khdrbs1  (Proteintech)

Journal: Communications Biology

Article Title: An in vitro CRISPR screen of cell-free DNA identifies apoptosis as the primary mediator of cell-free DNA release

doi: 10.1038/s42003-024-06129-1

Figure Lengend Snippet: a Immunoblot analysis of Sam68 and FADD after CRISPR-mediated knockout (KO) in the MCF-10A background. TWT = Targeted Wild-Type. b Quantification of DNA release from MCF-10A KO cell lines in culture. Individual cell lines shown, with data representing mean fold change ± SEM internally normalized to cell concentration for each cell line and then normalized to control; n = 3 biologically independent samples. Electropherograms were individually run at least n = 3 times and representative traces were selected. c Quantification of DNA release from Sam68 KO MCF-10A cell lines rescued by Sam68-GFP overexpression. Parental and TWT were grouped and labeled control, and two Sam68 KO3 and KO4 were grouped. Data represent mean fold change ± SEM internally normalized to cell concentration for each cell line and then normalized to control; n = 4 for all lines except n = 3 for Sam68 TWT before combining. Electropherograms were individually run at least n = 3 times and representative traces were selected. d Quantification of DNA release from FADD KO MCF-10A cell lines rescued by FADD-GFP overexpression. Parental and TWT were grouped and labeled control, and two FADD KO cell lines were grouped. Data represent mean fold change ± SEM internally normalized to cell concentration for each cell line and then normalized to control; n = 3 for all GFP-expressing lines and n = 4 for each FADD-GFP expressing line before combining. Electropherograms were individually run at least n = 3 times and representative traces were selected. e Cell growth assay of KO MCF-10A cell lines. Parental and TWT were grouped and labeled control. All four Sam68 KO cell lines and both FADD KO cell lines are respectively grouped. Data represent mean cell concentration ± SEM; n = 3 for each cell line before combining. f Annexin V and Propidium Iodide (PI) assay of KO MCF-10A cell lines. Parental and TWT were grouped and labeled control. All four Sam68 KO cell lines and both FADD KO cell were grouped, respectively. Data represent mean signal (RFU for PI; RLU for Annexin) ± SEM internally normalized to cell concentration for each cell line and then normalized to control; n = 6 biologically independent samples for each cell line before combining. g Cytotoxic assay of MCF-10A KO panel treated with 1 ng/mL TRAIL ligand. Parental and TWT were grouped and labeled control. All four Sam68 mutant cell lines and both FADD mutant cell lines were grouped, respectively. Data represent mean percent survival ± SEM as normalized to vehicle of each cell line condition; n = 3 biologically independent samples for each cell line before combining. h Quantification of DNA release from MCF-10A KO panel treated with 1 ng/mL TRAIL ligand. Parental and TWT were grouped and labeled control. All four Sam68 mutant cell lines and both FADD mutant cell lines were respectively grouped. Data represent mean fold change ± SEM internally normalized to cell concentration for each cell line and then normalized to control; n = 3 biologically independent samples for each cell line before combining. i Quantification of DNA release from MCF-10A KO cell lines treated with 20 μg/mL ZVAD-FM-K. Parental MCF-10As, Sam68 KO3/4 mutant cell lines, and both FADD mutant cell lines were respectively grouped. Data represent mean fold change ± SEM in DNA release internally normalized to cell concentration for each cell line and then normalized to control vehicle; n = 3 biologically independent samples for all untreated lines and FADD KO1, n = 4 for treated lines for each cell line before combining. All statistics were ANOVA with Dunnett’s multiple comparison test at endpoint.

Article Snippet: Lentiviral expression vectors with CMV promoters driving GFP-tagged human Sam68 and FADD were purchased from Origene (PS100093, RC200263L4, RC201805L4).

Techniques: Western Blot, CRISPR, Knock-Out, Concentration Assay, Control, Over Expression, Labeling, Expressing, Growth Assay, Mutagenesis, Comparison

Journal: Communications Biology

Article Title: An in vitro CRISPR screen of cell-free DNA identifies apoptosis as the primary mediator of cell-free DNA release

doi: 10.1038/s42003-024-06129-1

Figure Lengend Snippet: a Quantification of DNA release from MDA-MB-231, MDA-MB-468, and HCT116 cancer cell lines with overexpression of GFP-tagged Sam68 or FADD. Data represent mean fold change ± SEM in DNA release normalized to cell concentration for each cell line, then overall to GFP control; n = 4 biologically independent samples. b Fragmentation pattern of MDA-MB-231, MDA-MB-468, and HCT116 cancer cell lines with overexpression of GFP-tagged Sam68 and FADD. Electropherograms were individually run at least n = 3 times and representative traces were selected. c Quantification of Annexin V signal from MDA-MB-231, MDA-MB-468, and HCT116 cancer cell lines with overexpression of GFP-tagged Sam68 and FADD. Data represent mean fold change ± SEM in RLU signal normalized to cell concentration at collection for each cell line, then overall to GFP control; n = 4 biologically independent samples for MDA-MB-231 and HCT116, n = 6 for MDA-MB-468. d Quantification of DNA release from MDA-MB-231, MDA-MB-468, and HCT116 cancer cell lines treated with TRAIL ligand. Data represent mean fold change ± SEM in DNA release normalized to cell concentration for each treatment, then overall to vehicle control; n = 4 biologically independent samples. e Fragmentation pattern of MDA-MB-231, MDA-MB-468, and HCT116 cancer cell lines treated with TRAIL ligand. Electropherograms were individually run at least n = 3 times and representative traces were selected. f Quantification of Annexin V signal from MDA-MB-231, MDA-MB-468, and HCT116 cancer cell lines treated with TRAIL Ligand. Data represent mean fold change ± SEM RLU signal normalized to cell concentration for each treatment, then overall to vehicle control; n = 6 biologically independent samples. g Quantification of Propidium Iodide signal from MDA-MB-231, MDA-MB-468, and HCT116 cancer cell lines treated with TRAIL Ligand. Data represent mean fold change ± SEM RFU signal normalized to cell concentration for each treatment, then overall to vehicle control; n = 6 biologically independent samples. All statistics were ANOVA with Dunnett’s multiple comparison test at endpoint.

Article Snippet: Lentiviral expression vectors with CMV promoters driving GFP-tagged human Sam68 and FADD were purchased from Origene (PS100093, RC200263L4, RC201805L4).

Techniques: Over Expression, Concentration Assay, Control, Comparison

Journal: Human Molecular Genetics

Article Title: Arginine methylation of RNA-binding proteins is impaired in Huntington’s disease

doi: 10.1093/hmg/ddad125

Figure Lengend Snippet: Arginine methylation of HNRNPUL2, KHDRBS1(SAM68) and SRSF1 is reduced in HD ISPNs ( A ) Total cell lysates from non-differentiated control (33CAG) and HD (180CAG) ISPNs were prepared as described in the Experimental Section. IPs with a mixture of mono-methyl (MMA) and asymmetric dimethyl (ADMA) antibodies were performed followed by western blot analysis with indicated protein-specific antibodies. Protein bands were visualized and quantified using the Licor System and Image Studio software. ( B ) – ( D ) Graphs (left) show the mean intensity values (±SEM) of the signal detected with antibodies to total HNRNPUL2, KHDRBS1(SAM68) and SRSF1 in the IPs normalized to the signal obtained from input lysates with the same antibodies. Total protein levels normalized to β-tubulin were also quantified (right graphs). n = 3 (biological replicates), Normality Test (Shapiro–Wilk): Passed, Equal Variance Test: Passed. T -test with equal variances was performed: (B) * 33CAG versus 180CAG, P 0.04. (C) * 33CAG versus 180CAG, P 0.0046. * * 33CAG versus 180CAG, P 0.0039.

Article Snippet: FLAG-tagged HNRNPUL2 and KHDRBS1 expression plasmids were obtained from GenScript.

Techniques: Methylation, Control, Western Blot, Software

Journal: Human Molecular Genetics

Article Title: Arginine methylation of RNA-binding proteins is impaired in Huntington’s disease

doi: 10.1093/hmg/ddad125

Figure Lengend Snippet: RNA association of hypomethylated RBPs is decreased in HD cells. RNA-binding assay in ISPNs. RNA pull-downs were performed as described in the Experimental Section according to Castello et al . (2013) . ( A ) Validation of RNA-binding assay in ISPNs demonstrating detection of indicated RNA-bound proteins in cross-linked cells (but not in the eluates of nonirradiated control cells) with protein-specific antibodies. ( B ) The amounts of mRNA within RNA-protein complexes were estimated by absorbance at A260, and equal amounts were analyzed by western blotting with indicated protein-specific antibodies. Representative experiment is shown. ( C ) – ( G ) Quantitation of blots shown in (B). Graphs show the mean intensity values (±SD) of the signal with antibodies to indicated proteins detected in the RNA-bound fraction normalized to the signal obtained from input lysates with the same antibodies. n = 3 (biological replicates). Normality test (Shapiro–Wilk): Passed, equal variance test: Passed. One-way ANOVA was performed with pairwise multiple comparison procedures (Holm–Sidak method). (C) HNRNPUL2, * 33EE versus 180, P 0.002; * * 33DG versus 180, P 0.002. (D) KHDRBS1, * 33EE versus 180, P 0.004; * * 33DG versus 180, P < 0.001; * * * 33DG versus 33EE, P < 0.001. (E) FUS, * 33EE versus 180, P 0.003; * * 33DG versus 180, P 0.008. (F) SRSF1, * 33EE versus 180, P 0.003; * * 33DG versus 180, P 0.011. (G) TAF15, * 33EE versus 180, P < 0.001; * * 33DG versus 180, P < 0.001; * * * 33DG versus 33EE, P < 0.001.

Article Snippet: FLAG-tagged HNRNPUL2 and KHDRBS1 expression plasmids were obtained from GenScript.

Techniques: RNA Binding Assay, Biomarker Discovery, Control, Western Blot, Quantitation Assay, Comparison

Journal: Human Molecular Genetics

Article Title: Arginine methylation of RNA-binding proteins is impaired in Huntington’s disease

doi: 10.1093/hmg/ddad125

Figure Lengend Snippet: Arginine methylation of HNRNPUL2, KHDRBS1(SAM68) and SRSF1 is reduced in HD ISPNs ( A ) Total cell lysates from non-differentiated control (33CAG) and HD (180CAG) ISPNs were prepared as described in the Experimental Section. IPs with a mixture of mono-methyl (MMA) and asymmetric dimethyl (ADMA) antibodies were performed followed by western blot analysis with indicated protein-specific antibodies. Protein bands were visualized and quantified using the Licor System and Image Studio software. ( B ) – ( D ) Graphs (left) show the mean intensity values (±SEM) of the signal detected with antibodies to total HNRNPUL2, KHDRBS1(SAM68) and SRSF1 in the IPs normalized to the signal obtained from input lysates with the same antibodies. Total protein levels normalized to β-tubulin were also quantified (right graphs). n = 3 (biological replicates), Normality Test (Shapiro–Wilk): Passed, Equal Variance Test: Passed. T -test with equal variances was performed: (B) * 33CAG versus 180CAG, P 0.04. (C) * 33CAG versus 180CAG, P 0.0046. * * 33CAG versus 180CAG, P 0.0039.

Article Snippet: The following primary antibodies were used for staining: FUS/TLS (E3O81, #67840, Cell Signaling Technology), TAF15 (TAFII68, #A300-308A Bethyl Laboratories), KHDRBS1 (SAM68, #A302-110A, Bethyl Laboratories), HNRNPUL2 (E1B-AP5 #A300-863A, Bethyl Laboratories); SRSF1 (#32-4600, ThermoFisher); PRMT6 (rabbit #720142, and mouse 7B11 MA5-32944, ThermoFisher); HTT (#MCA2050, Bio-Rad), phospho-HTT pS1864 ( ).

Techniques: Methylation, Control, Western Blot, Software

Journal: Human Molecular Genetics

Article Title: Arginine methylation of RNA-binding proteins is impaired in Huntington’s disease

doi: 10.1093/hmg/ddad125

Figure Lengend Snippet: RNA association of hypomethylated RBPs is decreased in HD cells. RNA-binding assay in ISPNs. RNA pull-downs were performed as described in the Experimental Section according to Castello et al . (2013) . ( A ) Validation of RNA-binding assay in ISPNs demonstrating detection of indicated RNA-bound proteins in cross-linked cells (but not in the eluates of nonirradiated control cells) with protein-specific antibodies. ( B ) The amounts of mRNA within RNA-protein complexes were estimated by absorbance at A260, and equal amounts were analyzed by western blotting with indicated protein-specific antibodies. Representative experiment is shown. ( C ) – ( G ) Quantitation of blots shown in (B). Graphs show the mean intensity values (±SD) of the signal with antibodies to indicated proteins detected in the RNA-bound fraction normalized to the signal obtained from input lysates with the same antibodies. n = 3 (biological replicates). Normality test (Shapiro–Wilk): Passed, equal variance test: Passed. One-way ANOVA was performed with pairwise multiple comparison procedures (Holm–Sidak method). (C) HNRNPUL2, * 33EE versus 180, P 0.002; * * 33DG versus 180, P 0.002. (D) KHDRBS1, * 33EE versus 180, P 0.004; * * 33DG versus 180, P < 0.001; * * * 33DG versus 33EE, P < 0.001. (E) FUS, * 33EE versus 180, P 0.003; * * 33DG versus 180, P 0.008. (F) SRSF1, * 33EE versus 180, P 0.003; * * 33DG versus 180, P 0.011. (G) TAF15, * 33EE versus 180, P < 0.001; * * 33DG versus 180, P < 0.001; * * * 33DG versus 33EE, P < 0.001.

Article Snippet: The following primary antibodies were used for staining: FUS/TLS (E3O81, #67840, Cell Signaling Technology), TAF15 (TAFII68, #A300-308A Bethyl Laboratories), KHDRBS1 (SAM68, #A302-110A, Bethyl Laboratories), HNRNPUL2 (E1B-AP5 #A300-863A, Bethyl Laboratories); SRSF1 (#32-4600, ThermoFisher); PRMT6 (rabbit #720142, and mouse 7B11 MA5-32944, ThermoFisher); HTT (#MCA2050, Bio-Rad), phospho-HTT pS1864 ( ).

Techniques: RNA Binding Assay, Biomarker Discovery, Control, Western Blot, Quantitation Assay, Comparison