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Journal: International Journal of Molecular Sciences
Article Title: circSLC41A1 Resists Porcine Granulosa Cell Apoptosis and Follicular Atresia by Promoting SRSF1 through miR-9820-5p Sponging
doi: 10.3390/ijms23031509
Figure Lengend Snippet: SRSF1 resists GC apoptosis and follicular atresia. ( A ) Venn diagram of the DEGs (differentially expressed genes) between HFs and AFs and miR-9820-5p target genes predicted by PITA, Miranda, and TargetSpy. ( B ) Differentially expressed SRSF1 in HFs and AFs detected by qRT-PCR. ( C , D ) Effective knockdown of SRSF1 by si-SRSF1 qRT-PCR and western blotting (WB). ( E ) The shift of GC apoptosis rates after SRSF1 knockdown was detected by flow cytometry. Data are expressed as the mean ± SEM of three experiments; * p < 0.05, ** p < 0.01.
Article Snippet: The antibodies used in this study were
Techniques: Quantitative RT-PCR, Western Blot, Flow Cytometry
Journal: International Journal of Molecular Sciences
Article Title: circSLC41A1 Resists Porcine Granulosa Cell Apoptosis and Follicular Atresia by Promoting SRSF1 through miR-9820-5p Sponging
doi: 10.3390/ijms23031509
Figure Lengend Snippet: miR-9820-5p promotes pGCs apoptosis by targeting SRSF1 . ( A , B ) The binding of miR-9820-5p and SRSF1-WT was predicted by RNAhybrid and verified by dual-luciferase activity analysis. ( C , D ) SRSF1 mRNA and protein levels after transfection of miR-9820-5p mimics. ( E , F ) SRSF1 mRNA and protein levels after transfection of miR-9820-5p inhibitors. ( G ) The shift of GC apoptosis rates after co-transfection of miR-9820-5p inhibitors and si-SRSF1 detected by flow cytometry. Data are expressed as the mean ± SEM of three experiments; * p < 0.05, ** p < 0.01.
Article Snippet: The antibodies used in this study were
Techniques: Binding Assay, Luciferase, Activity Assay, Transfection, Cotransfection, Flow Cytometry
Journal: International Journal of Molecular Sciences
Article Title: circSLC41A1 Resists Porcine Granulosa Cell Apoptosis and Follicular Atresia by Promoting SRSF1 through miR-9820-5p Sponging
doi: 10.3390/ijms23031509
Figure Lengend Snippet: circSLC41A1 inhibits GCs apoptosis by upregulating SRSF1 through sponging miR-9820-5p. ( A ) The shift of SRSF1 mRNA levels after co-transfection of si-circSLC41A1 and miR-9820-5p inhibitors detected by qRT-PCR. ( B ) The shift of SRSF1 protein levels after co-transfection of the si-circSLC41A1 and miR-9820-5p inhibitors detected by WB. ( C ) Change of GC apoptosis rates after co-transfection of si-circSLC41A1 and miR-9820-5p inhibitors detected by flow cytometry. Data are expressed as the mean ± SEM of three experiments; * p < 0.05, ** p < 0.01.
Article Snippet: The antibodies used in this study were
Techniques: Cotransfection, Quantitative RT-PCR, Flow Cytometry
Journal: International Journal of Molecular Sciences
Article Title: circSLC41A1 Resists Porcine Granulosa Cell Apoptosis and Follicular Atresia by Promoting SRSF1 through miR-9820-5p Sponging
doi: 10.3390/ijms23031509
Figure Lengend Snippet: Schematic diagram of circSLC41A1 functions in GC apoptosis. circSLC41A1 can function by competing with SRSF1 mRNA for miR-9820-5p interactions, thereby alleviating the post-transcriptional repression of SRSF1 and thus resisting GC apoptosis and follicular atresia. circSLC41A1 also had the potential to encode small circSLC41A1-134aa peptides.
Article Snippet: The antibodies used in this study were
Techniques:
Journal: EMBO Molecular Medicine
Article Title: SRSF protein kinase 1 modulates RAN translation and suppresses CGG repeat toxicity
doi: 10.15252/emmm.202114163
Figure Lengend Snippet: A Hybridization chain reaction (HCR) detection of CGG repeat RNA along with immunocytochemistry (ICC) to detect SRSF1‐FLAG in U2OS cells. Expanded view of merged channels shows the CGG RNA foci (arrowhead) and an example of SRSF1‐CGG RNA foci co‐localization (arrow). DAPI marks the nucleus. Scale bars are 10 µm. B Co‐localization analysis of CGG RNA (red traces) and SRSF1 (green traces) showing overlaps of CGG RNA and SRSF1. (a.u. = arbitrary unit). C Co‐immunoprecipitation of indicated SRSF proteins with PP7‐tagged control sequence (partial nLUC sequence) to correct for non‐specific binding, AUG, and CGG reporter RNAs. SRSF1 and 2 specifically immunoprecipitated with PP7‐tagged CGG reporter. Source data are available online for this figure.
Article Snippet: For Western blots, following antibodies were used: FLAG‐M2 at 1:1,000 dilution (mouse, Sigma F1804), 1:2,500 β‐Actin (mouse, Sigma A1978), 1:1,000
Techniques: Hybridization, Immunocytochemistry, Immunoprecipitation, Sequencing, Binding Assay
Journal: EMBO Molecular Medicine
Article Title: SRSF protein kinase 1 modulates RAN translation and suppresses CGG repeat toxicity
doi: 10.15252/emmm.202114163
Figure Lengend Snippet: A Schematic of (CGG)90‐EGFP construct and experimental outline for rough eye phenotype screening. B Quantitation of GMR‐GAL4‐driven uas‐(CGG)90‐EGFP eye phenotype with candidate modifiers ( n ≥ 30 flies/genotype). siNTC = siRNA against a non‐targeting control gene (mCherry). Different siRNA lines for the same target gene are numbered (#1 and #2). Error bars represent mean ± SD. C Representative photographs of fly eyes expressing either GMR‐GAL4 driver alone or with uas‐(CGG)90‐EGFP construct, with fly SRSF1 (dSF2) and SRSF2 (dSC35) knockdown or disruptions (insertion). D Representative photographs of fly eyes and quantitation (below) of rough eye scores with fly SRSF1 overexpression (dSF2 OE); n = 20–32/genotype. Error bars represent mean ± SD. E Representative photographs of fly eyes expressing GMR‐GAL4‐driven (GGGGCC)28‐EGFP with indicated uas‐siRNAs against fly SRSF genes in comparison with non‐targeting control (NTC) siRNA against LUC/luciferase. F Quantitation of (GGGGCC)28‐EGFP rough eye phenotype with SRSF modifiers ( n ≥ 30 flies/genotype). Error bars represent mean ± SD. G Representative photographs of fly eyes expressing GMR‐GAL4‐driven (GGGGCC)28‐EGFP at 29°C along with the quantifications of necrosis and eye width. n = 28–30/genotype. Error bars represent mean ± SD. H, I Survival assays of flies expressing (CGG)90‐EGFP under Tub5‐GS (H) and ELAV‐GS (I) drivers with control or SRSF1 siRNAs. Expression of (CGG)90‐EGFP was initiated with addition of drug starting 1 day post‐eclosion and continued through experiment (Log‐rank Mantel–Cox test; n = 98–101/genotype for Tub‐GS and n = 120–141/genotype for ELAV‐GS flies); * P < 0.05, ** P < 0.01. J Survival assays of (GGGGCC)28‐EGFP expressing fly under Tub5‐GS driver (Log‐rank Mantel–Cox test; n = 71–93/genotype) with control or SRSF1 siRNAs. ** P < 0.01. Data information: For eye scoring, target siRNA lines were compared to non‐targeting control siRNA lines using a two‐tailed Student’s t ‐test with Welch’s correction for multiple comparisons. ** P < 0.01; *** P < 0.001; **** P < 0.0001. Human orthologs of fly genes are used for labeling. Details of fly genes are described in Appendix Table . Source data are available online for this figure.
Article Snippet: For Western blots, following antibodies were used: FLAG‐M2 at 1:1,000 dilution (mouse, Sigma F1804), 1:2,500 β‐Actin (mouse, Sigma A1978), 1:1,000
Techniques: Construct, Quantitation Assay, Expressing, Over Expression, Luciferase, Two Tailed Test, Labeling
Journal: EMBO Molecular Medicine
Article Title: SRSF protein kinase 1 modulates RAN translation and suppresses CGG repeat toxicity
doi: 10.15252/emmm.202114163
Figure Lengend Snippet: A–C Survival assays of (CGG)90‐EGFP and (G4C2)28‐EGFP expressing fly under Tub5‐GS driver with respective siRNAs as mentioned. Log‐rank Mantel–Cox test; n = 50–61 (A), 65–67 (B); and 65–73 (C). * P < 0.05. D Drosophila SRSF1 (dSF2, blue bar) and SRPK1 (gray) levels after siRNA knockdown as compared to non‐targeting siRNA (siNTC, black bars), quantified by qRT–PCR. Error bars represent mean ± SD RNAs from two ( n = 2 biological repeats) independent fly crosses (20–25 flies/genotype per cross) used to run RT–qPCR in triplicates (3 technical replicates). All data points presented in the graphs. E Western blot of FMR‐polyG‐EGFP RAN products in (CGG)90‐EGFP/ELAV‐GS flies with or without SRSF1 knockdown. Error bars represent mean ± SD. Total protein from three ( n = 3 biological repeats) independent fly crosses (20–22 flies/genotype per cross) and run in four replicates per gel (technical replicates). All data points presented in the graphs. Data information: Statistical analysis in (D) and (E) is performed using Student’s t ‐test with Welch’s correction. * P < 0.05, **** P < 0.0001.
Article Snippet: For Western blots, following antibodies were used: FLAG‐M2 at 1:1,000 dilution (mouse, Sigma F1804), 1:2,500 β‐Actin (mouse, Sigma A1978), 1:1,000
Techniques: Expressing, Quantitative RT-PCR, Western Blot
Journal: EMBO Molecular Medicine
Article Title: SRSF protein kinase 1 modulates RAN translation and suppresses CGG repeat toxicity
doi: 10.15252/emmm.202114163
Figure Lengend Snippet: A, B Representative (A) photographs of fly eyes and (B) quantitation with siRNA‐mediated knockdown of SRPK1 (dSRPK1); n = 30–34/genotype. Error bars represent mean ± SD. C Representative photographs of fly eyes expressing GMR‐GAL4‐driven (GGGGCC)28‐EGFP with siRNA‐mediated knockdown of SRPK1 or disruption by insertion. D Quantitation of rough eye phenotypes. t ‐test with Welch corrections for comparisons with the control; n = 31–34 flies/ genotype. Error bars represent mean ± SD. E Representative external eye imaging for the detection of GFP aggregates caused by (CGG)90‐EGFP transgene expression (top). Converted images used to quantify total intensity of GFP puncta (bottom). F Depletion of SRSF1 or SRPK1 by RNAi results in reduced (CGG)90‐EGFP puncta compared to control siRNA as quantified by total intensity (a.u. = arbitrary unit). n = 13–15 flies/genotype. Error bars represent mean ± SD. Data information: Statistical analysis was performed using two‐tailed Student’s t ‐test with Welch’s correction, *** P < 0.001; **** P < 0.0001. Source data are available online for this figure.
Article Snippet: For Western blots, following antibodies were used: FLAG‐M2 at 1:1,000 dilution (mouse, Sigma F1804), 1:2,500 β‐Actin (mouse, Sigma A1978), 1:1,000
Techniques: Quantitation Assay, Expressing, Imaging, Two Tailed Test
Journal: EMBO Molecular Medicine
Article Title: SRSF protein kinase 1 modulates RAN translation and suppresses CGG repeat toxicity
doi: 10.15252/emmm.202114163
Figure Lengend Snippet: A Schematic of SRPK1 signaling pathway that regulates subcellular SRSF1 localization, which in turns can impact export and translation of repeat RNAs in the cytoplasm. SRPK1 may also act directly on protein translation pathways (dotted arrow). Known pharmacological compounds that inhibit SRPK1 can disrupt this pathway. B Anti‐FLAG immunoblot of DMSO and SRPIN340 pre‐treated HEK293T cells expressing AUG‐nLuc‐3xFLAG control or CGG‐nLuc‐3xFLAG RAN translation reporters. β‐Actin is used as a loading control. To prevent signal saturation, AUG‐nLuc lysate was diluted 1:3 in sample buffer prior to loading ( n = 3 biological replicates). Schematics of the AUG‐nLUC‐3xFLAG and +1CGG(100)‐nLuc‐3xFLAG reporters presented on top. C Relative expression of AUG‐nLuc and CGG‐nLuc reporters in HEK293T cells ( n = 8–9 biological replicates) following treatment with DMSO and SRPIN340. D Anti‐FLAG immunoblot of DMSO and SRPIN340 pre‐treated HEK293T cells expressing GGGGCC‐nLuc‐3xFLAG (GA70) and +2CGG‐nLuc‐3xFLAG (FMRpolyA) RAN translation reporters ( n = 3 biological replicates). Schematics of the GA70 (GGGGCCx70) and +2CGG reporters presented on top. E Immunoblot of DMSO and SPHINX31 pre‐treated HEK293T cells expressing AUG‐nLuc‐3xFLAG control or CGG‐nLuc‐3xFLAG RAN translation reporters ( n = 3 biological replicates). F Anti‐FLAG of DMSO and SPHINX31 pre‐treated HEK293T cells expressing GGGGCC‐nLuc‐3xFLAG (GA70) and +2CGG‐nLuc‐3xFLAG (FMRpolyA) RAN translation reporters ( n = 3 biological replicates). Data information: Error bars represent mean ± SD. Statistical analysis was performed using two‐tailed Student’s t ‐test with Welch’s correction. * P < 0.05; ** P < 0.01, *** P < 0.001, and **** P < 0.0001. To prevent over‐exposure, the AUG‐nLuc lysate was diluted 1:3 in sample buffer. Source data are available online for this figure.
Article Snippet: For Western blots, following antibodies were used: FLAG‐M2 at 1:1,000 dilution (mouse, Sigma F1804), 1:2,500 β‐Actin (mouse, Sigma A1978), 1:1,000
Techniques: Western Blot, Expressing, Two Tailed Test
Journal: EMBO Molecular Medicine
Article Title: SRSF protein kinase 1 modulates RAN translation and suppresses CGG repeat toxicity
doi: 10.15252/emmm.202114163
Figure Lengend Snippet: A Immunoblot of cytoplasmic phospho‐SRSF1/2 after SRPIN340 treatment. GAPDH is used as the loading control. Error bars represent mean ± SD ( n = 3 biological replicates). Statistical analysis was performed using Student’s t ‐test with Welch’s correction. * P < 0.05. Full images of the same blot showing levels of all phospho SR proteins (pan SRSF phosphorylation) after SRPIN340 treatment are presented in Fig . B ICC images of FLAG‐tagged SRSF1 after SRPIN340 treatment compared to vehicle (DMSO). Quantification shows the ratio of nuclear and cytoplasmic intensity of SRSF1 signal (see methods for details). Error bars indicate mean ± 95% CI ( n = 85–126 cells/condition). Statistical analysis was performed using t ‐test with Welch’s correction, **** P < 0.0001. C Nucleocytoplasmic distribution CGG repeat RNA after SRPIN340 treatment compared to vehicle (DMSO) as detected by HCR. Quantification shows the ratio of nuclear and cytoplasmic intensity of CGG RNA signal (see methods for details) as parts of whole. Error bars indicate mean ± 95% CI ( n = 124–151 cells/condition). Statistical analysis was performed using t ‐test with Welch’s correction, **** P < 0.0001. D Anti‐FLAG immunoblot blot of DMSO and SRPIN340 pre‐treated HEK293T cells transfected with in vitro transcribed CGG‐nLuc‐3xFLAG reporter RNA. β‐Actin is used as a loading control. Error bars represent mean ± SD ( n = 6 biological replicates). Statistical analysis was performed using Student’s t ‐test with Welch’s correction. * P < 0.05. E Relative expression of in vitro transcribed CGG‐nLuc reporter in HEK293T cells pre‐treated with DMSO and SRPIN340. Error bars represent mean ± SD ( n = 9 biological replicates). F Expression of in vitro transcribed Aug‐nLuc and CGG‐nLuc reporters in rabbit reticulocyte lysate (RRL) in vitro translation system following pre‐treatment with DMSO or SRPIN340. Error bars represent mean ± SD ( n = 9 biological replicates). Data information: For E and F, statistical analysis was performed using Student’s t ‐test with Welch’s correction. * P < 0.05; ** P < 0.01. Source data are available online for this figure.
Article Snippet: For Western blots, following antibodies were used: FLAG‐M2 at 1:1,000 dilution (mouse, Sigma F1804), 1:2,500 β‐Actin (mouse, Sigma A1978), 1:1,000
Techniques: Western Blot, Transfection, In Vitro, Expressing
Journal: EMBO Molecular Medicine
Article Title: SRSF protein kinase 1 modulates RAN translation and suppresses CGG repeat toxicity
doi: 10.15252/emmm.202114163
Figure Lengend Snippet: A Subcellular levels of phosphorylated SRSF proteins with or without SRPIN340 treatment measured by Western blot using an antibody, which detects phosphorylated SR proteins (p‐SRSFs). GAPDH is used as cytoplasmic marker and loading control for cytoplasmic fraction of phosphorylated SRSFs ( n = 3 biological repeats). Detected p‐SRSFs are labeled/color‐coded. p‐SRSF1/2 data presented in Fig are outlined in red dots. B Relative nanoluciferase (nLuc) expression of +1 CGG‐nLuc‐3xF reporter transfected as plasmid or as in vitro transcribed RNA in presence or absence of SRPIN340 treatment ( n = 9). Error bars represent mean ± SD ( n = 12 biological repeats). Statistical analysis is performed using two‐tailed Student’s t ‐test with Welch’s correction. ** P < 0.01, *** P < 0.001.
Article Snippet: For Western blots, following antibodies were used: FLAG‐M2 at 1:1,000 dilution (mouse, Sigma F1804), 1:2,500 β‐Actin (mouse, Sigma A1978), 1:1,000
Techniques: Western Blot, Marker, Labeling, Expressing, Transfection, Plasmid Preparation, In Vitro, Two Tailed Test
Journal: bioRxiv
Article Title: In vivo CGG repeat RNA binding protein capture identifies RAN translation modifiers and suppressors of repeat toxicity
doi: 10.1101/2021.01.08.425998
Figure Lengend Snippet: (A) Schematic of PP7-tagged RNA reporters and PCP-NLS-FLAG constructs used in this study. (B) Relative expression from PP7-tagged CGG-nLuc reporters compared to AUG-driven reporters in HEK293T cells (n=9). (C) Expression of AUG-control and CGG-nLuc RAN translation reporters in HEK293T cells treated with the ER stress agent thapsigargin (TG, 2 μM) (n=9) normalized to vehicle (DMSO). (D) Schematic of immunoprecipitation and mass spectrometry experiments aimed at identifying CGG repeat RNA interacting proteins (see Methods for details). (E-F) Log 2 fold-change of the CGG-interacting protein enrichment compared AUG-reporter (n=2 independent experiments; error bars represent range between repeats) under normal (E) and after integrated stress response activation by TG (F). (G) GO term analysis of manually curated differentially enriched CGG-interacting proteins. (H) Log 2 fold-change of SRSF proteins: 1, 2, 3 and 6 in CGG-reporter enrichment compared to AUG-reporter basally and after TG treatment (n=2 independent experiment; error bars represent range between two repeats). (I) Co-immunoprecipitation of indicated SRSF proteins with scrambled (scr), AUG and CGG-tagged reporter constructs. SRSF1 and 2 specifically immunoprecipitated with CGG-tagged reporter. For graphs in (B-C) error bar represents +/− SD. Statistical analysis was performed using: Two-tailed Student’s t test with Welch’s correction, ***p < 0.001; ****p < 0.0001.
Article Snippet: Cells were stained with primary
Techniques: Construct, Expressing, Immunoprecipitation, Mass Spectrometry, Protein Enrichment, Activation Assay, Two Tailed Test
Journal: bioRxiv
Article Title: In vivo CGG repeat RNA binding protein capture identifies RAN translation modifiers and suppressors of repeat toxicity
doi: 10.1101/2021.01.08.425998
Figure Lengend Snippet: (A) Schematic of (CGG)90-EGFP construct and experimental outline for rough eye phenotype. (B) Quantitation of GMR-GAL4 driven uas-(CGG)90-EGFP eye phenotype with candidate modifiers (t-test with Welch corrections for comparisons with the control; n ≥ 30 flies/genotype). **p < 0.01; ***p < 0.001; ****p < 0.0001. [NTC = non targeting control] (C) Representative photographs of fly eyes expressing either GMR-GAL4 driver alone or with uas- (CGG)90-EGFP construct, with fly SRSF1 (dSF2) and SRSF2 (dSC35) knockdown or disruptions. (D) Representative photographs of fly eyes as in (C) and quantitation as in (B) with SRSF1/dSF2 overexpression (dSF2 OE). (E) Survival assay on flies expressing (CGG)90-EGFP under Tub5-GS driver with drug initiation starting 1 day post eclosion and continuing through experiment (Log-rank Mantel–Cox test; n = 78-80/genotype) with SRSF1 knockdown *p < 0.05. (F) Representative photographs of fly eyes as in (C) and quantitation as in (B) with siRNA mediated knockdown of SRPK1 (dSRPK1). Details of fly genotypes described in supplementary Table 2.
Article Snippet: Cells were stained with primary
Techniques: Construct, Quantitation Assay, Expressing, Over Expression, Clonogenic Cell Survival Assay
Journal: bioRxiv
Article Title: In vivo CGG repeat RNA binding protein capture identifies RAN translation modifiers and suppressors of repeat toxicity
doi: 10.1101/2021.01.08.425998
Figure Lengend Snippet: (A) Representative photographs of fly eyes expressing GMR-GAL4 driven (GGGGCC)28-EGFP with indicated uas-siRNAs to fly SRSF proteins at 25°C. (B) Quantitation of GMR-GAL4 driven (GGGGCC)28-EGFP eye phenotype with SRSF modifiers (t-test with Welch corrections for comparisons with the control; n ≥ 30 flies/ genotype). ****p < 0.0001 (C) Representative photographs of fly eyes expressing either GMR-GAL4 driven (GGGGCC)28-EGFP at 29°C with siRNA against SRSF1. Quantification of eye necrosis (D) and eye diameter (E) at 29°C in GMR-GAL4 driven (GGGGCC)28-EGFP flies. (F) Survival assays of (GGGGCC)28-EGFP expressing fly under Tub5-GS driver (Log-rank Mantel–Cox test; n = 71-90/genotype) with control or SRSF1 siRNA. **p < 0.01 (G) Representative photographs of fly eyes expressing GMR-GAL4 driven (GGGGCC)28-EGFP with siRNA mediated knockdown of SRPK1 or disruption by insertion. (H) Quantitation of rough eye phenotypes.. t-test with Welch corrections for comparisons with the control; n ≥ 30 flies/ genotype). ***p < 0.001; ****p < 0.0001
Article Snippet: Cells were stained with primary
Techniques: Expressing, Quantitation Assay
Journal: bioRxiv
Article Title: In vivo CGG repeat RNA binding protein capture identifies RAN translation modifiers and suppressors of repeat toxicity
doi: 10.1101/2021.01.08.425998
Figure Lengend Snippet: (A) Schematic of +1CGG RAN translation nLuc-3xFLAG and AUG-driven control nLuc-3xFLAG reporters. (B) Relative expression of AUG-nLuc and CGG-nLuc reporters in HEK293T cells (n=10-14) following knockdown of SRSF1. Comparisons between siEGFP and siSRSF1 treated cells. (C) Anti-FLAG Western blot of FMRpolyG nLUC-3xFLAG with and without SRSF1 knockdown in HEK293T cells (n=3). Error bars represent mean +/−SEM. t-test with Welch corrections. *p < 0.05 (D) Representative images of subcellular distribution of CGG-nLuc-3xFLAG reporter RNAs using HCR with and without SRSF1 knockdown in HEK293T cells. n= # of cells used for quantification for each condition as mentioned in the table. Quantification of CGG-nLuc-3xFLAG reporter RNA intensity (E) and nuclear/cytoplasmic distribution (F). Error bars represent mean +/−SEM. t-test with Bonferroni and Welch’s correction *p < 0.05, ****p < 0.0001.
Article Snippet: Cells were stained with primary
Techniques: Expressing, Western Blot
Journal: bioRxiv
Article Title: In vivo CGG repeat RNA binding protein capture identifies RAN translation modifiers and suppressors of repeat toxicity
doi: 10.1101/2021.01.08.425998
Figure Lengend Snippet: (A) Schematic of AKT/SRPK1 signaling pathway that regulate SRSF1 localization with example of known pharmacological compounds that inhibit SRPK1. (B) Anti-FLAG Western blot of DMSO and SRPIN340 pre-treated HEK293T cells expressing AUG-nLUC-3xFLAG control or CGG-nLuc-3xFLAG RAN translation reporters. β-Actin is used as a loading control. To prevent signal saturation, AUG-nLuc lysate was diluted 1:3 in sample buffer prior to loading (n=3). (C) Relative expression of AUG-nLuc and CGG-nLuc reporters in HEK293T cells (n=8-9) following treatment with DMSO and SRPIN340. (D) Anti-FLAG Western blot of DMSO and SRPIN340 pre-treated HEK293T cells expressing GGGGCC-nLuc-3xFLAG (GA70) and +2CGG-nLuc-3xFLAG (FMRpolyA) RAN translation reporters (n=3). Schematics of the GA70 (GGGGCCx70) and +2CGG reporters presented on top. (E) Western blot of DMSO and SPHINX31 pre-treated HEK293T cells expressing AUG-nLUC-3xFLAG control or CGG-nLuc-3xFLAG RAN translation reporters. (n=3). (F) Anti-FLAG Western blot of DMSO and SPHINX31 pre-treated HEK293T cells expressing GGGGCC-nLuc-3xFLAG (GA70) and +2CGG-nLuc-3xFLAG (FMRpolyA) RAN translation reporters (n=3). Error bars represent mean +/− SD. *p < 0.05; **p < 0.01 and ***p < 0.001. To prevent over-exposure, the AUG-nLuc lysate was diluted 1:3 in the sample buffer.
Article Snippet: Cells were stained with primary
Techniques: Western Blot, Expressing
Journal: bioRxiv
Article Title: In vivo CGG repeat RNA binding protein capture identifies RAN translation modifiers and suppressors of repeat toxicity
doi: 10.1101/2021.01.08.425998
Figure Lengend Snippet: (A) Schematic of PP7-tagged RNA reporters and PCP-NLS-FLAG constructs used in this study. (B) Relative expression from PP7-tagged CGG-nLuc reporters compared to AUG-driven reporters in HEK293T cells (n=9). (C) Expression of AUG-control and CGG-nLuc RAN translation reporters in HEK293T cells treated with the ER stress agent thapsigargin (TG, 2 μM) (n=9) normalized to vehicle (DMSO). (D) Schematic of immunoprecipitation and mass spectrometry experiments aimed at identifying CGG repeat RNA interacting proteins (see Methods for details). (E-F) Log 2 fold-change of the CGG-interacting protein enrichment compared AUG-reporter (n=2 independent experiments; error bars represent range between repeats) under normal (E) and after integrated stress response activation by TG (F). (G) GO term analysis of manually curated differentially enriched CGG-interacting proteins. (H) Log 2 fold-change of SRSF proteins: 1, 2, 3 and 6 in CGG-reporter enrichment compared to AUG-reporter basally and after TG treatment (n=2 independent experiment; error bars represent range between two repeats). (I) Co-immunoprecipitation of indicated SRSF proteins with scrambled (scr), AUG and CGG-tagged reporter constructs. SRSF1 and 2 specifically immunoprecipitated with CGG-tagged reporter. For graphs in (B-C) error bar represents +/− SD. Statistical analysis was performed using: Two-tailed Student’s t test with Welch’s correction, ***p < 0.001; ****p < 0.0001.
Article Snippet: For HCR: - Primary Antibodies for
Techniques: Construct, Expressing, Immunoprecipitation, Mass Spectrometry, Protein Enrichment, Activation Assay, Two Tailed Test
Journal: bioRxiv
Article Title: In vivo CGG repeat RNA binding protein capture identifies RAN translation modifiers and suppressors of repeat toxicity
doi: 10.1101/2021.01.08.425998
Figure Lengend Snippet: (A) Schematic of (CGG)90-EGFP construct and experimental outline for rough eye phenotype. (B) Quantitation of GMR-GAL4 driven uas-(CGG)90-EGFP eye phenotype with candidate modifiers (t-test with Welch corrections for comparisons with the control; n ≥ 30 flies/genotype). **p < 0.01; ***p < 0.001; ****p < 0.0001. [NTC = non targeting control] (C) Representative photographs of fly eyes expressing either GMR-GAL4 driver alone or with uas- (CGG)90-EGFP construct, with fly SRSF1 (dSF2) and SRSF2 (dSC35) knockdown or disruptions. (D) Representative photographs of fly eyes as in (C) and quantitation as in (B) with SRSF1/dSF2 overexpression (dSF2 OE). (E) Survival assay on flies expressing (CGG)90-EGFP under Tub5-GS driver with drug initiation starting 1 day post eclosion and continuing through experiment (Log-rank Mantel–Cox test; n = 78-80/genotype) with SRSF1 knockdown *p < 0.05. (F) Representative photographs of fly eyes as in (C) and quantitation as in (B) with siRNA mediated knockdown of SRPK1 (dSRPK1). Details of fly genotypes described in supplementary Table 2.
Article Snippet: For HCR: - Primary Antibodies for
Techniques: Construct, Quantitation Assay, Expressing, Over Expression, Clonogenic Cell Survival Assay
Journal: bioRxiv
Article Title: In vivo CGG repeat RNA binding protein capture identifies RAN translation modifiers and suppressors of repeat toxicity
doi: 10.1101/2021.01.08.425998
Figure Lengend Snippet: (A) Representative photographs of fly eyes expressing GMR-GAL4 driven (GGGGCC)28-EGFP with indicated uas-siRNAs to fly SRSF proteins at 25°C. (B) Quantitation of GMR-GAL4 driven (GGGGCC)28-EGFP eye phenotype with SRSF modifiers (t-test with Welch corrections for comparisons with the control; n ≥ 30 flies/ genotype). ****p < 0.0001 (C) Representative photographs of fly eyes expressing either GMR-GAL4 driven (GGGGCC)28-EGFP at 29°C with siRNA against SRSF1. Quantification of eye necrosis (D) and eye diameter (E) at 29°C in GMR-GAL4 driven (GGGGCC)28-EGFP flies. (F) Survival assays of (GGGGCC)28-EGFP expressing fly under Tub5-GS driver (Log-rank Mantel–Cox test; n = 71-90/genotype) with control or SRSF1 siRNA. **p < 0.01 (G) Representative photographs of fly eyes expressing GMR-GAL4 driven (GGGGCC)28-EGFP with siRNA mediated knockdown of SRPK1 or disruption by insertion. (H) Quantitation of rough eye phenotypes.. t-test with Welch corrections for comparisons with the control; n ≥ 30 flies/ genotype). ***p < 0.001; ****p < 0.0001
Article Snippet: For HCR: - Primary Antibodies for
Techniques: Expressing, Quantitation Assay
Journal: bioRxiv
Article Title: In vivo CGG repeat RNA binding protein capture identifies RAN translation modifiers and suppressors of repeat toxicity
doi: 10.1101/2021.01.08.425998
Figure Lengend Snippet: (A) Schematic of +1CGG RAN translation nLuc-3xFLAG and AUG-driven control nLuc-3xFLAG reporters. (B) Relative expression of AUG-nLuc and CGG-nLuc reporters in HEK293T cells (n=10-14) following knockdown of SRSF1. Comparisons between siEGFP and siSRSF1 treated cells. (C) Anti-FLAG Western blot of FMRpolyG nLUC-3xFLAG with and without SRSF1 knockdown in HEK293T cells (n=3). Error bars represent mean +/−SEM. t-test with Welch corrections. *p < 0.05 (D) Representative images of subcellular distribution of CGG-nLuc-3xFLAG reporter RNAs using HCR with and without SRSF1 knockdown in HEK293T cells. n= # of cells used for quantification for each condition as mentioned in the table. Quantification of CGG-nLuc-3xFLAG reporter RNA intensity (E) and nuclear/cytoplasmic distribution (F). Error bars represent mean +/−SEM. t-test with Bonferroni and Welch’s correction *p < 0.05, ****p < 0.0001.
Article Snippet: For HCR: - Primary Antibodies for
Techniques: Expressing, Western Blot
Journal: bioRxiv
Article Title: In vivo CGG repeat RNA binding protein capture identifies RAN translation modifiers and suppressors of repeat toxicity
doi: 10.1101/2021.01.08.425998
Figure Lengend Snippet: (A) Schematic of AKT/SRPK1 signaling pathway that regulate SRSF1 localization with example of known pharmacological compounds that inhibit SRPK1. (B) Anti-FLAG Western blot of DMSO and SRPIN340 pre-treated HEK293T cells expressing AUG-nLUC-3xFLAG control or CGG-nLuc-3xFLAG RAN translation reporters. β-Actin is used as a loading control. To prevent signal saturation, AUG-nLuc lysate was diluted 1:3 in sample buffer prior to loading (n=3). (C) Relative expression of AUG-nLuc and CGG-nLuc reporters in HEK293T cells (n=8-9) following treatment with DMSO and SRPIN340. (D) Anti-FLAG Western blot of DMSO and SRPIN340 pre-treated HEK293T cells expressing GGGGCC-nLuc-3xFLAG (GA70) and +2CGG-nLuc-3xFLAG (FMRpolyA) RAN translation reporters (n=3). Schematics of the GA70 (GGGGCCx70) and +2CGG reporters presented on top. (E) Western blot of DMSO and SPHINX31 pre-treated HEK293T cells expressing AUG-nLUC-3xFLAG control or CGG-nLuc-3xFLAG RAN translation reporters. (n=3). (F) Anti-FLAG Western blot of DMSO and SPHINX31 pre-treated HEK293T cells expressing GGGGCC-nLuc-3xFLAG (GA70) and +2CGG-nLuc-3xFLAG (FMRpolyA) RAN translation reporters (n=3). Error bars represent mean +/− SD. *p < 0.05; **p < 0.01 and ***p < 0.001. To prevent over-exposure, the AUG-nLuc lysate was diluted 1:3 in the sample buffer.
Article Snippet: For HCR: - Primary Antibodies for
Techniques: Western Blot, Expressing
Journal: The Journal of Cell Biology
Article Title: An APEX2 proximity ligation method for mapping interactions with the nuclear lamina
doi: 10.1083/jcb.202002129
Figure Lengend Snippet: Related to : Additional analyses of our APEX2-lamin-B1–identified proteome. (A) A heatmap matrix representing the percentage overlap between the APEX2-lamin-B1 identified proteome and previously identified NL or nuclear membrane proteomes. We calculated the percentage between two datasets by dividing the overlapping number over the pooled, nonredundant datasets. HPA represents nuclear membrane localized proteins identified by the Human Protein Atlas Project. (B) Ranking of APEX2-lamin-B1 proteome normalized to tyrosine content. Red circles indicate known NL proteins, and blue circles represent PCBP proteins. (C) Density line plot of protein tyrosine content for nuclear protein (red line), cytosolic proteins (blue line), and our APEX2-lamin-B1–identified proteins (black line). Nuclear and cytosolic proteins were defined by the Human Protein Atlas Project. (D) Pie charts of our APEX2-lamin-B1 proteome showing the number of transmembrane (left) and secretory (right) proteins as defined by the Human Protein Atlas Project. (E) Western blot validation of SRSF1, SRSF2, and HNRNPA1 identified in our APEX2-lamin-B1 proteome. PrA is a protein-A dynabead control. (F) Immunostaining for PCBP2 and Nup153 in WT and lamin TKO mESCs. (G) Immunostaining of PCBP2 and lamin-A/C in WT, lamin-B1/B2-null (DKO), and DKO plus lamin-A/C siRNA SV-40–transformed MEFs. (H) Boxplot displaying the nuclear/cytosol ratio for the indicated MEF genotypes ( n ≥ 31). Notches represent the 95% confidence interval around the median, and blue dots represent the individual measurements. *, a two-tailed t test P value of <0.05. Int. dens, integrated density; MS, mass spectrometry.
Article Snippet: Other primary antibodies used for immunofluorescence include rabbit anti-HNRNPA1 (1:2,500; ProteinTech; 11176-1-AP),
Techniques: Western Blot, Immunostaining, Transformation Assay, Two Tailed Test, Mass Spectrometry