EMBO/soda-nel huggingface dataset:
The dual role of c-src in cell-to-cell transmission of α-synuclein (2020)
Figure 6

Figure 6. Brain permeable c-src inhibitor, saracatinib efficiently inhibits the propagation of α-syn in A53T TG mice

Two to three-month-old hemizygous M83 mice were injected into the striatum with sterile PBS or 10 μg of recombinant α-syn fibrils. The mice were treated with saracatinib at 10 mg/kg/day by oral gavage for 4 weeks.

A. Schematic diagram showing the in vivo experiment.

B. Fixed brain tissues containing the frontal cortex and the striatum were lysed. Western blot was performed with indicated antibodies. Values were derived from three individual mice (n=3).

C. Immunohistochemistry of pSer129 α-syn was performed. Arrow indicates pSer129 α-syn positive inclusion. Scale bar indicates 50 μm.

D. Scoring of pSer129 immunoreactivity in the cortex and the striatum of both regions. Values were derived from eight individual mice (n=8). *** P < 0.001, ** P < 0.01, * P < 0.05 compared to PBS group, unpaired t-test. Data are expressed as mean ± SEM.

E. Schematic representation of the scoring of α-syn inclusion pathology detected by pSer129 staining.
The dual role of c-src in cell-to-cell transmission of α-synuclein (2020)
Figure 7

Figure 7. Saracatinib efficiently inhibits the micro/astrogliosis in A53T TG mice

A. Immunohistochemistry of GFAP was performed. GFAP intensities in the cortex and the striatum were analyzed. Values were derived from three individual mice (n=3). *** P < 0.001, ** P < 0.01, * P < 0.05, one-way ANOVA.

B. Immunohistochemistry of IBA-1 and MHC Class II were performed. Arrow indicates MHC class II positive cells. Numbers of MCH class II positive cells in the cortex and the striatum were analyzed. Values were derived from three individual mice (n=3). *** P < 0.001, ** P < 0.01, one-way ANOVA.

Data information: Data are expressed as mean ± SEM. Scale bar indicated 50 μm.
A role for Mog1 in H2Bub1 and H3K4me3 regulation affecting RNAPII transcription and mRNA export (2018)
Figure 1

Figure 1. Monoubiquitination of H2B requires Mog1. (A) MOG1 interacts genetically with the H2B deubiquitination machinery. Ten-fold serial dilutions of the indicated strains were spotted on YPD and incubated for 2 days at the indicated temperatures. (B) Expression levels of total H2B and H2Bub1 were analyzed in wild-type (WT), mog1Δ, ubp8Δ and ubp8Δmog1Δ whole cell lysates by western blotting using anti-total H2B and anti-H2Bub1 antibodies. Bar graphs of H2Bub1 levels after H2B normalization show the mean and standard deviations of at least three independent experiments. The P-value was calculated using Student´s t-test. Error bars represent SD. The P-value was calculated using Student´s t-test (*, P = 0.01-0.05; **, P = 0.001-0.01; ***, P < 0.001). (C), (D) and (E) The indicated strains were diluted as described in (A) and were then spotted on YPD or YPGal and incubated for 2 days.
A role for Mog1 in H2Bub1 and H3K4me3 regulation affecting RNAPII transcription and mRNA export (2018)
Figure 2

Figure 2. Mog1 affects trimethylation of H3K4 (A) The expression levels of H3K4me3 and H3 in total extracts of wild-type (WT), mog1Δ, set1Δ and spp1Δ strains were analyzed by western blotting using the indicated antibodies. Bar graphs of H3K4me3 levels after H3 normalization show the mean and standard deviations of at least three independent experiments. The P-value was calculated using Student´s t-test (*, P = 0.01-0.05; **, P = 0.001-0.01; ***, P < 0.001). (B) ChIP analysis of H3K4me3 presence at ADH1, PMA1 and YEF3 promoter (P) and 5'ORF in wild-type (WT) or mog1Δ strains relative to total H3. The bar graphs indicate the mean and standard deviation for at least three independent experiments. The significance of the differences was calculated using Student´s t-test (*, P value = 0.01-0.05; **, P value = 0.001-0.01; ***, P < 0.001). (C) MOG1 interacts genetically with the H3 methylases Set1 and Set2. Ten-fold serial dilutions of the indicated strains were spotted on YPD and incubated for 2 days at the indicated temperatures. (D) histone H3K4A and H3K4Amog1Δ strains were compared with their isogenic strains as in (C).
A role for Mog1 in H2Bub1 and H3K4me3 regulation affecting RNAPII transcription and mRNA export (2018)
Figure 3

Figure 3. MOG1 deletion affects sensitivity to hydroxyurea and RNAPII-dependent transcription. (A) Ten-fold serial dilutions of the indicated strains were spotted on YPD containing the indicated concentrations of hydroxyurea (HU) and were incubated for 2-3 days. (B) mRNA synthesis rates (SR) as determined in Genomic Run-On analysis and (C) mRNA abundance (RA), in mog1∆ compared with wild type (WT) cells. Bar graphs showing the average level and standard deviation of three experiments (n=3) for SR and RA values obtained as the median of the whole gene dataset values in arbitrary units (x 107) from image analysis quantification. Error bars represent SD. Significance of the differences was obtained using Student´s t-test and statistical difference is considered as *, P value = 0.01-0.05; **, P value = 0.001-0.01; ***, P value < 0.001. The scatter plots (right panels) show the variation of the transcription (B) or mRNA (C) levels of individual genes. Pearson correlation values are: -0.039 (SR) and -0.042 (RA). This low correlation and the flat shape of the clouds on scatter plots (see red tendency line) indicates that there is no bias of the transcriptional effect with regard to expression level. (D) Box plots of SR and RA median levels of mog1Δ cells compared with WT for all genes (Total), SAGA and TFIID-dominated genes. These data were obtained from three independent replicates (n=3) of the RA and SR data that were averaged as explained in the main text. The median and standard deviation for the whole data set (Total) and for 351 (for RA) or 316 (SR) SAGA-dominated genes and for the 3903 (RA) or 3403 (SR) TFIID-dominated genes are shown. Significance of the differences was calculated using Student´s t-test. No significant difference between the different groups was found. The similar variations in SRs and RAs indicate that there are no changes in mRNA half-lives upon MOG1 deletion (shown in Fig EV4). The total number of genes with confident value in the analyses was 3953 for RA and 3989 for SR.
A role for Mog1 in H2Bub1 and H3K4me3 regulation affecting RNAPII transcription and mRNA export (2018)
Figure 4

Figure 4. Mog1 affects the recruitment of Rad6, Bre1 and Rtf1 to 5'ORFs (5') and 3'ORFs (3') of genes. (A) ChIP analysis of TAP-tagged Rad6 presence at ADH1, PMA1 and YEF3 coding regions (5' & 3') in WT or mog1Δ strains. Bar charts indicate the mean and standard deviation for at least three independent experiments. Significance of the differences was obtained using Student´s t-test (*, P value = 0.01-0.05; **, P value = 0.001-0.01, ***, P value = 0.0001-0.001, ****, P value <0.0001). (B), (C) and (D) Bre1-TAP, Rtf1-TAP and Rpb3 respectively, were analyzed as described in (A). (E) Bar charts showing the relative chromatin occupancy to 5'ORFs (left panel) or 3'ORFs (right panel) of each factor relative to Rpb3 occupancy at these gene positions. Inputs were analyzed by western blotting using anti-TAP and anti-PGK (loading control) antibodies to monitor protein levels (Fig EV5).
A role for Mog1 in H2Bub1 and H3K4me3 regulation affecting RNAPII transcription and mRNA export (2018)
Figure 5

Figure 5. Mog1 co-purifies with factors involved in transcription and associates with chromatin (A) Co-purification of Rtf1-PK in cells expressing Mog1-TAP (+,+). A non-tagged strain (-,-) and cells expressing only Rtf1-PK (+,-) were included as negative controls for co-purification. Inputs and IPs are depicted for each strain. (B) ChIP analysis of the presence of HA-tagged Mog1 compared to a non-tagged strain (WT) at PMA1 and YEF3 promoter (P) and coding regions (5'ORF). The occupancy level was calculated as the signal ratio of the IP samples in relation to the Input signal normalized to an Intergenic region. Bar charts indicate the mean and standard deviation for at least three independent experiments. Significance of the differences was obtained using Student´s t-test and is presented as a P-value (*, P<0.05). (C) List of proteins identified from at least two independent LC-MS/MS analyses of Mog1-TAP purifications. Numbers indicate peptides of matching amino acids from identified peptides with a confidence greater or equal to 95% divided by the total number of amino acids in the sample (coverage 95%). An identifier of function or complex for each protein is listed. (D) Mog1-HA immunoprecipitation in cells expressing Bre1-PK (upper panel) and Mog1-TAP co-precipitation in cells expressing Ada2-HA (lower panel) are indicate as in (A) Inputs and IPs are depicted for each strain. (E) Representation of the different fragments of the MOG1 gene identified in genome-wide Shg1 yeast two-hybrid screening. The region common to all identified MOG1 clones between residues 114 and 154 is depicted in grey as the SHG1 ID (interaction domain). The start and end positions in the MOG1 gene are indicated as well as the number of clones containing this fragment.
A role for Mog1 in H2Bub1 and H3K4me3 regulation affecting RNAPII transcription and mRNA export (2018)
Figure 7

Figure 7. Mog1 binding to Ran is dispensable for its function in epigenetic modification. (A) The Mog1E65K Ran-binding mutant complements the slow growth of mog1ΔsetΔ mutants. Ten-fold serial dilutions of wild-type (WT), mog1Δ, set1Δ, and mog1ΔsetΔ strains transformed with wild-type MOG1 (pMOG1) or with the Ran-binding mutant (pMOG1E65K) as indicated were spotted on SC-LEU and incubated for 2 days at the indicated temperatures. (B) The Mog1E65K Ran-binding mutant complements the low H3K4me3 and H2Bub1 levels of mog1Δ. The expression levels of H3K4me3, total H3, H2Bub1, total H2B and PGK1 in whole cell lysates from wild type (WT) or from mog1Δ cells transformed with an empty plasmid (+pEmpty), wild type MOG1 (pMOG1) or a Ran-binding mutant (pMOG1E65K) were analyzed by western blotting using the corresponding antibodies. (C) Gsp2 is not involved in H2Bub1 regulation. The expression levels of total H2B and of H2Bub1 were analyzed in whole cell lysates from wild type (WT) and gsp2Δ strains by western blotting using anti-total H2B and anti-H2Bub1 antibodies.

EXPANDED VIEW FIGURE LEGENDS
CG7630 is the D. melanogaster homolog of the cytochrome c oxidase subunit COX7B (2022)
Figure 3

Fig. 3: CG7630 is a bona fide COX subunit

(a) Relative quantification (RQ) of CG7630 transcript after RNAi with a 120-bp inverted-repeat sequence (act5c-gal4>UAS-CG7630RNAi) compared to control (act5c-gal4>+). Data are plotted as mean ± S.D. (n = 3 biological replicates, Student's t test ***p = 0.0005). (b) Relative percentage of egg to adult viability of CG7630 RNAi cross (act5c-gal4/CyO.GFP x UAS-CG7630RNAi) and control cross (act5c-gal4/CyO.GFP x w1118), calculated at three developmental stages (eggs, pupae and adults), (n > 250, Chi-square test 9.920 df(2), **p = 0.007). (c) Mendelian frequencies of adults obtained by crossing heterozygous act5c-gal4>CyO.GFP flies with w1118 genetic background flies (+/+) (n = 388 individuals, Chi-square test 0.2527, df(1), p=0.6152), and by crossing heterozygous act5c-gal4>CyO.GFP flies with homozygous UAS-CG7630RNAi flies (n = 340 individuals, Chi-square test 226.7, df(1), ****p < 0.0001). (d) Kinetic enzyme activity of individual MRC complexes in CG7630 RNAi and control individuals normalized by citrate synthase (CS) activity. Data are plotted as mean ± S.D. (n = 3 biological replicates, 2-way ANOVA with Sidak's multiple comparisons, *p ≤ 0.05, ****p ≤ 0.0001). (e) In gel-activity assays for MRC complexes II and IV in DDM-solubilized mitochondria from RNAi (act5c-gal4>UAS-CG7630RNAi) and control larvae (act5c-gal4>+). (f) BN-PAGE, Western blot immunodetection of MRC complexes from CG7630 RNAi (act5c-gal4>UAS-CG7630RNAi) and control (act5c-gal4>+) larvae using antibodies against specific subunits: anti-UQCRC2 (complex III), anti-NDUFS3 (complex I), anti-COX4 (complex IV) and anti-SDHA (complex II). (g) Western blot analysis of steady state levels (SDS-PAGE) of MRC subunits COX4 (complex IV), SDHA (complex II) and UQCR-C2 (complex III) in whole lysates from CG7630 RNAi (act5c-gal4>UAS-CG7630RNAi) and control (act5c-gal4>+) larvae. (h) Western blot analysis of anti-HA affinity purified samples from S2R+ D. melanogaster cells. EV = samples from cells carrying the empty expression vector. CG7630-HA = samples from cells expressing HA-tagged CG7630. Tot = total mitochondrial-enriched fractions; unb = unbound material; IP:HA = immunoprecipitated material. Samples were probed with antibodies against MRC subunits of complexes I (NDUFS3), II (SDHA), III (UQCR-C2), IV (COX4) and V (ATP5A).
CG7630 is the D. melanogaster homolog of the cytochrome c oxidase subunit COX7B (2022)
Figure 6

Fig. 6: Human COX7B can rescue COX deficiency caused by loss of CG7630.

(a) Relative quantification (RQ) of CG7630 transcript in wild-type D. melanogaster S2R+ cells treated with a dsRNA targeting CG7630 (CG7630 KD) and a mock control dsRNA (mock). Data are plotted as mean ± S.D. (n = 3 biological replicates, Student's t test ***p = 0.0005). (b) Relative quantification (RQ) of CG7630 and COX7B transcripts in stable S2R+ cell lines carrying the empty expression vector (EV:GFP;neo), and expression vectors carrying HA-tagged forms of human COX7B (hCOX7B-HA;neo) and CG7630 (CG7630-HA;neo). Cells were treated with a dsRNA targeting CG7630 (striped bars, CG7630 KD) and a mock control dsRNA (solid bars, mock). N/D = not detected. Data are plotted as mean ± S.D. (n = 3 biological replicates, 1-way ANOVA with Dunnet's multiple comparisons, *p = 0.0370, **p = 0.0074, ***p = 0.0003). (c) Immunoblot analysis of stable S2R+ cell lines carrying the empty expression vector (EV:GFP;neo), and expression vectors carrying HA-tagged forms of human COX7B (hCOX7B-HA;neo) and CG7630 (CG7630-HA;neo). The CG7630 transcript was knocked-down (KD) in cells by a dsRNA targeting CG7630 and compared with cells treated with a mock control dsRNA (mock). Samples were probed with an antibody anti HA tag and with an antibody against Hsp70 as an endogenous control. (d) Kinetic enzyme activity of COX normalized by citrate synthase activity (CS) in stable S2R+ cell lines carrying the empty expression vector (EV:GFP;neo), and expression vectors carrying HA-tagged forms of human COX7B (hCOX7B-HA;neo) and CG7630 (CG7630-HA;neo). Cells were treated with a dsRNA targeting CG7630 (striped bars, CG7630 KD) and a mock control dsRNA (solid bars, mock). Data are plotted as mean ± S.D. (n = 3 biological replicates, 2-way ANOVA with Sidak's multiple comparisons, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001). (e) Steady-state levels of MRC subunits in samples from rescue experiment performed as in (d).

EXPANDED VIEW FIGURE LEGENDS
LncRNA-MIF, a c-Myc-activated long non-coding RNA, suppresses glycolysis by promoting Fbxw7-mediated c-Myc degradation (2016)
Figure 1

Figure 1. LncRNA-MIF inhibits c-Myc expression.

A. HeLa, H1299 and A549 cells were infected with lentiviruses expressing control shRNA or lncRNA-MIF shRNA. Forty-eight hours after infection, cell lysates were subjected to Western blot analysis with anti-c-Myc antibody.

B. Schematic illustration of lncRNA-MIF genomic localization and three putative c-Myc binding sites.

C. Total RNA from the indicated cell lines was subjected to Northern blot analysis to determine the molecular size of lncRNA-MIF. Actin was also included as a positive control.

D. HeLa cells were infected with lentiviruses expressing control RNA, lncRNA-MIF, control shRNA, or lncRNA-MIF shRNA as indicated. Forty-eight hours after infection, cell lysates were analyzed by Western blotting with anti-c-Myc and anti-GAPDH antibodies.

E. HeLa cells were infected with lentiviruses expressing control RNA, lncRNA-MIF, control shRNA, or lncRNA-MIF shRNA as indicated. Forty-eight hours after infection, total RNA was extracted from these cells and subjected to real-time RT-PCR analysis. Data shown are mean ± SD (n = 3; *P < 0.05, two-tailed t-test).

F. HeLa cells expressing either control RNA or lncRNA-MIF were treated with or without MG132 (20 μM) for 6 h. Cell lysates were analyzed by Western blot with the indicated antibodies.

G. HeLa cells expressing either control shRNA or lncRNA-MIF shRNA were treated with MG132 for 6 h. Cell lysates were analyzed by Western blotting with the indicated antibodies.

H. HeLa cells were infected with lentiviruses expressing control RNA, lncRNA-MIF or lncRNA-MIF-AS as indicated. Forty-eight hours after infection, cells were treated with cycloheximide (CHX, 50 μg/ml) for the indicated periods of time. Cell lysates were then analyzed by Western blotting to examine the c-Myc protein half-life. Protein band intensity were analyzed by ImageJ.

I. Band intensity of c-Myc in Fig 1H was analyzed by ImageJ.

J. HeLa cells were infected with lentiviruses expressing control shRNA, lncRNA-MIF shRNA-1, -2 or -3 as indicated. Forty-eight hours after infection, cells were treated with CHX for the indicated periods of time. Cell lysates were then analyzed by Western blotting to examine the half-life of c-Myc protein.

K. Band intensity of c-Myc in Fig 1J was analyzed by ImageJ.
LncRNA-MIF, a c-Myc-activated long non-coding RNA, suppresses glycolysis by promoting Fbxw7-mediated c-Myc degradation (2016)
Figure 2

Figure 2. LncRNA-MIF is a direct transcriptional target of c-Myc.

A. P493-6 cells (human B cell lymphoma cell line) carrying a c-Myc tet-off system were treated with Doxycycline (1 μg/ml) for the indicated periods of time. Total RNA was subjected to real-time RT-PCR analysis. Data shown are mean ± SD (n = 3; *P < 0.05, **P < 0.01, two-tailed t-test). Cell lysates were also analyzed by Western blotting with the indicated antibodies.

B. HeLa, HCT116 and MCF10A cells were infected with lentiviruses expressing control shRNA or c-Myc shRNA. Forty-eight hours after infection, total RNA and cell lysates were analyzed by real-time RT-PCR and Western blotting, respectively. Data shown are mean ± SD (n = 3; *P < 0.05, **P < 0.01, two-tailed t-test).

C. HeLa, HCT116 and MCF10A cells were transfected with control vector or Flag-c-Myc. Twenty-four hours after transfection, total RNA was extracted from these cells and subjected to real-time RT-PCR analysis. Data shown are mean ± SD (n = 3; *P < 0.05, **P < 0.01, two-tailed t-test). Cell lysates were also analyzed by Western blotting.

D. Schematic illustration of consensus c-Myc binding sites in lncRNA-MIF gene promoter. The indicated pGL3-based luciferase reporter constructs were generated to examine transcriptional activities of three putative c-Myc binding sites MIF1, MIF2 and MIF3 in response to c-Myc induction. MIF1-M, MIF2-M and MIF3-M indicate their corresponding mutant binding sites which are shown in the open boxes.

E. HeLa cells were co-transfected with either Flag-c-Myc or control vector plus the indicated reporter constructs and Renilla luciferase plasmid. Twenty-four hours after transfection, reporter activity was measured and plotted after normalizing with respect to Renilla luciferase activity. Data shown are mean ± SD (n = 3; **P < 0.01, two-tailed t-test).

F. HeLa cells expressing control shRNA or c-Myc shRNA were co-transfected with the indicated reporter constructs and Renilla luciferase plasmid. Twenty-four hours after transfection, reporter activity was measured and plotted after normalizing with respect to Renilla luciferase activity. Data shown are mean ± SD (n = 3; **P < 0.01, two-tailed t-test).

G. P493-6 cells were co-transfected with the indicated reporter constructs and Renilla luciferase plasmid. Twenty-four hours after trasfection, cells were treated with doxycycline for 0 hour and 12 hours respectively. Then reporter activity was measured and plotted after normalizing with respect to Renilla luciferase activity. Data shown are mean ± SD (n = 3; **P < 0.01, two-tailed t-test).

H. Lysates from HeLa cells were subjected to ChIP assay. ChIP products were amplified by qPCR reaction with the indicated pairs of primers. Data shown are mean ± SD (n = 3; *P < 0.05, two-tailed t-test).
LncRNA-MIF, a c-Myc-activated long non-coding RNA, suppresses glycolysis by promoting Fbxw7-mediated c-Myc degradation (2016)
Figure 4

Figure 4. LncRNA-MIF regulates Fbxw7 and c-Myc expression via miR-586.

A. Schematic illustration of psicheck2-based luciferase reporter constructs used for examining the effect of miR-586 on the 3’ UTR of Fbxw7. Mutated bases are in red color in open box.

B. miR-586 mimics or negative control mimics (NC mimics) were transfected into HeLa cells together with the indicated pSICHECK2-based luciferase reporter construct. Twenty-four hours after transfection, reporter activity was measured and plotted after normalizing with respect to Renilla luciferase activity. Data shown are mean ± SD (n = 3; *P < 0.05, two-tailed t-test).

C. miR-586 inhibitor or negative control inhibitor (NC inhibitor)was transfected into HeLa cells together with the indicated pSICHECK2-based luciferase reporter construct. Twenty-four hours after transfection, reporter activity was measured and plotted after normalizing with respect to Renilla luciferase activity. Data shown are mean ± SD (n = 3; *P < 0.05, two-tailed t-test).

D. HeLa cells were transfected with miR-586 mimics or inhibitors as indicated. Forty-eight hours after transfection, cell lysates were analyzed by Western blotting.

E. HeLa cells were transfected with miR-586 mimics or inhibitors as indicated. Forty-eight hours after transfection, total RNA was extracted and subjected to real-time RT-PCR analysis. Data shown are mean ± SD (n = 3; **P < 0.01, two-tailed t-test).

F. The copy numbers of miR-586 and lncRNA-MIF transcript per cell in the indicated cells were quantified using a quantitative real-time RT-PCR assay. Data shown are mean ± SD (n = 3).

G. HeLa cells were infected with lentiviruses expressing control shRNA or lncRNA-MIF shRNA. Forty-eight hours after infection, total RNA was subjected to real-time RT-PCR analysis. HeLa cells were infected with lentiviruses expressing lncRNA-MIF or control RNA. Forty-eight hours after infection, total RNA was analyzed by real-time RT-PCR. Data shown are mean ± SD (n = 3; *P < 0.05, **P < 0.01, two-tailed t-test).

H. HeLa cells expressing lncRNA-MIF or control RNA were transfected with miR-586 mimics or NC mimics. Twenty-four hours after transfection, cell lysates were analyzed by Western blotting.

I. HeLa cells expressing lncRNA-MIF or control shRNA were transfected with miR-586 inhibitor or NC inhibitor. Twenty-four hours after transfection, cell lysates were analyzed by Western blotting.

J. Mutant bases (in red color) in nonsense mutant lncRNA-MIF (negative control, NC) and in miR-586 binding defective lncRNA-MIF are shown in boxes.

K. HeLa cells were infected with lentiviruses expressing control RNA, lncRNA-MIF, lncRNA-MIF harboring nonsense mutation or miR586 binding defective lncRNA-MIF. Forty-eight hours after transfection, cell lysates were analyzed by Western blotting.