transfected with either control (si-SCR) or lnc-SMaRT siRNAs (si-SMaRT-1, si-SMaRT-2) fixed after at two days of differentiation. Histograms represent MHC-positive mononucleated cells/total MHC-positive cells ratio and fusion index quantification (F.I.). At least 5 randomly chosen microscope fields of two independent biological samples were analyzed (n>600 cells for each field). Data are presented as the mean. of the two biological replicates (dots).
Figure 2A Gene expression heatmap showing the hierarchical clustering of protein-coding genes whose expression is altered upon lnc-SMaRT depletion. Expression values, calculated as RPKMs, were log2-transformed and mean centered. Only genes with an average RPKM between 10 and 1000 were plotted.B Volcano plot describing the differential gene expression between si-SCR and si-SMaRT treated C2C12 samples. For each gene, log2 of fold-change versus -log10 of the unadjusted p-value is plotted. Points in red represent differentially expressed genes (FDR adjusted p-value < 0.05).D Western blot on protein extract from control and lnc-SMaRT overexpressing C2C12 stable cell lines collected after one day of differentiation. CASP3 antibody was used. ACTN was used as loading control. Representative results from three independent experiments are shown.
Figure 4A Immunofluorescence for total MLX protein detection (red signal) performed on C2C12 cells treated with siRNAs against Mlx-γ (si-Mlx-γ), lnc-SMaRT (si-SMaRT), DHX36 (si-DHX36) or with a control siRNA (si-SCR). Cells were fixed two days after the switch to differentiation medium. Representative binary images of single confocal planes for MLX immunofluorescence are shown for each treatment. The inserts on the topside of the images sketch the MLX signal peaks in the nuclei (white stars) to highlight the variation of MLX staining in each condition. Dashed lines indicate the edge of the nucleus.B Histogram represents the fluorescence intensity ratio of the MLX protein signals in the nuclear (Nuc) and cytoplasmic (Cyt) compartment in the indicated conditions. About 50 cells from two independent experiments were analyzed Data are presented as the mean of two biological replicates (dots).
Figure 5A Left panel: schematic representation of the luciferase constructs produced for Mlx. Mlx 5'- and 3'UTR-interacting regions were cloned respectively upstream and downstream the Renilla luciferase coding region (RLuc-Mlx 5'-3') and mutant derivatives devoid of the 5' interacting region (RLuc-Mlx-3') or the 3' UTR interacting region (RLuc-Mlx-5') were obtained. These constructs were co-transfected in C2C12 myoblasts in growth conditions together with plasmids expressing lnc-SMaRT (+) or with a control vector (-). Right panel: Luciferase activity data, presented as the mean ± s.e.m. of three biological replicates (dots), are shown with respect to each RLuc control vector (RLuc-Mlx 5'-3', RLuc-Mlx-3', RLuc-Mlx-5') set to a value of 1. Statistical analysis was performed with ordinary analysis of variance (ANOVA) followed by Dunnett's multiple comparison test. *P < 0.05.B The RLuc-Mlx-5' construct was co-transfected in N2a cells together with plasmids expressing lnc-SMaRT (+) or with a control vector (-) and treated with control siRNA (+) or siRNA against DHX36 (-). Luciferase activity data, presented as the mean ± s.e.m. of three biological replicates (dots), are shown with respect to RLuc-Mlx-5' vector set to a value of 1. Statistical analysis was performed with ordinary analysis of variance (ANOVA) followed by Dunnett's multiple comparison test. **P < 0.01, ***P < 0.001.C Left panel: schematic representation of the luciferase constructs used. The RLuc-Mlx-5' construct and its derived mutants (RLuc-Mlxmut30, RLuc-Mlx Δ75) were transfected in N2a cells in growth conditions together with lnc-SMaRT-expressing plasmids (lnc-SMaRT) or with its derived mutant (lnc-SMaRTmut) in the indicated combinations. Right panel: Luciferase activity data, presented as the mean ± s.e.m. of three biological replicates (dots), are shown with respect to each RLuc control vector (RLuc-Mlx-5', RLuc-Mlxmut30, RLuc-Mlx Δ75) set to a value of 1. Statistical analysis was performed with ordinary analysis of variance (ANOVA) followed by Dunnett's multiple comparison test. ***P < 0.001.
Figure 6A Upper panel: schematic representation of the predicted base pairing between lnc-SMaRT (region A) and Mlx γ mRNA; the predicted G-4 structure is boxed in red while the synthetic oligos sequences are underlined in red. Lower panel: Lane 1 and 2: 5 pmol of lnc-SMaRT and Mlx synthetic oligos were loaded on a denaturing polyacrylamide gel and stained for total RNA (SYBR GOLD, indicated as SYBR in the figure). Lane 3 and 4: 5 pmol of lnc-SMaRT and Mlx synthetic oligos were loaded on a native polyacrylamide gel and stained for total RNA (SYBR GOLD). Lane 5 and 6: 200 pmol of lnc-SMaRT and Mlx synthetic oligos were loaded on a native polyacrylamide gel and stained with the selective G-quadruplex staining N-methyl mesoporphyrin IX (NMM). Lane 7 and 8: 5 pmol of Mlx oligo alone (7) or in combination with 5pmol of lnc-SMaRT oligo (8) were heated at 100°C for 10 min and, after slow cooling, loaded on a native polyacrylamide gel and stained for total RNA (SYBR GOLD). Lane 9 and 10: 200 pmol of Mlx oligo alone (7) or in combination with 200 pmol of lnc-SMaRT oligo (8) were heated at 100°C for 10 min and, after slow cooling, loaded on a native polyacrylamide gel and stained with the selective G-quadruplex staining N-methyl mesoporphyrin IX. Asterisk (*) indicates the second NMM-positive band obtained upon SMaRT and γ-oligo interaction. Data information: Representative gels from at least three independent experiments are shown.B 5 pmol of indicated oligos were loaded on a native polyacrylamide gel. Oligos were treated as described. °C = oligos were heated (+) or were not heated (-) at 100 °C for 10 min and loaded after slow cooling. KCl= oligos were incubated (+) or were not (-) in a buffer containing 100mM KCl (for G-quadruplex stabilization). The gel was stained with total RNA staining (SYBR GOLD). Data information: Representative gels from at least three independent experiments are shown.C 200 pmol of indicated oligos treated as in (B) were loaded on a native polyacrylamide gel with the selective G-quadruplex staining N-methyl mesoporphyrin IX. Asterisk (*) indicates the second NMM-positive band obtained upon SMaRT and γ-oligo interaction. Data information: Representative gels from at least three independent experiments are shown.
Figure 2C) ykt6ts pho8∆60 cells containing an empty plasmid, 3HA-Ykt6 or 3HA-tagged Ykt6 mutant variants as indicated were analyzed as in A).D) ykt6ts pho8∆60 cells containing an empty plasmid, 3HA-Ykt6, or ADH1 promoter driven 3HA-Ykt6 wild type or 3HA-tagged Ykt6 mutant variants as indicated were grown and Pho8∆60 activity was monitored as described in A).E) Ykt6 levels were analyzed from the strains shown in D) and corresponding cell extracts were analyzed by anti-HA and anti-Pgk1 Western blotting.
Figure 3A) ykt6ts cells containing centromeric plasmids as indicated were grown at permissive temperature (23 ºC), shifted to the restrictive temperature at 37 ºC for 1 hour before starvation for 1 hour at 37 ºC. Cells were lysed and the total extract (T) separated into a cytoplasmic (S) and a 100'000x g membrane pellet (P) fraction. Fractions were analyzed by anti-HA, anti-Tom70 and anti-Pgk1 Western blotting. One representative experiment out of three biological replicates is shown.B) vam3∆ or ykt6ts vam3∆ cells containing the indicated centromeric plasmids were grown at permissive temperature (23 ºC), shifted to the restrictive temperature at 37 °C for 1 hour followed by 1 hour starvation in SD-N at 37 ºC. After cell lysis, samples were subjected to proteinase K (ProtK) and Triton X-100 (TX100) treatment as indicated and analyzed by anti-Ape1 Western blotting. Ape1*: proteinase K-resistant fragment of Ape1. One representative experiment out of three biological replicates is shown.C) Trichloroacetic acid extracts were prepared from ykt6ts cells containing an empty plasmid, 3HA-Ykt6 wild type, or 3HA-tagged Ykt6 mutant variants. Cells were grown at the permissive temperature (23 ºC) to late exponential phase in selective medium, shifted to the restrictive temperature (37 °C) for 1 h preincubation and continued to grow at the restrictive temperature for another 4 hours before harvesting. Samples were analyzed by anti-CPY and anti-Pgk1 Western blotting. One representative experiment out of three biological replicates is shown.D) Exponentially growing ykt6ts cells at permissive temperature (23 ºC) containing centromeric plasmids as indicated were shifted to the restrictive temperature (37 ºC) for 1 hour before starvation for 4 hours in SD-N. Trichloroacetic acid extracts were prepared. Lipidation of Atg8 was analyzed on a 15 % SDS-PAGE containing 6 M urea by anti-Atg8 and anti-Pgk1 Western blotting. One representative experiment out of three biological replicates is shown.
Figure 4A and B) GFP-Atg8 atg1∆, GFP-Atg8 atg1∆ vam3∆, GFP-Atg8 wild type, GFP-Atg8 vam3∆, GFP-Atg8 ykt6ts or GFP-Atg8 ykt6ts vam3∆ cell containing centromeric plasmids as indicated were grown at permissive temperature (23 ºC), shifted to the restrictive temperature at 37 ºC for 2 hours before starvation for 1 hour at 37 ºC in SD-N. Representative microscopy images are shown in A). GFP-Atg8 puncta per cell were quantified from 3 biological replicates (n≥100 cells) and the mean was plotted in B). Error bars are SD. Scale bar: 5 µm.Autophagosomes were prepared from GFP-Atg8 vam3∆ pep4∆ or GFP-Atg8 ykt6ts vam3∆ pep4∆ cells containing centromeric plasmids as indicated. Cells were starved for 16 hours at the permissive temperature (23 °C) to allow formation of mature, closed autophagosomes and shifted for 1 hour to the restrictive temperature before harvesting. Vacuoles were isolated from Vph1-4xmCherry atg15∆ pep4∆ cells grown under rich conditions at 30 °C. Fusion reactions were incubated at the restrictive temperature (30 °C) for 2 hours. Representative images are shown in C).Autophagosomes were prepared from GFP-Atg8 vam3∆ pep4∆ or GFP-Atg8 ykt6ts vam3∆ pep4∆ cells containing centromeric plasmids as indicated. Cells were starved for 16 hours at the permissive temperature (23 °C) to allow formation of mature, closed autophagosomes and shifted for 1 hour to the restrictive temperature before harvesting. Vacuoles were isolated from Vph1-4xmCherry atg15∆ pep4∆ cells grown under rich conditions at 30 °C. Fusion reactions were incubated at the restrictive temperature (30 °C) for 2 hours. The graph in D) shows the mean from at least three independent biological experiments. Error bars represent the standard deviation.The indicated strains were grown to late exponential phase at permissive temperature (23 ºC), shifted to the restrictive temperature at 37 °C for 1 hour followed by starvation in SD-N for 1 hour at 37 ºC. 3HA-Ykt6 and its mutant variants expressed from a centromeric plasmid were immunoprecipitated (IP) and analyzed by anti-HA Western blotting. Co-precipitating proteins were analyzed by anti-Vam3 and anti-Vti1 Western blotting. One representative experiment out of three biological replicates is shown.
Figure 1X-ray analysis of 6-months-old H2-c-fosLTR/Egfrwa/+ and H2-c-fosLTR/Egfrwa/wa littermates. Scale bars: 1cmBone tumor number per mouse at 5-6 months of age (n=23 wa/+, 15 wa/wa)Quantification of tumor size in tibiae at 5-6 months of age (n=23 wa/+, 15 wa/wa)Alkaline Phosphatase (ALP) levels in the serum at endpoint (age = 5-6 months; n=16 wa/+, 11 wa/wa)X-ray analysis before (age = 2 months) and after (age = 7 months) vehicle or Erlotinib treatmentQuantification of tumor number during treatment (n=6 Vehicle, 5 Erlotinib)Tumor size during treatment (n=6 Vehicle, 5 Erlotinib)Analysis of serum ALP levels during treatment (n=5)PET summation images (0-90 min) in horizontal (upper panel) and coronal view (lower panel) depicting [11C]Erlotinib distribution in one H2-c-fosLTR/Egfrwt mouse (M125). Anatomical structures are labelled with arrows (T, tumor; L, liver; H, heart; B, brain). Scale bars: 1cm Concentration-time curves of [11C]Erlotinib in bone tumors in right scapula of three H2-c-fosLTR/Egfrwt mice measured with PET. Broken line indicates threshold for in vitro effect of Erlotinib (1 µM)
Figure 2X-ray analysis of 7-months-old H2-c-fosLTR/Egfrwt and H2-c-fosLTR/Egfr∆Ob littermates. Scale bars: 1cmBone tumor number per mouse at 6-7 months of age (n=22 wt, 11 ∆Ob) Quantification of tumor size in tibiae (n=22 wt, 11 ∆Ob)ALP levels in the serum at 7-month endpoint (n=17 wt, 8 ∆Ob)µPET/CT analysis of 7-months-old H2-c-fosLTR/Egfrwt and H2-c-fosLTR/Egfr∆Ob littermates Standardized uptake values (SUV) of the µPET-tracer Na[18F]F in the pelvic osteosarcoma of 4- and 7-months-old H2-c-fosLTR/Egfrwt and H2-c-fosLTR/Egfr∆Ob mice. n=6 wt, 4 ∆Ob for 4 month time-point, n=6 wt, 3 ∆Ob for 7 month time-point
Figure 3PCNA and cleaved Caspase 3 IHC staining and quantification shown as % positive cells (for PCNA) and as positive cells per mm² (for cleaved Caspase 3) in OS from H2-c-fosLTR/Egfrwt (n=6) and H2-c-fosLTR/Egfr∆Ob (n=5) mice. Scale bars: 100µmEgfr, Ccnd1, c-fos and transgenic c-fos (c-fostg) mRNA expression levels in tumors of H2-c-fosLTR/Egfrwt and H2-c-fosLTR/Egfr∆Ob mice normalized to Tbp. n=17 wt, 14 ∆Ob (Egfr, Ccnd1), n=16 wt, 13 ∆Ob (c-fos), n=16 wt, 14 ∆Ob (c-fostgIHC staining and quantification showing pRSK2 (n=4), pCREB (n=7 wt, 6∆Ob) and c-Fos (n=5) positive cells (%) in OS from H2-c-fosLTR/Egfrwt and H2-c-fosLTR/Egfr∆Ob littermates. Scale bars: 100µm
Figure 4Western Blot analysis of primary OS cells isolated from H2‑c‑fosLTR/Egfrwt and H2-c-fosLTR/Egfr∆Ob micec-fos and transgenic c-fos (c fostg) mRNA expression levels in primary H2-c-fosLTR OS cells after 4x in vitro passages, cultured under standard conditions (n=3 independent cell lines)Western Blot analysis of H2-c-fosLTR/Egfrwt OS cells treated for 24h with Erlotinibc-fos and c-fostg mRNA expression levels in H2-c-fosLTR/Egfrwt OS cells treated for 24h with Erlotinib (10µM) or DMSO as control (n=4 independent cell lines)Western Blot analysis of starved H2-c-fosLTR/Egfrwt OS cells, pre-treated with DMSO (1:1000), Afatinib (5µM), GSK2233470 (10µM), Rapamycin (10nM) or U0126 (10µM) for 30 minutes and stimulated with EGF (50ng/ml) as indicatedWestern Blot analysis of primary OS cells isolated from a p53f/f Rb1f/f Osx-Cre mouse after 24h Erlotinib treatmentc-fos mRNA expression levels in p53f/f Rb1f/f Osx-Cre OS cells treated for 24h with Erlotinib (10µM) (n=3)Western Blot analysis of starved p53f/f Rb1f/f Osx-Cre OS cells, pre-treated with DMSO (1:1000) or Afatinib (5µM) for 30 minutes and stimulated with EGF (50ng/ml) as indicated
Figure 5X-ray analysis of 6-months-old H2-c-fosLTR and H2-c-fosLTR/ColAREG littermates. Scale bars: 1cmBone tumor number per mouse at 5-6 months of age (n=22 wt, 13 ColAREG)Quantification of tumor size in tibiae at 5-6 months of age (n=22 wt, 13 ColAREG)Alkaline Phosphatase (ALP) levels in the serum at endpoint (age = 5-6 months; n=29 wt, 19 ColAREG)c-fos mRNA expression levels in osteosarcomas of H2-c-fosLTR (n=11) and H2-c-fosLTR/ColAREG mice (n=14)Western Blot analysis of bone tumor protein lysates from H2-c-fosLTR and H2-c-fosLTR/ColAREG mice
Figure 6Representative images of OS biopsies stained with antibodies against EGFR and c‑Fos. Scale bars: 50µmKaplan-Meier survival curve comparing the survival of patients with EGFR and c-Fos double positive osteosarcomas against patients without co-expression of both proteins. (n=52)
Figure 7Western Blot analysis showing EGFR and c-Fos protein levels in human OS cell lines cultured under standard conditionsCell viability of 143b or LM7 cells cultured for 24h in medium (+10% FCS) with DMSO (1:2000) or Erlotinib (10µM) (n=3, representative result from 3 independent experiments, shown as fold change, normalized to DMSO)Cell viability of 143b or LM7 cells cultured for 24h in medium (+0.5% FCS) containing 1xPBS (contr., 1:200) or EGF (50ng/ml) (n=3, representative result from 3 independent experiments, shown as fold change, normalized to control)143b xenograft growth curve during therapeutic regime (n=8 vehicle, 6 Erlotinib; two independent experiments) 143b tumor weight at endpoint (n=8 Vehicle, 6 Erlotinib; two independent experiments)LM7 xenograft growth curve during therapeutic regime (n=7 Vehicle, 5 Erlotinib; two independent experiments) LM7 tumor weight at endpoint. n=7 vehicle, 5 Erlotinib, two independent experimentIHC analysis of PCNA and cleaved Caspase 3 in 143b-derived tumors (n=4). Scale bars: 50µm IHC analysis of pCREB and c-Fos in 143b-derived tumors. (n=5 for pCREB and n=4 for c-Fos analysis). Scale bars: 50µmWestern Blot analysis of pCREB/CREB and p-c-Fos/c-Fos protein expression in lysates directly isolated from 143b xenografts at endpoint, after 14 days of vehicle or Erlotinib treatment
Figure 1 (B) Automatic cluster-mapping of the temperature-dependent splicing changes in CHX, based on changes in PSI (percent spliced in). Categories (right) were manually assigned.  (C) Examples for cold-induced (blue) or heat-induced (red) AS-decay via exon inclusion or exon skipping. In each example, on top a simplified exon-intron structure is given, PTCs and the length of frameshift inducing exons are highlighted. Predicted splicing changes were validated by radioactive RT-PCR with primers binding to the surrounding constitutive exons. Box plots show median PSI of validation PCRs of four different samples from at least two different mice, whiskers show min to max. Individual data points are shown as circles.