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Proteintech 1 ap anti sfpq rabbit
1 Ap Anti Sfpq Rabbit, supplied by Proteintech, used in various techniques. Bioz Stars score: 95/100, based on 66 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/1+ap+anti+sfpq+rabbit/NONO+Antibody/pmc11955919__SC___016___D5SC00596E___s002-302-13-18
Average 95 stars, based on 66 article reviews

1 ap anti sfpq rabbit - by Bioz Stars, 2026-09

95/100 stars

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Western Blot:

Article Title: Interrogation of mirror-image l -RNA–protein interactions reveals key mechanisms of single-stranded G-rich l -RNA cytotoxicity and a potential mitigation strategy
Article Snippet: .. Antibody Host Dilution (WB) Dilution (IF) Source anti-p54nrb Rabbit 1:500 1:50 Proteintech; cat.# 11058-1-AP anti-SFPQ Rabbit 1:2000 1:50 Proteintech; cat.# 15585-1-AP anti-HNRNPR Rabbit 1:500 1:50 Invitrogen; cat.# PA5-109826 anti-RBM15 Mouse 1:500 1:200 Proteintech; cat.# 66059-1-IG anti-FUS Mouse 1:5000 1:20 Proteintech; cat.# 60160-1-IG anti-NCL Rabbit 1:5000 1:500 Proteintech; cat.# 10556-1-AP anti-FBL Mouse 1:100 antibodies.com; cat.# A85370 anti-ALYREF Rabbit 1:500 Proteintech; cat.# 16690-1-AP anti-HIST3 Rabbit 1:1000 Invitrogen; cat.# PA5-16183 anti-G3BP1 Mouse 1:5000 Proteintech; cat.# 66486-1-IG anti-RBMX Rabbit 1:1000 Invitrogen; cat.# PA5-49468 Cy3 anti-mouse IgG Goat 1:500 Invitrogen; cat.# A10521 Cy3 anti-rabbit IgG Goat 1:500 Invitrogen; cat.# A10520 Alexa FluorTM488 anti-mouse IgG Goat 1:2000 Invitrogen; cat.# A-11001 Alexa FluorTM Plus 647 anti-rabbit IgG Goat 1:10000 Invitrogen; cat.# A32733 Alexa FluorTM Plus 647 anti-mouse IgG Goat 1:10000 Invitrogen; cat.# A32728 Table S3. ..

Image Search Results

(A) Cross-linking network between paraspeckle and viral proteins. Pink – essential for paraspeckle formation, blue – important, yellow – localised to paraspeckles but dispensable , gray – other proteins cross-linked to both paraspeckle and orange - viral proteins. (B) Paraspeckle structure, showing the interaction between NEAT1 long noncoding RNA and proteins such as SFPQ and NONO (left) and the NEAT1 isoforms, NEAT1_1 and NEAT1_2, and the position of FISH probes/qPCR primers on NEAT1 used in this study (right). Created in bioRender. https://BioRender.com/b92y974 (C) AP-MS of NONO versus IgG controls from WSN-infected A549 cells (14 hpi, n=3). NONO and its known interactors are coloured pink and viral proteins - orange. (D) AP-MS of NP versus IgG controls from WSN-infected A549 cells (14 hpi, n=3). Proteins that were also identified in SHVIP are highlighted. Proteins coloured as in E. (E) Maximum projection of confocal microscopy images of A549 cells infected with WSN (MOI 3) at 4, 8, and 12 hpi. NEAT1 - magenta, vRNA (PB2 fragment) - green, and DNA (DAPI) - grey. (F) CV of NEAT1_2 (NEAT1_1 in case of MEF) in the nucleus across different cell lines infected with WSN. (G) Number of paraspeckles per nucleus across different cell lines infected with WSN. (F-G) Data are represented as mean + SD. Statistical analysis was performed using ordinary one-way ANOVA. Statistical significance: *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Journal: bioRxiv

Article Title: Snapshot of in-cell protein contact sites reveals new host factors and hijacking of paraspeckles during influenza A virus infection

doi: 10.1101/2025.03.09.642134

Figure Lengend Snippet: (A) Cross-linking network between paraspeckle and viral proteins. Pink – essential for paraspeckle formation, blue – important, yellow – localised to paraspeckles but dispensable , gray – other proteins cross-linked to both paraspeckle and orange - viral proteins. (B) Paraspeckle structure, showing the interaction between NEAT1 long noncoding RNA and proteins such as SFPQ and NONO (left) and the NEAT1 isoforms, NEAT1_1 and NEAT1_2, and the position of FISH probes/qPCR primers on NEAT1 used in this study (right). Created in bioRender. https://BioRender.com/b92y974 (C) AP-MS of NONO versus IgG controls from WSN-infected A549 cells (14 hpi, n=3). NONO and its known interactors are coloured pink and viral proteins - orange. (D) AP-MS of NP versus IgG controls from WSN-infected A549 cells (14 hpi, n=3). Proteins that were also identified in SHVIP are highlighted. Proteins coloured as in E. (E) Maximum projection of confocal microscopy images of A549 cells infected with WSN (MOI 3) at 4, 8, and 12 hpi. NEAT1 - magenta, vRNA (PB2 fragment) - green, and DNA (DAPI) - grey. (F) CV of NEAT1_2 (NEAT1_1 in case of MEF) in the nucleus across different cell lines infected with WSN. (G) Number of paraspeckles per nucleus across different cell lines infected with WSN. (F-G) Data are represented as mean + SD. Statistical analysis was performed using ordinary one-way ANOVA. Statistical significance: *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Article Snippet: The following primary antibodies were used for western blot analysis: NONO mouse monoclonal (1:1,000, Proteintech, clone 2A2B10, catalogue no. 66361-1-Ig); NONO rabbit polyclonal (1:1,000, Proteintech, catalogue no. 11058-1-AP); SFPQ mouse monoclonal (1:1,000, Proteintech, clone 1G4A5, catalogue no. 67129-1-Ig); SFPQ rabbit polyclonal (1:1,000, Proteintech, catalogue no. 15585-1-AP); PSPC1 rabbit polyclonal (1:1,000, Proteintech, catalogue no. 16714-1-AP); SLC7A5 rabbit polyclonal (1:1,000, Proteintech, catalogue no. 28670-1-AP); SLC7A5 mouse monoclonal (1:1,000, Proteintech, clone 2G5H3, catalogue no. 67951-1-Ig); SLC3A2 rabbit polyclonal (1:1,000, Proteintech, catalogue no 15193-1-AP); SLC3A2 mouse monoclonal (1:1,000, Proteintech, clone 2B10F5, catalogue no. 66883-1-Ig); NP mouse monoclonal (1:1,000, Abcam, clone C43, catalogue no. ab128193); M2 mouse (1:1,000, ThermoFisher, clone 14C2, catalogue no. MA1-082); PB1, PB2, M1 (1: 1000, Abcam ab22396), NS1 (1:1,000, ThermoFisher, MA5-35909); Vinculin mouse (1:5000, Merck, catalogue no. V9131); Cyclophilin B rabbit monoclonal (1:5,000, CST, clone D1V5J, catalogue no. 43603).

Techniques: Infection, Confocal Microscopy

(A) Luciferase activity in A549 cells transfected with siRNAs targeting paraspeckle components. Data are represented as mean ± SD (n = 3). siNEAT1_1 and 2 indicate two different siRNAs both targeting the whole NEAT1.The data for RBM14, RBMX, RBM5, HNRNPA1, HNRNPA2B1, HNRNPK and PSPC1 were reproduced from Figure S4C. (B) Quantitative PCR analysis of viral RNA species of fragment NP normalised to GAPDH in A549 cells with knockdowns of paraspeckle proteins, infected with WSN (MOI 3) at 6hpi. Data are presented as log-fold-change of as mean ± SD relative to the siNT condition (n=3). (C) Schematic of NONO knockout and rescue workflow. CRISPR-Cas9 with two sgRNAs generated NONO KO1 and KO2 A549 cell lines. Lentiviral overexpression of mEGFP-NONO or mEGFP (control) in KO cells was used for rescue. Wild-type, NONO KO, and rescued cells were analysed for paraspeckle function and viral replication. Created in BioRender. https://BioRender.com/t41h404 . (D) Luciferase activity in wt, NONO KO1, and KO2 in A549 cells (WSN with PB2-T2A-NanoLuc, MOI 0.01, 48 hpi). Data are represented as mean ± SD (n = 3). (E) Luciferase activity in wt, NONO KO1, and KO2 A549 cells lentivirally overexpressing mEGFP (control) or mEGFP-NONO (WSN with PB2-T2A-NanoLuc, MOI 0.01, 48 hpi). Data are represented as mean ± SD (n = 3). (F) Density plot of log2-fold-changes of proteins cross-linked to NONO, NP and NS1 identified by AP-MS (NONO infected vs. NONO mock). Proteins cross-linked to NP and NS1 (orange) are co-depleted in infected cells compared to proteins not linked to NP or NS1 (grey). (G) Log2-fold changes of interactors identified by AP-MS against NONO (infected vs. mock) and NP (infected vs. infected isotype control). Proteins cross-linked to NONO (pink), NP (orange), and non-associated proteins (grey) are shown. (H) Selected protein categories enriched in both NP and NONO-mock AP-MS datasets from G (see also Table S3). (I) Schematic representation of paraspeckles disruption: IAV proteins, particularly NP and NS1, disrupt paraspeckle integrity by binding core proteins like SFPQ and NONO, and possibly NEAT1, initiating paraspeckle disassembly. As the infection progresses, PA-X promotes NEAT1 degradation, while POL II inhibition further destabilises paraspeckles, leading to their complete disruption. Created in BioRender. https://BioRender.com/y54j344 . (A-B, D-E) Data are represented as mean ± SD. Statistical analysis was performed using ordinary one-way ANOVA. Statistical significance: *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Journal: bioRxiv

Article Title: Snapshot of in-cell protein contact sites reveals new host factors and hijacking of paraspeckles during influenza A virus infection

doi: 10.1101/2025.03.09.642134

Figure Lengend Snippet: (A) Luciferase activity in A549 cells transfected with siRNAs targeting paraspeckle components. Data are represented as mean ± SD (n = 3). siNEAT1_1 and 2 indicate two different siRNAs both targeting the whole NEAT1.The data for RBM14, RBMX, RBM5, HNRNPA1, HNRNPA2B1, HNRNPK and PSPC1 were reproduced from Figure S4C. (B) Quantitative PCR analysis of viral RNA species of fragment NP normalised to GAPDH in A549 cells with knockdowns of paraspeckle proteins, infected with WSN (MOI 3) at 6hpi. Data are presented as log-fold-change of as mean ± SD relative to the siNT condition (n=3). (C) Schematic of NONO knockout and rescue workflow. CRISPR-Cas9 with two sgRNAs generated NONO KO1 and KO2 A549 cell lines. Lentiviral overexpression of mEGFP-NONO or mEGFP (control) in KO cells was used for rescue. Wild-type, NONO KO, and rescued cells were analysed for paraspeckle function and viral replication. Created in BioRender. https://BioRender.com/t41h404 . (D) Luciferase activity in wt, NONO KO1, and KO2 in A549 cells (WSN with PB2-T2A-NanoLuc, MOI 0.01, 48 hpi). Data are represented as mean ± SD (n = 3). (E) Luciferase activity in wt, NONO KO1, and KO2 A549 cells lentivirally overexpressing mEGFP (control) or mEGFP-NONO (WSN with PB2-T2A-NanoLuc, MOI 0.01, 48 hpi). Data are represented as mean ± SD (n = 3). (F) Density plot of log2-fold-changes of proteins cross-linked to NONO, NP and NS1 identified by AP-MS (NONO infected vs. NONO mock). Proteins cross-linked to NP and NS1 (orange) are co-depleted in infected cells compared to proteins not linked to NP or NS1 (grey). (G) Log2-fold changes of interactors identified by AP-MS against NONO (infected vs. mock) and NP (infected vs. infected isotype control). Proteins cross-linked to NONO (pink), NP (orange), and non-associated proteins (grey) are shown. (H) Selected protein categories enriched in both NP and NONO-mock AP-MS datasets from G (see also Table S3). (I) Schematic representation of paraspeckles disruption: IAV proteins, particularly NP and NS1, disrupt paraspeckle integrity by binding core proteins like SFPQ and NONO, and possibly NEAT1, initiating paraspeckle disassembly. As the infection progresses, PA-X promotes NEAT1 degradation, while POL II inhibition further destabilises paraspeckles, leading to their complete disruption. Created in BioRender. https://BioRender.com/y54j344 . (A-B, D-E) Data are represented as mean ± SD. Statistical analysis was performed using ordinary one-way ANOVA. Statistical significance: *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Techniques: Luciferase, Activity Assay, Transfection, Real-time Polymerase Chain Reaction, Infection, Knock-Out, CRISPR, Generated, Over Expression, Control, Disruption, Binding Assay, Inhibition

( A ) Co-IP assay with antibody (Ab) against PTBP1 (top) or PSF (bottom) detecting the PTBP1/PSF interaction within whole-cell extracts of HEK293 cells and the PTBP1/PSF interaction after RNase T1 or DNase I treatment. The dose of RNase T1 or DNase I was increased as indicated. ( B ) Native RNA IP (RIP) assay followed by reverse transcription quantitative polymerase chain reaction (RT-qPCR) detecting MALAT1 (red bars) and 7SK RNA (black bars) retrieved by PTBP1-, PSF-, or hnRNP L–specific Ab or by normal immunoglobulin G (IgG) in HEK293 cells. Data are shown as means ± standard deviation (SD) of n = 3 independent experiments. ( C ) Immunoblotting using the indicated Abs detecting IP efficiency of the native RIP assay in (B). ( D ) Co-IP assay with Ab against PTBP1 (left) or PSF (right) detecting the PTBP1/PSF interaction in HEK293 cells and the PTBP1/PSF interaction after MALAT1 knockdown by two different short hairpin RNAs (shRNAs). shNC, a nontargeting control shRNA; shMALAT1, a shRNA targeting MALAT1. ( E and F ) Confocal images of MALAT1 labeled by RNAscope in situ hybridization (ISH) and PTBP1 and PSF proteins concurrently stained by immunofluorescence (IF) in HEK293 cells (E) and in control and MALAT1-depleted HEK293 cells (F). Scale bars, 10 μm. ( G ) Co-IP assay with Ab against PTBP1 (left) or PSF (right) in HEK293 cells detecting the PTBP1/PSF interaction in response to MALAT1 knockdown with or without complementation with lacZ or MALAT1 RNA.

Journal: Science Advances

Article Title: MALAT1 modulates alternative splicing by cooperating with the splicing factors PTBP1 and PSF

doi: 10.1126/sciadv.abq7289

Figure Lengend Snippet: ( A ) Co-IP assay with antibody (Ab) against PTBP1 (top) or PSF (bottom) detecting the PTBP1/PSF interaction within whole-cell extracts of HEK293 cells and the PTBP1/PSF interaction after RNase T1 or DNase I treatment. The dose of RNase T1 or DNase I was increased as indicated. ( B ) Native RNA IP (RIP) assay followed by reverse transcription quantitative polymerase chain reaction (RT-qPCR) detecting MALAT1 (red bars) and 7SK RNA (black bars) retrieved by PTBP1-, PSF-, or hnRNP L–specific Ab or by normal immunoglobulin G (IgG) in HEK293 cells. Data are shown as means ± standard deviation (SD) of n = 3 independent experiments. ( C ) Immunoblotting using the indicated Abs detecting IP efficiency of the native RIP assay in (B). ( D ) Co-IP assay with Ab against PTBP1 (left) or PSF (right) detecting the PTBP1/PSF interaction in HEK293 cells and the PTBP1/PSF interaction after MALAT1 knockdown by two different short hairpin RNAs (shRNAs). shNC, a nontargeting control shRNA; shMALAT1, a shRNA targeting MALAT1. ( E and F ) Confocal images of MALAT1 labeled by RNAscope in situ hybridization (ISH) and PTBP1 and PSF proteins concurrently stained by immunofluorescence (IF) in HEK293 cells (E) and in control and MALAT1-depleted HEK293 cells (F). Scale bars, 10 μm. ( G ) Co-IP assay with Ab against PTBP1 (left) or PSF (right) in HEK293 cells detecting the PTBP1/PSF interaction in response to MALAT1 knockdown with or without complementation with lacZ or MALAT1 RNA.

Article Snippet: The Abs used for immunoblotting were rabbit anti-PTBP1 Ab (382421, Zen Bioscience), rabbit anti-PSF Ab (15585-1-ap, Proteintech), rabbit anti-H3 Ab (17168-1-ap, Proteintech), mouse anti–hRNP L Ab (ab6106, Abcam), rabbit anti–hRNP U Ab (ab172608, Abcam), rabbit anti–hRNP A1 Ab (ab208026, Abcam), mouse anti–hRNP F Ab (04-1462, Sigma-Aldrich), and mouse anti-actin Ab (66009-1-Ig, Proteintech).

Techniques: Co-Immunoprecipitation Assay, Reverse Transcription, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Standard Deviation, Western Blot, Knockdown, Control, shRNA, Labeling, RNAscope, In Situ Hybridization, Staining, Immunofluorescence

( A ) Heatmaps showing the changes in percent spliced-in (ΔPSI) compared with the negative control after knockdown of MALAT1 , PTBP1 , and PSF in HEK293 cells with two independent shRNAs. AS events with significant changes in PSI ( P < 0.05) are presented. ( B ) Correlation plots showing ΔPSI from two independent MALAT1-, PTBP1-, or PSF-specific shRNAs in HEK293 cells. ( C ) Pie charts showing types of AS events regulated by MALAT1, PTBP1, or PSF depletion as detected by RNA-seq in HEK293 cells. The size of each pie chart reflects the number of events detected. ( D ) Venn diagram showing overlap of cassette exons regulated by MALAT1, PTBP1, and PSF in HEK293 cells.

Journal: Science Advances

Article Title: MALAT1 modulates alternative splicing by cooperating with the splicing factors PTBP1 and PSF

doi: 10.1126/sciadv.abq7289

Figure Lengend Snippet: ( A ) Heatmaps showing the changes in percent spliced-in (ΔPSI) compared with the negative control after knockdown of MALAT1 , PTBP1 , and PSF in HEK293 cells with two independent shRNAs. AS events with significant changes in PSI ( P < 0.05) are presented. ( B ) Correlation plots showing ΔPSI from two independent MALAT1-, PTBP1-, or PSF-specific shRNAs in HEK293 cells. ( C ) Pie charts showing types of AS events regulated by MALAT1, PTBP1, or PSF depletion as detected by RNA-seq in HEK293 cells. The size of each pie chart reflects the number of events detected. ( D ) Venn diagram showing overlap of cassette exons regulated by MALAT1, PTBP1, and PSF in HEK293 cells.

Techniques: Negative Control, Knockdown, RNA Sequencing

( A ) Co-IP assay with anti-PSF Ab detecting the PTBP1/PSF interaction within cell extracts of the indicated HCC cell lines and the PTBP1/PSF interaction after DNase I or RNase T1 treatment. The dose of DNase I and RNase T1 was 15 U per reaction. ( B ) Co-IP assay with anti-PSF Ab detecting the PTBP1/PSF interaction in the indicated HCC cell lines and the PTBP1/PSF interaction after MALAT1 knockdown by two different shRNAs. Depletion of 7SK RNA and nuclear paraspeckle assembly transcript 1 (NEAT1), two other nuclear-retained lncRNAs used as controls, was found to have no effect on the PTBP1/PSF interaction (top). The knockdown efficiency of 7SK RNA, NEAT1, and MALAT1 was detected by RT-qPCR (bottom). Data are shown as means ± SD of n = 3 independent experiments. ** P < 0.01 and *** P < 0.001 by Student’s t test. ( C and D ) Confocal images of MALAT1 labeled by RNAscope ISH and PTBP1 and PSF proteins concurrently stained by IF in HCCLM3 cells (C) and in control and MALAT1-depleted HCCLM3 cells (D). Scale bars, 10 μm.

Journal: Science Advances

Article Title: MALAT1 modulates alternative splicing by cooperating with the splicing factors PTBP1 and PSF

doi: 10.1126/sciadv.abq7289

Figure Lengend Snippet: ( A ) Co-IP assay with anti-PSF Ab detecting the PTBP1/PSF interaction within cell extracts of the indicated HCC cell lines and the PTBP1/PSF interaction after DNase I or RNase T1 treatment. The dose of DNase I and RNase T1 was 15 U per reaction. ( B ) Co-IP assay with anti-PSF Ab detecting the PTBP1/PSF interaction in the indicated HCC cell lines and the PTBP1/PSF interaction after MALAT1 knockdown by two different shRNAs. Depletion of 7SK RNA and nuclear paraspeckle assembly transcript 1 (NEAT1), two other nuclear-retained lncRNAs used as controls, was found to have no effect on the PTBP1/PSF interaction (top). The knockdown efficiency of 7SK RNA, NEAT1, and MALAT1 was detected by RT-qPCR (bottom). Data are shown as means ± SD of n = 3 independent experiments. ** P < 0.01 and *** P < 0.001 by Student’s t test. ( C and D ) Confocal images of MALAT1 labeled by RNAscope ISH and PTBP1 and PSF proteins concurrently stained by IF in HCCLM3 cells (C) and in control and MALAT1-depleted HCCLM3 cells (D). Scale bars, 10 μm.

Techniques: Co-Immunoprecipitation Assay, Knockdown, Quantitative RT-PCR, Labeling, RNAscope, Staining, Control

( A ) Native RIP followed by RT-qPCR detecting MALAT1 and 7SK RNA retrieved by hnRNP A1–, hnRNP F–, or hnRNP U–specific Ab or by normal IgG in HEK293 cells. Data are shown as means ± SD of n = 3 independent experiments. ( B ) Immunoblotting using the indicated Abs detecting IP efficiency of the native RIP assay in (A). ( C ) Co-IP assay with anti-PSF Ab detecting the effect of RNase T1 treatment, DNase I treatment, or MALAT1 knockdown on PSF interaction with the indicated hnRNP proteins in HEK293 cells. The dose of RNase T1 and DNase I was 15 U per reaction.

Journal: Science Advances

Article Title: MALAT1 modulates alternative splicing by cooperating with the splicing factors PTBP1 and PSF

doi: 10.1126/sciadv.abq7289

Figure Lengend Snippet: ( A ) Native RIP followed by RT-qPCR detecting MALAT1 and 7SK RNA retrieved by hnRNP A1–, hnRNP F–, or hnRNP U–specific Ab or by normal IgG in HEK293 cells. Data are shown as means ± SD of n = 3 independent experiments. ( B ) Immunoblotting using the indicated Abs detecting IP efficiency of the native RIP assay in (A). ( C ) Co-IP assay with anti-PSF Ab detecting the effect of RNase T1 treatment, DNase I treatment, or MALAT1 knockdown on PSF interaction with the indicated hnRNP proteins in HEK293 cells. The dose of RNase T1 and DNase I was 15 U per reaction.

Techniques: Quantitative RT-PCR, Western Blot, Co-Immunoprecipitation Assay, Knockdown

( A ) Correlation plots showing expression levels between MALAT1, PTBP1, and PSF in HCC from the TCGA HCC datasets. TPM, transcripts per million. ( B ) Relative expression levels of MALAT1, PTBP1, and PSF in HCC and paired no cancerous hepatic tissue samples from TCGA RNA-seq dataset (top) and GEO GSE76297 dataset (bottom). P < 0.001 by Wilcoxon matched-paired signed rank test. ( C ) Kaplan-Meier analysis of the disease-free survival between patients with HCC with high MALAT, PTBP1, and PSF expression levels and others from the TCGA HCC panel. The median expression level was used as the cutoff. The P value was determined by log-rank test.

Journal: Science Advances

Article Title: MALAT1 modulates alternative splicing by cooperating with the splicing factors PTBP1 and PSF

doi: 10.1126/sciadv.abq7289

Figure Lengend Snippet: ( A ) Correlation plots showing expression levels between MALAT1, PTBP1, and PSF in HCC from the TCGA HCC datasets. TPM, transcripts per million. ( B ) Relative expression levels of MALAT1, PTBP1, and PSF in HCC and paired no cancerous hepatic tissue samples from TCGA RNA-seq dataset (top) and GEO GSE76297 dataset (bottom). P < 0.001 by Wilcoxon matched-paired signed rank test. ( C ) Kaplan-Meier analysis of the disease-free survival between patients with HCC with high MALAT, PTBP1, and PSF expression levels and others from the TCGA HCC panel. The median expression level was used as the cutoff. The P value was determined by log-rank test.

Techniques: Expressing, RNA Sequencing

( A ) Heatmaps showing the ΔPSI compared with the negative control after knockdown of MALAT1 , PTBP1 , and PSF in HepG2 cells with two independent shRNAs. Cassette exons with significant changes in PSI ( P < 0.05) are presented. ( B ) Venn diagram showing overlap of cassette exons regulated by MALAT1, PTBP1, and PSF in HepG2 cells. ( C ) Correlation plots showing ΔPSI for cassette MPP-regulated exons in HepG2 cells. ( D ) Pie chart showing percentage inclusion and exclusion of the MPP-regulated exons in HepG2 cells. ( E ) Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of the genes containing MPP-regulated exons. NOD, nonobese diabetic.

Journal: Science Advances

Article Title: MALAT1 modulates alternative splicing by cooperating with the splicing factors PTBP1 and PSF

doi: 10.1126/sciadv.abq7289

Figure Lengend Snippet: ( A ) Heatmaps showing the ΔPSI compared with the negative control after knockdown of MALAT1 , PTBP1 , and PSF in HepG2 cells with two independent shRNAs. Cassette exons with significant changes in PSI ( P < 0.05) are presented. ( B ) Venn diagram showing overlap of cassette exons regulated by MALAT1, PTBP1, and PSF in HepG2 cells. ( C ) Correlation plots showing ΔPSI for cassette MPP-regulated exons in HepG2 cells. ( D ) Pie chart showing percentage inclusion and exclusion of the MPP-regulated exons in HepG2 cells. ( E ) Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of the genes containing MPP-regulated exons. NOD, nonobese diabetic.

Techniques: Negative Control, Knockdown

( A ) Pie chart showing percentage of MPP-regulated exons with or without nearby PTBP1- and PSF-binding sites. ( B ) Native RIP assay followed by RT-qPCR validating the binding of PTBP1 and PSF to the indicated MPP-regulated pre-mRNAs. SRPK1, SRSF protein kinase 1; PGBD1, piggyBac transposable element derived; POMK, protein O-mannose kinase; NAV2, neuron navigator 2; ATG13, autophagy related 13; ATP9A, ATPase phospholipid transporting 9A; DICER1, dicer 1, ribonuclease III; EPB41L2, erythrocyte membrane protein band 4.1 like 2; THRB, thyroid hormone receptor beta; PIK3C2A, phosphatidylinositol-4-phosphate 3-kinase catalytic subunit type 2 alpha. Data are shown as means ± SD of n = 3 independent experiments. ** P < 0.01 and *** P < 0.001 by Student’s t test. ( C ) RNA pull-down followed by RT-qPCR detecting the indicated MPP-regulated pre-mRNAs coprecipitated with MALAT1 in HepG2 cells. Data are shown as means ± SD of n = 3 independent experiments. ** P < 0.01 and *** P < 0.001 by Student’s t test. ( D ) ChIRP assay followed by qPCR detecting the MALAT1 interaction with specific chromatin regions, from which the pre-mRNAs in (C) are produced, in HepG2 cells. Data are shown as means ± SD of n = 3 independent experiments. * P < 0.05 and ** P < 0.01 by Student’s t test. ( E ) 2C analysis detecting the association of PTBP1 and PSF with cellular RNA and the effect of MALAT1 knockdown on the association. The PTBP1 and PSF association with cellular RNA was dependent on UV cross-link. Histone H3 was used as a negative control that exhibits no association with cellular RNA. ( F ) Native RIP assay followed by RT-qPCR detecting the binding of PTBP1 and PSF to the indicated MPP-regulated pre-mRNAs in control and MALAT1 -knockdown HepG2 cells. Data are shown as means ± SD of n = 3 independent experiments. * P < 0.05 and ** P < 0.01 by Student’s t test. ns, not significant.

Journal: Science Advances

Article Title: MALAT1 modulates alternative splicing by cooperating with the splicing factors PTBP1 and PSF

doi: 10.1126/sciadv.abq7289

Figure Lengend Snippet: ( A ) Pie chart showing percentage of MPP-regulated exons with or without nearby PTBP1- and PSF-binding sites. ( B ) Native RIP assay followed by RT-qPCR validating the binding of PTBP1 and PSF to the indicated MPP-regulated pre-mRNAs. SRPK1, SRSF protein kinase 1; PGBD1, piggyBac transposable element derived; POMK, protein O-mannose kinase; NAV2, neuron navigator 2; ATG13, autophagy related 13; ATP9A, ATPase phospholipid transporting 9A; DICER1, dicer 1, ribonuclease III; EPB41L2, erythrocyte membrane protein band 4.1 like 2; THRB, thyroid hormone receptor beta; PIK3C2A, phosphatidylinositol-4-phosphate 3-kinase catalytic subunit type 2 alpha. Data are shown as means ± SD of n = 3 independent experiments. ** P < 0.01 and *** P < 0.001 by Student’s t test. ( C ) RNA pull-down followed by RT-qPCR detecting the indicated MPP-regulated pre-mRNAs coprecipitated with MALAT1 in HepG2 cells. Data are shown as means ± SD of n = 3 independent experiments. ** P < 0.01 and *** P < 0.001 by Student’s t test. ( D ) ChIRP assay followed by qPCR detecting the MALAT1 interaction with specific chromatin regions, from which the pre-mRNAs in (C) are produced, in HepG2 cells. Data are shown as means ± SD of n = 3 independent experiments. * P < 0.05 and ** P < 0.01 by Student’s t test. ( E ) 2C analysis detecting the association of PTBP1 and PSF with cellular RNA and the effect of MALAT1 knockdown on the association. The PTBP1 and PSF association with cellular RNA was dependent on UV cross-link. Histone H3 was used as a negative control that exhibits no association with cellular RNA. ( F ) Native RIP assay followed by RT-qPCR detecting the binding of PTBP1 and PSF to the indicated MPP-regulated pre-mRNAs in control and MALAT1 -knockdown HepG2 cells. Data are shown as means ± SD of n = 3 independent experiments. * P < 0.05 and ** P < 0.01 by Student’s t test. ns, not significant.

Techniques: Binding Assay, Quantitative RT-PCR, Derivative Assay, Membrane, Produced, Knockdown, Negative Control, Control

( A ) CCK-8 assays detecting implication of PTBP1 and PSF in the regulatory effect of MALAT1 on proliferation of HepG2 cells. Data are shown as means ± SD of n = 3 independent experiments. * P < 0.05 and ** P < 0.01 by one-way analysis of variance (ANOVA). ( B and C ) Transwell assays detecting implication of PTBP1 and PSF in the regulatory effect of MALAT1 on migration [B (top) and C] and invasion [B (bottom) and C] of HepG2 cells. Scale bars, 100 μm. Data in (C) are shown as means ± SD of n = 3 independent experiments. ** P < 0.01 by one-way ANOVA. ( D ) CCK-8 assays detecting contribution of MALAT1 to the regulatory effect of PTBP1 and PSF on proliferation of HepG2 cells. Data are shown as means ± SD of n = 3 independent experiments. * P < 0.05 and ** P < 0.01 by one-way ANOVA. ( E and F ) Transwell assays detecting contribution of MALAT1 to the regulatory effect of PTBP1 and PSF on migration [E (top) and F] and invasion [E (bottom) and F] of HepG2 cells. Scale bars, 100 μm. Data in (F) are shown as means ± SD of n = 3 independent experiments. ** P < 0.01 by one-way ANOVA. ( G ) RT-qPCR detecting contribution of MALAT1 to the regulatory effect of PTBP1 and PSF on pre-mRNA AS in HepG2 cells. Primers used for the RT-qPCR detection of long (L) and short (S) transcript variants, as well as shRNAs targeting long and short variants used in fig. S9G, are illustrated. Data are shown as means ± SD of n = 3 independent experiments. * P < 0.05, ** P < 0.01, and *** P < 0.001 by Student’s t test.

Journal: Science Advances

Article Title: MALAT1 modulates alternative splicing by cooperating with the splicing factors PTBP1 and PSF

doi: 10.1126/sciadv.abq7289

Figure Lengend Snippet: ( A ) CCK-8 assays detecting implication of PTBP1 and PSF in the regulatory effect of MALAT1 on proliferation of HepG2 cells. Data are shown as means ± SD of n = 3 independent experiments. * P < 0.05 and ** P < 0.01 by one-way analysis of variance (ANOVA). ( B and C ) Transwell assays detecting implication of PTBP1 and PSF in the regulatory effect of MALAT1 on migration [B (top) and C] and invasion [B (bottom) and C] of HepG2 cells. Scale bars, 100 μm. Data in (C) are shown as means ± SD of n = 3 independent experiments. ** P < 0.01 by one-way ANOVA. ( D ) CCK-8 assays detecting contribution of MALAT1 to the regulatory effect of PTBP1 and PSF on proliferation of HepG2 cells. Data are shown as means ± SD of n = 3 independent experiments. * P < 0.05 and ** P < 0.01 by one-way ANOVA. ( E and F ) Transwell assays detecting contribution of MALAT1 to the regulatory effect of PTBP1 and PSF on migration [E (top) and F] and invasion [E (bottom) and F] of HepG2 cells. Scale bars, 100 μm. Data in (F) are shown as means ± SD of n = 3 independent experiments. ** P < 0.01 by one-way ANOVA. ( G ) RT-qPCR detecting contribution of MALAT1 to the regulatory effect of PTBP1 and PSF on pre-mRNA AS in HepG2 cells. Primers used for the RT-qPCR detection of long (L) and short (S) transcript variants, as well as shRNAs targeting long and short variants used in fig. S9G, are illustrated. Data are shown as means ± SD of n = 3 independent experiments. * P < 0.05, ** P < 0.01, and *** P < 0.001 by Student’s t test.

Techniques: CCK-8 Assay, Migration, Quantitative RT-PCR

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