Journal: Communications Chemistry

Article Title: A Hybrid compound H93 treats prostate cancer by directly binding UHRF1 and promoting protein dimerization

doi: 10.1038/s42004-025-01744-3

Figure Lengend Snippet: a Co-immunoprecipitation (Co-IP) showed that H93 promotes the protein dimerization of exogenously expressed UHRF1. HEK293T cells were co-transfected with His-UHRF1 and Flag-UHRF1 plasmids, followed by treatment with 0, 5, 10, or 20 μM of H93 for 8 h. The cell lysates were incubated with protein A/G bead-bound anti-His antibody, then subjected to immunoblotting with anti-His or anti-Flag antibodies. b HEK293T cells were co-transfected with VC155-UHRF1, UHRF1-VN155 and dsRed plasmids for 48 h and then treated with H93 (0, 5, 10 or 20 μM) for 2, 4 or 6 h. The protein interaction between VC155-UHRF1 and UHRF1-VN155 was assessed by bimolecular fluorescence complementation (BiFC) assays. The length of the scale bar is 20 μm. c HEK293T cells were co-transfected with His-UHRF1 and Myc-DNMT1 plasmids, and then treated with H93 (0, 5, 10 or 20 μM) for 8 h. Cell lysates were subjected to Co-IP using an anti-His antibody, and DNMT1 protein was assessed by immunoblotting. d DU145 (Left) or VCaP (Right) cells were treated with H93 (0, 5, 10 or 20 μM) for 8 h, and the endogenous protein interaction between UHRF1 and DNMT1 was measured by Co-IP. e H93 had no significant effect on the interaction of UHRF1 and DNMT1 when the W527, R528, and R581 residues were mutated to alanine (UHRF1 3A ). HEK293T cells were co-transfected with myc-DNMT1 and His-UHRF1 WT or His-UHRF1 3A plasmids for 48 h, followed by treatment with 0 or 20 μM H93 for 8 h and subjected to Co-IP. Experiments were performed in at least triplicate, and representative images are presented.

Article Snippet: DU145 (PCa, Cat# HTB-81, ATCC, Bethesda, MA, USA), VCaP (PCa, Cat#CRL-2876, ATCC), PC3 (PCa, Cat#CRL-1435, ATCC), CWR22Rv1 (PCa, Cat#CRL-2505, ATCC), C4-2 (PCa, Cat#CRL-3314, ATCC), LNCaP (PCa, Cat#CRL-1740, ATCC), HUVEC (human umbilical venous endothelial cells, Cat#PCS-100-013, ATCC) and HEK293T (embryonic kidney cells, Cat#CRL-1573, ATCC) cell lines were used in the experiments.

Techniques: Immunoprecipitation, Co-Immunoprecipitation Assay, Transfection, Incubation, Western Blot, Fluorescence

Journal: Communications Chemistry

Article Title: A Hybrid compound H93 treats prostate cancer by directly binding UHRF1 and promoting protein dimerization

doi: 10.1038/s42004-025-01744-3

Figure Lengend Snippet: a DU145 (Left) or VCaP (Right) cells were treated with indicated concentrations of H93 for 48 h, and the total DNA methylation level was measured by 5-methylcytosine (5mC) DNA dot-blot assays. Experiments were performed in triplicate. b DU145 (Left) or VCaP (Right) cells were treated with 0 (black dots), 10 (black squares) or 20 μM (black triangles) of H93 for 48 h, and the total DNA methylation level was measured by 5mC ELISA assays. The data are represented as the mean ± SD, n = 3 independent experiments. c Quantitated gene expression changes of representative tumor suppressor genes in DU145 (Upper) and VCaP (Lower) cells treated with 0 (empty black circles), 2.5 (black squares), 5 (black triangles), 10 (black rhombus) or 20 μM (black dots) of H93. n = 3 independent experiments. d–f DU145 subcutaneous xenografts were induced in immune-deficient nude mice, and then treated with vehicle (20 ml kg −1 day -1 ) or H93 (50 or 100 mg kg −1 day −1 ) by oral administration. n = 8 per group. The tumor nodes were harvested 28 days after treatment. d The tumor volumes (Left) and weights (Right) were compared with vehicle 20 ml kg −1 day −1 group depicted as black dots, H93 50 mg kg −1 day −1 group as black squares, H93 100 mg kg −1 day −1 group as black triangles. n = 8 animals. e The body weights of nude mice were continuously monitored and vehicle 20 ml kg −1 day −1 group was depicted as black line, H93 50 mg kg −1 day −1 group as red line, H93 100 mg kg −1 day −1 group as glaucous line. n = 8 animals. f Representative images of Hematoxylin-Eosin (HE), Ki67 immunochemistry (IHC) and transferase-mediated deoxyuridine triphosphate-biotin nick end labeling (TUNEL) staining in DU145 xenografts subjected to different concentrations of H93 (Left), and the quantitative data of Ki67 (Middle) and TUNEL (Right) staining, with vehicle 20 ml kg −1 day −1 group depicted as black dots, H93 50 mg kg −1 day −1 group as black squares, H93 100 mg kg −1 day −1 group as black triangles, and n = 40 views. The length of scale bars is 200 μm (×100) or 50 μm (×400). * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001. The data are shown as mean ± SD.

Article Snippet: DU145 (PCa, Cat# HTB-81, ATCC, Bethesda, MA, USA), VCaP (PCa, Cat#CRL-2876, ATCC), PC3 (PCa, Cat#CRL-1435, ATCC), CWR22Rv1 (PCa, Cat#CRL-2505, ATCC), C4-2 (PCa, Cat#CRL-3314, ATCC), LNCaP (PCa, Cat#CRL-1740, ATCC), HUVEC (human umbilical venous endothelial cells, Cat#PCS-100-013, ATCC) and HEK293T (embryonic kidney cells, Cat#CRL-1573, ATCC) cell lines were used in the experiments.

Techniques: DNA Methylation Assay, Dot Blot, Enzyme-linked Immunosorbent Assay, Gene Expression, End Labeling, TUNEL Assay, Staining

Journal:

Article Title: Analysis of an Autographa californica Multicapsid Nucleopolyhedrovirus lef-6 -Null Virus: LEF-6 Is Not Essential for Viral Replication but Appears To Accelerate Late Gene Transcription

doi: 10.1128/JVI.76.11.5503-5514.2002

Figure Lengend Snippet: Construction of a lef-6-null virus. (A) The strategy for construction of a lef-6-null BACmid containing a deletion of the AcMNPV lef-6 gene is shown in the diagram. The structure of the lef-6 locus is shown above that of the linear DNA fragment excised from transfer vector (pBluIAPGUSorf29CAT) and used for recombination in E. coli. In pBluIAPGUSorf29CAT, the lef-6 ORF was excised and replaced with a p6.9 promoter-driven GUS gene plus a chloramphenicol resistance gene cassette (cat). The structure of the resulting lef-6 locus in BACmid vAclef6KO is shown in the lower portion of the diagram. The positions of primers used for PCR analysis of the resulting AcMNPV BACmid (vAclef6KO) are indicated by small arrows, and the locations and sizes of predicted PCR products are indicated by brackets below the diagrams. (B) The results of PCR analysis of the lef-6 locus of the lef-6-null BACmid (vAclef6KO) are shown on an ethidium bromide-stained agarose gel. Templates (vAclef6KO and wild-type AcMNPV) and primer pairs (A+B, C+D, and E+F) are shown above the lanes, and the sizes of PCR amplification products (in kilobase pairs) are indicated on the right and left. M, marker DNAs. Primer pairs (A+B, C+D, and E+F) correspond to those shown in panel A. (C) Southern blot analysis was used to confirm the absence of lef-6 in the lef-6-null BACmid (vAclef6KO). For hybridization analysis, a lef-6-specific DNA probe was hybridized to XhoI-digested virus or the BACmid DNAs indicated above each lane.

Article Snippet: The lef-6 ORF was amplified from Ac M NPV DNA by using Vent DNA polymerase (New England Biolabs) and two oligonucleotide primers containing flanking Bam HI sites: 5′lef6pPet (5′-CG GGATCC AATGGTGTTCAACGTGTACTACAAC-3′) and 3′lef6pPet (5′-CG GGATCC GCTTGTTTTTCTAATACATTCAAGTC-3′).

Techniques: Plasmid Preparation, Staining, Agarose Gel Electrophoresis, Amplification, Marker, Southern Blot, Hybridization

Journal:

Article Title: Analysis of an Autographa californica Multicapsid Nucleopolyhedrovirus lef-6 -Null Virus: LEF-6 Is Not Essential for Viral Replication but Appears To Accelerate Late Gene Transcription

doi: 10.1128/JVI.76.11.5503-5514.2002

Figure Lengend Snippet: Analysis of viral DNA replication in Sf9 cells infected with lef-6-null and control viruses. (A) Sf9 cells were infected with viruses derived from either wild-type AcMNPV, control BACmid (vAc64−/+GUS), lef-6-null BACmid (vAclef6KO), or lef-6 repair BACmids (vAclef6KO-REP-P or vAclef6KO-REP-ie1P), and total cellular DNAs were isolated at various times posttransfection (6 to 96 h) and examined by Southern dot blot hybridization. Total AcMNPV DNA was labeled with [32P]dATP as a hybridization probe. A standard curve of AcMNPV DNA is shown on the right (10 to 400 ng of AcMNPV DNA). (B) Quantitative analysis of viral DNA replication by Southern dot blot analysis. Three replicates of each virus infection and time point were examined as in panel A, and DNA was quantified by phosphorimager analysis. Bars represent the average of three dot blot samples, and error bars represent the standard deviation.

Article Snippet: The lef-6 ORF was amplified from Ac M NPV DNA by using Vent DNA polymerase (New England Biolabs) and two oligonucleotide primers containing flanking Bam HI sites: 5′lef6pPet (5′-CG GGATCC AATGGTGTTCAACGTGTACTACAAC-3′) and 3′lef6pPet (5′-CG GGATCC GCTTGTTTTTCTAATACATTCAAGTC-3′).

Techniques: Infection, Derivative Assay, Isolation, Dot Blot, Hybridization, Labeling, Standard Deviation

Journal: Nature Communications

Article Title: The ULK3 kinase is a determinant of keratinocyte self-renewal and tumorigenesis targeting the arginine methylome

doi: 10.1038/s41467-023-36410-6

Figure Lengend Snippet: a ULK3 expression in TCGA profiles of Head/Neck (H&N), Lung, and Cervical Squamous Cell Carcinomas (SCCs) versus matched normal tissues updated to 2018. Individual z-scores values, mean ± SEM, P < 0.01, two-tailed unpaired t-test. Box plots show ULK3 levels in the indicated SCCs versus normal tissues in TCGA datasets updated to 2016 as in ref. . GEPIA-generated median, box (25–75%) and whiskers (5–95%), log(transcript per million+1), value cutoff P < 0.01. b Quantification of Skin, H&N and Cervical SCCs tissue arrays ULK3 immunostaining with unmatched (Skin and H&N) or matched (Cervical) non-affected tissues. Skin: n (Normal = 8, SCC = 24); H&N: n (Normal = 8, SCC = 26); Cervical: n (Normal = 14, SCC = 14), mean ± SEM, P < 0.001, two-tailed unpaired t -test. c ULK3 (green) and VIMENTIN (magenta) immunofluorescence and quantification of nuclear ULK3 in normal skin (NS) and skin SCC. Representative low and high magnifications, n (NSs/SCCs) = 6, mean ± SEM, P < 0.0001, two-tailed unpaired t-test. Scale bar 10 µm. ULK3 immunofluorescence of additional NSs/SCCs is in Supplementary Fig. . d ULK3 (green) and Ki67 (magenta) or TP63 (magenta) immunofuorescence of skin SCCs (SCC#3 and SCC#4). Representative low and high magnifications with quantification of double nuclear ULK3/TP63 (yellow) or ULK3/Ki67 (yellow) positivity. n (SCCs) = 4, mean ± SEM, Scale bar 20 µm. e ULK3 (green) and pankeratin (magenta) immunostaining with quantification of ULK3 in actinic keratosis lesions (AKs). Representative low and high magnifications, n (AKs) = 3, mean ± SEM. Scale bar 5 µm. f ULK3 (green) and TP63 (magenta) immunostaining with quantification of ULK3/TP63 positive cells (yellow) in AKs. Representative low and high magnifications, n (AKs) = 3, mean ± SEM. Scale Bar 5 µm. Additional AK ULK3/TP63 immunostaining is in Supplementary Fig. . g RT-qPCR analysis of ULK family members ( ULK1-4 ), normalized to 36β4 , in SCC cells versus average level (solid line) in human keratinocytes (HKCs) (strains #GB2- #GB10). n (HKCs) = 9, n (SCCs) = 7. Representative experiment of two independent biological replicates. RT-qPCR of ULK1-4 in each HKCs and SCCs are in Supplementary Fig. . h Immunoblot and densitometric quantification of ULK3 in HKCs (#GB11- #GB13) and SCC cells (normalized to ß-ACTIN). n (HKCs) = 3, n (SCCs) = 8, mean ± SEM, P < 0.05, two-tailed unpaired t -test. The researcher’s initials and a number identify each primary HKC strain. A number identifies patient-derived SCC samples .

Article Snippet: Slides were processed with routine histology procedures as in refs. , using rabbit anti ULK3 ATLAS antibody (AB_2677003, 1:100 dilution).

Techniques: Expressing, Two Tailed Test, Generated, Immunostaining, Immunofluorescence, Quantitative RT-PCR, Western Blot, Derivative Assay

Journal: Nature Communications

Article Title: The ULK3 kinase is a determinant of keratinocyte self-renewal and tumorigenesis targeting the arginine methylome

doi: 10.1038/s41467-023-36410-6

Figure Lengend Snippet: a Cell growth on days 3 and 5 of HKC (strain #TP26) and of three SCC cell lines infected with two ULK3 silencing lentiviruses (shULK3 #1, #2) versus empty control vector (shCTR) plated in triplicate at low-density. Representative experiment of two independent biological replicates. The efficient downregulation of ULK3 in HKCs and SCC cells is in Supplementary Fig. . b Live fluorescence images of SCC13 and Cal27 cells infected with two ULK3 silencing lentiviruses, or control virus, and stained for early- (phosphatidylserine-FITC, green), late- apoptosis (7-aminoactinomycin D, magenta) and for all viable cells (CytoCalcein, violet-blue). Quantification, in duplicate cultures, SCC13 n (fields/shCTR) = 6, n (fields/shULK3) = 25; Cal27 n (fields/shCTR) = 7, n (fields/shULK3) = 24, n (cells/field) > 90, mean ± SEM, P < 0.001 or P < 0.0001 one-way ANOVA. Scale bar 10 µm. c Colony formation of HKCs (strain # TP26), five SCC cell lines and HeLa cells infected with two ULK3 silencing lentiviruses, or a control virus, and plated at limited cell density for one week. Crystal violet stained dishes were quantified using ImageJ. n (dishes) = 3, mean ± SEM, P < 0.0001, one-way ANOVA. Representative images of the stained colonies are in Supplementary Fig. . Supplementary Fig. show similar assays with SCC cells treated with the ULK3 inhibitor SU6668 . d Surveyor Assay of SCC13 cells one week after the infection with lentiviruses co-expressing the Cas9 enzyme and one of the three RNA guides targeting ULK3 (gRNA ULK3 #1-3), or a scrambled control gRNA (SCR gRNA). Each targeted genomic region (amplified using corresponding oligo pairs (#1-3) (-)) was tested for DNA mismatch by PCR by incubation with single-strand DNAse. (+). In control reactions, we used genomic DNA from SCR-infected SCC13 cells. The arrows indicate the amplified region and the asterisks the DNAase cleavage products. e ULK3 and ß-ACTIN immunoblotting of SCC13 cells one week after the infection with ULK3 gRNA (#1-3), versus a scrambled gRNA (SCR) lentiviruses. f Colony formation of SCC13 cells, plus/minus ULK3 deletion as above, plated at limited dilution for six days. n (dishes) = 3, mean ± SEM, P < 0.0001, one-way ANOVA.

Article Snippet: Slides were processed with routine histology procedures as in refs. , using rabbit anti ULK3 ATLAS antibody (AB_2677003, 1:100 dilution).

Techniques: Infection, Control, Plasmid Preparation, Fluorescence, Virus, Staining, Expressing, Amplification, Incubation, Western Blot

Journal: Nature Communications

Article Title: The ULK3 kinase is a determinant of keratinocyte self-renewal and tumorigenesis targeting the arginine methylome

doi: 10.1038/s41467-023-36410-6

Figure Lengend Snippet: a ULK3, TP63 and ß−ACTIN immunoblotting of SCC13 cells infected with three ULK3 silencing lentiviruses (shULK3#1-3), or an empty vector (shCTR), for one week. b TP63 (green) immunostaining and quantification of fluorescence signal intensity of SCC13 cells infected with two ULK3 -silencing (shULK3 #1 and #2) versus a control (shCTR) lentivirus for one week. Individual cell values and mean ± SD, n (cells) > 40, P < 0.0001, one-way ANOVA. Scale bar 7 µm. c RT-qPCR analysis of GLI1 and GLI2 expression, normalized to 36β4 , in SCC13 cells plus/minus infection with ULK3 silencing or control lentiviruses as in panel ( a ). n (dishes) = 3, mean ± SEM, P < 0.01, one-way ANOVA. d RT-qPCR analysis of the indicated genes, normalized to 36β4 , in SCC cells infected with two ULK3 silencing lentiviruses or a control virus. n (dishes) = 3, mean ± SEM, P < 0.001, one-way ANOVA. e Keratin 10 (KRT10), INVOLUCRIN (INV) and ß−ACTIN (ACTIN) immunoblotting of SCC13 cells with two ULK3 silencing lentiviruses for one week versus a control virus. f RT-qPCR analysis of CDKN1A , normalized to 36ß4 , of HKCs (strains # TP24, # TP25, # TP26) and three SCC cell lines plus/minus ULK3 silencing for one week. n (HKCs) = 3, n (SCCs) = 3. g p21, ULK3 and ß−ACTIN immunoblotting of three SCC cell lines plus/minus ULK3 silencing for one week. n (SCC lines) = 3. h Colony formation assays of SCC cell lines infected with a ULK3 overexpressing lentivirus (ULK3), or an empty virus (CTR), and plated at low density for one week. n (dishes) = 4, mean ± SEM, P < 0.001, two-tailed unpaired t-test. i Sphere-forming capability of SCC13 cells infected with a ULK3 overexpressing virus (ULK3) or a control virus (CTR) and cultured for one week in Matrigel suspension as in . Representative phase contrast images and quantification of the number of spheres in four fields per dish. n (dishes/condition) = 4, mean ± SEM, P < 0.0001, two-tailed unpaired t-test. j RT-qPCR analysis of the indicated genes, normalized to 36β4 , in SCC13 cells plus/minus ULK3 overexpression (white and green respectively) as tested in panel ( i ). n (dishes) = 3, mean ± SEM, P < 0.001, two-tailed unpaired t-test.

Article Snippet: Slides were processed with routine histology procedures as in refs. , using rabbit anti ULK3 ATLAS antibody (AB_2677003, 1:100 dilution).

Techniques: Western Blot, Infection, Plasmid Preparation, Immunostaining, Fluorescence, Control, Quantitative RT-PCR, Expressing, Virus, Two Tailed Test, Cell Culture, Suspension, Over Expression

Journal: Nature Communications

Article Title: The ULK3 kinase is a determinant of keratinocyte self-renewal and tumorigenesis targeting the arginine methylome

doi: 10.1038/s41467-023-36410-6

Figure Lengend Snippet: a Volcano plot of genes regulated in HKCs (strains #TP24, #TP25, #TP31) infected with two ULK3 -silencing versus control lentiviruses (GSE: 183084). Marked are among most downregulated (magenta) and upregulated (green) genes. Clariom D analysis chip was made with Transcriptomic Analysis Console software (TAC) and plotted using Prism. Blue and red lines indicate the threshold of significance and ±1 fold change respectively, n (HKCs) = 3, P = 0.05, two-tailed t -test. b Gene set enrichment analysis (GSEA) of HKCs profiles with ULK3 gene silencing versus control as in (a). Enrichment plots of ∆Np63 and MYC -related signatures, and list of most significant signatures negatively associated with profiles of ULK3 -silenced HKCs, divided as related to growth/differentiation (blue) or cellular metabolism (green). n (strains) = 3, P < 0.001, permutation-based p values. c Gene ontology (GO) analysis, by database for annotation, visualization and integrated discovery (DAVID), of the processes consistently regulated by ULK3 silencing in HKCs and SCC13 cells (GSE: 183084 and 183085), divided as pertinent to cell cycle (orange), translation (blue) and metabolism (green). d Heat map of expression values for components of Glycolytic (Glu) or Glutaminolytic (Gln) pathways, from the profiles of HKCs and SCC13 cells plus/minus ULK3 downregulation. Values visualized with Morpheus software. e RT-qPCR analysis of the indicated genes, normalized to 36β4 , in SCC13 cells with or without ULK3 silencing. n (dishes) = 3, mean ± SEM, P < 0.0001, one-way ANOVA. f Glutaminase 1 (GLS1), Pyruvate Kinase M1 (PKM1) and ß−ACTIN immunoblots of SCC cells infected with two ULK3 (shULK3 #1, #2) versus control silencing (shCTR) lentiviruses. The arrow indicates upregulated GLS1. g Steady-state metabolite profile of SCC13 infected with two ULK3 - versus control- silencing lentiviruses (MTBL S5155). After MetaboAnalyst software analysis, significantly modulated metabolites were grouped according to metabolic processes, ranked by fold changes (logFC). The values and the statistical analysis for glutathione and glutamate are in Supplementary Fig. . h Pathways affected by ULK3 downregulation in SCC13 cells as identified by metabolite set enrichment analysis . Fold enrichment of the top 11 most significantly affected pathways ranked according to p values. n (samples/condition) = 3, P < 0.0, two-tailed t-test.

Article Snippet: Slides were processed with routine histology procedures as in refs. , using rabbit anti ULK3 ATLAS antibody (AB_2677003, 1:100 dilution).

Techniques: Infection, Control, Software, Two Tailed Test, Expressing, Quantitative RT-PCR, Western Blot

Journal: Nature Communications

Article Title: The ULK3 kinase is a determinant of keratinocyte self-renewal and tumorigenesis targeting the arginine methylome

doi: 10.1038/s41467-023-36410-6

Figure Lengend Snippet: a ULK3-bound peaks distribution as found by ChIP seq (chromatin immunoprecipitation followed by DNA sequencing) analysis of SCC13 cells with anti-ULK3 antibodies (GSE: 183933). Peaks number (count) per distance (bp) from the Transcriptional Start Site (TSS) of genes. Supplementary Fig. shows the GO using DAVID software of biological processes related to the ULK3-bound genes. b Peaks distribution as found by ChIP-seq analysis with antibodies against acetylated lysine 27 of histone 3 (H3K27ac) in SCC13 cells plus/minus ULK3 silencing (GSE: 183933). Peaks number (count) per distance (bp) from the Transcriptional Start Site (TSS) of genes. ChIP-seq analysis was made using the Galaxy web platform. The magenta dashed lines indicate as a reference between control versus ULK3 silenced (shRNA) cells the 10-peaks value. c GO using DAVID software of most significant processes of ULK3-bound genes having downregulated H3K27ac peaks upon ULK3 silencing in SCC13 cells, as determined by ChIP seq analysis (GSE: 183933), P < 0.01, Fisher’s exact test. Supplementary Fig. shows the Venn diagram of ULK3-bound genes having downregulated H3K27ac peaks upon ULK3 silencing. d – f Graphic illustration of ULK3- and H3K27ac -binding peaks localization on two metabolic genes ( LDHA and PKM ) and a transcription factor ( FOXM1 ) in SCC13 cells infected with control versus ULK3 silencing lentiviruses, aligned using the integrative genomic viewer software (IGV). Position of the TSS, exons (boxes) and introns-transcribed regions from ENCODE. Blue squares mark the main areas with H3K27ac down-modulation upon ULK3 silencing. Supplementary Fig. shows a similar analysis made on EP300 promoter. ChIP with antibodies against the indicated proteins of SCC13 cells infected with two ULK3 silencing (red and blue) versus control (white) lentiviruses, followed by qPCR amplification of the two regions indicated in the scheme of the PKM , LDHA and FOXM1 genes, to which ULK3 was found to bind by CHIP-seq analysis. Results are expressed as enrichment fold relative to non-immune antibodies, n (experiments) = 2. Supplementary Fig. provides a similar analysis for the specificity control gene TUBA1A .

Article Snippet: Slides were processed with routine histology procedures as in refs. , using rabbit anti ULK3 ATLAS antibody (AB_2677003, 1:100 dilution).

Techniques: ChIP-sequencing, Chromatin Immunoprecipitation, DNA Sequencing, Software, Control, shRNA, Binding Assay, Infection, Amplification

Journal: Nature Communications

Article Title: The ULK3 kinase is a determinant of keratinocyte self-renewal and tumorigenesis targeting the arginine methylome

doi: 10.1038/s41467-023-36410-6

Figure Lengend Snippet: a GSEA of HKCs profiles with PRMT1 silencing by siRNA versus up- or down- regulated signatures from HKCs with ULK3 silencing, P < 0.001, permutation-based p. The Venn diagram of genes modulated by ULK3 silencing and bound by ULK3 in ChIP-seq in SCC13 cells is in Supplementary Fig. . The list of the genes modulated by siPRMT1 and shULK3 in HKCs is in Supplementary Fig. . b Proximity ligation assays (PLA) of two SCC cell lines with anti -ULK3 and -PRMT1 or -PRMT5 antibodies, with anti-ULK3 alone or IgG as controls. Representative images and quantification of the magenta puncta from the juxtaposition of ULK3 and PRMT1 or PRMT5 antibodies, n (cells/condition) = 75, mean ± SEM, P < 0.01, two-tailed unpaired t-test. Scale bar 5 µm. c Immunoprecipitations (IP) with anti -ULK3 (ULK3) or IgG antibodies from SCC13 cells followed by PRMT1 and PRMT5 immunoblotting (left), and IPs with anti -PRMT1, -PRMT5, or IgG antibodies followed by ULK3 immunoblotting (right). Inputs were run on the same gel. d ULK3 protein map with indicated the kinase and the microtubule-interacting (MIT1/2) domains, the positions of inactivating mutations (K139R and K44H) , , C-terminus truncation (∆C) and epitope tags (FLAG or V5). Analysis of HEK293T cells transfected with the indicated expression vectors followed by IP with anti-epitope antibodies (IP TAG) and immunoblotting with the indicated antibodies. e Analysis of HeLa cells mock-transfected or transfected with the indicated vectors followed by IP with FLAG antibodies (IP FLAG) and immunoblotting with the indicated antibodies. f PLA with anti -PRMT1 or -PRMT5 and anti -phosphorylated Ser/Thr (P-S/T) antibodies in HeLa cells infected with an ULK3 -silencing (shULK3#1) versus control (shCTRL) lentivirus. IgGs and anti-PRMT antibodies alone served as controls. Representative images and quantification of the magenta puncta from the juxtaposition of the antibodies. n (fields/condition) = 6, mean ± SEM, P < 0.01, two-tailed unpaired t -test. Scale bar 10 µm. g HeLa cells were either mock transfected (M) or transfected with expression vectors for FLAG-PRMT1 or FLAG-PRMT5 alone or in combination with wild-type ULK3 (ULK3 + PRMT1 and ULK3 + PRMT5). Total extracts (Inputs) and the proteins eluted (fractions E1–E3) from a phosphoprotein-binding resin (Talon), were analyzed by FLAG immunoblotting and quantified.

Article Snippet: Slides were processed with routine histology procedures as in refs. , using rabbit anti ULK3 ATLAS antibody (AB_2677003, 1:100 dilution).

Techniques: ChIP-sequencing, Ligation, Two Tailed Test, Western Blot, Transfection, Expressing, Infection, Control, Binding Assay

Journal: Nature Communications

Article Title: The ULK3 kinase is a determinant of keratinocyte self-renewal and tumorigenesis targeting the arginine methylome

doi: 10.1038/s41467-023-36410-6

Figure Lengend Snippet: a Immunoblot analysis of recombinant FLAG-tagged -PRMT1 (200 ng) or -PRMT5 (200 ng) admixed with His-tagged ULK3 (200 ng) and pulled-down with a Ni-NTA resin (NTA). The same amounts of proteins were incubated with the resin singularly as controls. Sequential FLAG and ULK3 immunoblots. b Recombinant PRMT1 and PRMT5 were incubated with ULK3 as above before PRMT pull-down with anti-FLAG antibodies. Immunoblots were probed as indicated. c Recombinant PRMT1 and PRMT5 plus/minus heating for 20 min at 90 °C ( h ) before incubation with ULK3, NTA pull-down, and sequential immunoblot analysis as in ( a ). d Recombinant PRMT1 and PRMT5 were incubated with recombinant ULK3 for 30 minutes at 37°C in kinase buffer containing 200 µM ATP. Proteins were separated on a Phos-TAG gel, which delays the migration of phosphorylated proteins , followed by immunoblot analysis with the indicated antibodies. Parallel kinase reactions contained 1 and 10 µM ULK3 inhibitor SU6668 . The immunoblot analysis of the same samples in Fig. 7d separated by SDS-PAGE is in Supplementary Fig. . A kinase assay with PRMT1 plus/minus heat treatment prior to ULK3 kinase assay is in Supplementary Fig. . e In vitro ULK3 kinase assay using recombinant GFP as substrate. Proteins were admixed as in (d) before western blot analysis using the indicated antibodies. f , g Impact of ULK3 on PRTM1 and PRMT5 activity. Increasing amounts of PRMT1 or PRMT5 (0, 50, 100, 200, 400 ng) were incubated with (red bars) or without ULK3 (white bars) as in ( d ), followed by the methylation reaction (with 50µM S-adenosyl-methionine) of recombinant histone 4 (H4) (0.5 µg). Quantification of H4 arginine 3 asymmetric-, for PRMT1 activity ( f ), or symmetric-, for PRMT5 activity ( g ), dimethylation by dot blots analysis with antibodies specific for these epigenetic modifications. Mean ± SD, n (dot blot/condition) = 3, P < 0.001, two-tailed unpaired t -test. Supplementary Fig. shows symmetric and asymmetric dimethylated arginine immunoblottings of methylation assays using ULK3 as a substrate. Supplementary Fig. shows an immunoblot with the same antibodies of ULK3 and truncated ULK3 IP from transfected HEK293 cells.

Article Snippet: Slides were processed with routine histology procedures as in refs. , using rabbit anti ULK3 ATLAS antibody (AB_2677003, 1:100 dilution).

Techniques: Western Blot, Recombinant, Incubation, Migration, SDS Page, Kinase Assay, In Vitro, Activity Assay, Methylation, Dot Blot, Two Tailed Test, Transfection

Journal: Nature Communications

Article Title: The ULK3 kinase is a determinant of keratinocyte self-renewal and tumorigenesis targeting the arginine methylome

doi: 10.1038/s41467-023-36410-6

Figure Lengend Snippet: a Immunoblots against the indicated proteins of SCC13 cells with ULK3 silencing by two shRNAs (shULK3 #1, #2) for 1 week or with two siRNAs (siULK3#1, 2) for 48 h, with corresponding controls. b Immunoblots of nuclear (N) and cytosolic (C) fractions from SCC13 cells with ULK3 silencing by two shRNA-specific versus control lentiviruses. The blots were sequentially probed with the indicated antibodies. c PRMT1, PRMT5 and ULK3 immunostaining (green) of SCC13 cells plus/minus ULK3 silencing with two lentiviruses versus control virus. Phalloidin labeled actin cytoskeleton (magenta). Scale bar 5 µm. d ChIP with anti -PRMT1 and -PRMT5 antibodies from SCC13 cells plus/minus ULK3 silencing followed by qPCR amplification of four regions of the PKM and LDHA genes. The position of amplified regions (magenta lines) and TSS (arrow) are shown. Results are expressed as enrichment fold relative to IgGs as in . e ChIPs with anti-histone 3 (H3) antibodies followed by PRMT1, PRMT5 and H3 immunoblotting from SCC13 cells plus/minus ULK3 silencing. For H3, the immunoblots of inputs and immunoprecipitations are shown at different exposure times. f Asymmetrically (H4R3DA) and symmetrically (H4R3DS) dimethylated arginine 3 of histone 4 immunoblotting in SCC cells plus/minus ULK3 silencing for one week, with anti-histone H4 as a loading control. An additional H4R3DS and H4R3DA immunoblot upon ULK3 silencing is in Supplementary Fig. . g H4R3DA (green) immunofluorescence of two SCC cell lines plus/minus ULK3 silencing, with DAPI (blue) as nuclear staining. Representative images and quantification of individual cells fluorescence signal, Cal27: n (cell/condition) = 35; SCC13: n (cells/condition) = 30, mean ± SD, P < 0.0001, one-way ANOVA. Scale bar 5 µm. An additional experiment is in Supplementary Fig. . Supplementary Fig. show immunoblot and immunostaining with anti -H4R3DS, -H4R3DA, and H4 antibodies of HeLa cells plus/minus ULK3 silencing. h H4R3DA, H4R3DS, total H4 and ULK3 immunoblotting of SCC13 cells with CRISPR/Cas9-mediated ULK3 gene deletion with three guide RNA (gRNA) versus control (SCR), as in Fig. . A similar analysis of HEK293T cells plus/minus CRISPR/Cas9-mediated ULK3 gene deletion is in Supplementary Fig. .

Article Snippet: Slides were processed with routine histology procedures as in refs. , using rabbit anti ULK3 ATLAS antibody (AB_2677003, 1:100 dilution).

Techniques: Western Blot, shRNA, Control, Immunostaining, Virus, Labeling, Amplification, Immunofluorescence, Staining, Fluorescence, CRISPR

Journal: Nature Communications

Article Title: The ULK3 kinase is a determinant of keratinocyte self-renewal and tumorigenesis targeting the arginine methylome

doi: 10.1038/s41467-023-36410-6

Figure Lengend Snippet: a, b Ear injection skin SCC model. SCC13 ( a ) and SCCO28 ( b ) cells expressing an enhanced green fluorescence protein (EGFP) were infected with two ULK3 -silencing or control lentiviruses. Mice (NOD/SCID-Prkdc scid ; CB-17) received contralateral ear injections of either ULK3- or control - silenced cells. Representative fluorescence with phase-contrast image superimposition of two ear pairs three weeks after injection and quantification of all tumor volumes (V = (length × width 2 ) × 0.5)) for SCC13 cells ( a ). For SCCO28 cells ( b ) the quantification of all tumor volumes was as (integrated density × area of GFP fluorescence, normalized to Day 1). n (mice/SCC cell line) = 20, mean ± SEM, P < 0.01, two-tailed unpaired t -test. Green histograms show the number of tumors obtained with each lentivirus after three weeks. c-g Intradermal backskin injection SCC model. Cal27 and SCC13 cells plus/minus ULK3 -silencing were injected (8/condition) with Matrigel in contralateral back sites of mice (NOD/SCID IL2Rγ −/− ). c Tumor volumes were measured as in (a). Mean ± SEM, SCC13 n (tumors/shCTR) = 7, n (tumors/shULK3) = 7; Cal27 n (tumors/shCTR) = 8, n (tumors/shULK3) = 5, P < 0.001, two-tailed unpaired t-test. d Quantification of Ki67 immunofluorescence of tumors formed by SCC cells plus/minus ULK3 silencing. Anti-pankeratin (panKRT) antibodies identified injected cells and DAPI stained the nuclei. Mean ± SEM, three fields per tumor, n (tumors/condition) = 4, P < 0.001, two-tailed unpaired t-test. Scale bar 30 µm. e Keratin 10 (KRT10, green) and panKRT (magenta) immunofluorescence of the SCC tumors as above. Representative images and quantification of fluorescence signal (area (%)/field), mean ± SEM, three fields per tumor, SCC13: n (tumors/condition) = 5, Cal27: n (tumors/condition) = 3, P < 0.001, two-tailed unpaired t-test. Scale bar 30 µm. f TP63 (magenta) and panKRT (green) immunofluorescence of tumors formed by SCC13 cells plus/minus ULK3 silencing. Representative images and quantification of fluorescence signal (% TP63 + panKRT positive cells/section), mean ± SEM, four fields per tumor, n (tumors/condition) = 3, P < 0.001, two-tailed unpaired t-test. Scale bar 30 µm. g H4R3DA (green) and panKRT (magenta) immunofluorescence, with DAPI nuclear staining, of SCC13 cell tumors plus/minus ULK3 silencing. Representative images and quantification of single cell nuclear fluorescent signal, shCTR: n (cells/ tumor #1) = 66, n (cells/ tumor #2) = 72; shULK3#1: n (cells/ tumor #1) = 72, n (cells/ tumor #2) = 58, n (cells/ tumor #3) = 66; shULK3#2: n (cells/ tumor)#1 = 68, n (cells/ tumor #2) = 58, n (cells/ tumor #3) = 67, mean ± SEM, P < 0.001, one-way ANOVA. Scale bar 5 µm.

Article Snippet: Slides were processed with routine histology procedures as in refs. , using rabbit anti ULK3 ATLAS antibody (AB_2677003, 1:100 dilution).

Techniques: Injection, Expressing, Fluorescence, Infection, Control, Two Tailed Test, Immunofluorescence, Staining

Journal: Nature Communications

Article Title: The ULK3 kinase is a determinant of keratinocyte self-renewal and tumorigenesis targeting the arginine methylome

doi: 10.1038/s41467-023-36410-6

Figure Lengend Snippet: a RT-qPCR analysis of ULK family members ( ULK1-4 ) in SCC13 cells 72 h after transfection with three locked antisense nucleic acids (LNAs) targeting ULK3 (#1-3) or scrambled control (SCR). Mean ± SEM, n (experiments) = 3, P < 0.001, P < 0.0001, two-tailed unpaired t-test. b ULK3 and ß-ACTIN immunoblots of SCC13 cells treated as in ( a ). c Colony forming assays of SCC cell lines transfected as in (a) and plated at low-density for one week, n (dishes) = 3, mean ± SEM, P < 0.0001, one-way ANOVA. d Sphere-forming capability of SCC13 cells transfected with ULK3-targeting LNAs (#2, #3) or control (SCR), grown one week on Matrigel. Representative phase-contrast images and quantification of sphere number, n (dishes) = 4, mean ± SEM, P < 0.0001, one-way ANOVA. e Cell cycle distribution of SCC13 cells expressing f luorescence u biquitin c ell c ycle i ndicators (FUCCI) after transfection with ULK3-targeting LNAs (#2, #3) or scrambled control (SCR). Day 3 representative images and quantification of G1/S (magenta) versus G2/M (green) reporter ratio over time (days). Mean ± SEM, n (dishes) = 3. Day 1: n (cells SCR) = 1405: n (cells LNA#2) = 1099, n (cells LNA#3) = 1551; Day 2: n (cells SCR) = 2205, n (cells LNA#2) = 2667, n (cells LNA#3) = 1266; Day3: n (cells SCR) = 1215, n (cells LNA#2) = 940, n (cells LNA#3) = 1726, P < 0.0001, one-way ANOVA. Scale bar 5 µm. f Percentage of cells treated as in ( e ) with nuclear damages (cytosolic DNA) or multinucleated, as detected by DAPI staining. Mean ± SEM, n (dishes/condition) = 3, n (cells SCR) = 860, n (cells LNA#2) = 906, n (cells LNA#3) = 911, ns: not significant, one-way ANOVA. g SCC13 cells 24 h after transfection with ULK3-targeting LNAs (#2, #3) or scrambled control were injected with Matrigel intradermally into the back of NOD/SCID (IL2Rγ -/- ) mice for 10 days. Tumor size was calculated as (V = (length × width 2 ) × 0.5)). Mean ± SEM, n (tumors/SCR) = 5, n (tumors/LNA) = 6, P < 0.001, and P < 0.0001, two-tailed unpaired t -test. h Ki67 and panKRT (to identify SCC cells) immunofluorescence of tumors described in ( g ), and DAPI staining. Percentage of Ki67 positive SCC13 cells, 4 fields/tumor, n (tumors/SCR) = 2, n (tumors/LNA) = 3, mean ± SEM, P < 0.01, P < 0.001, one-way ANOVA. i TP63 (magenta) and KRT10 (green) immunostaining of tumors described in ( g ). Low and high magnification representative images and quantification of TP63-positive cells and KRT10 area (%), normalized to the number of cells, 4 fields/tumor, n (tumors/SCR) = 2, n (tumors/LNA) = 3, mean ± SEM, P < 0.0001, one-way ANOVA. Scale bar 30 µm.

Article Snippet: Slides were processed with routine histology procedures as in refs. , using rabbit anti ULK3 ATLAS antibody (AB_2677003, 1:100 dilution).

Techniques: Quantitative RT-PCR, Transfection, Control, Two Tailed Test, Western Blot, Expressing, Staining, Injection, Immunofluorescence, Immunostaining

Journal: The Journal of Biological Chemistry

Article Title: Signal Transducer and Activator of Transcription 5b (Stat5b) Serine 193 Is a Novel Cytokine-induced Phospho-regulatory Site That Is Constitutively Activated in Primary Hematopoietic Malignancies *

doi: 10.1074/jbc.M111.319756

Figure Lengend Snippet: Identification of Ser-193 as a novel proline-flanked phosphorylation site in human Stat5b. YT cells were stimulated without (−) or with (+) IL-2 for 15 min, and Stat5b proteins were immunoprecipitated (IP) from soluble cell lysates with α-Stat5b antibodies. Two sets of immunoprecipitations were separated by SDS-PAGE. One set was Coomassie Blue-stained (A), and the other was Western blotted (WB) (B) with α-phospho-Tyr (α-pY), α-phospho-Ser (pS726/731), or α-Stat5b antibodies. HC, heavy chain; LC, light chain. C, tandem mass spectra of a monophosphorylated peptide showing site localization of Ser-193, as indicated by asterisks. D, amino acid sequence alignment of the region surrounding Ser-193 (asterisk) from each human Stat protein using the ClustalW program (progressive alignment) (31). E, domain architecture of human Stat5 with known and newly identified (asterisk) serine and tyrosine phosphorylation sites. Numbers indicate amino acid residues of human Stat5 (a/b).

Article Snippet: Human Stat5b cDNA (OriGene) was PCR-amplified and subcloned into the pCMV-Tag2 (Stratagene).

Techniques: Immunoprecipitation, SDS Page, Staining, Western Blot, Sequencing

Journal: The Journal of Biological Chemistry

Article Title: Signal Transducer and Activator of Transcription 5b (Stat5b) Serine 193 Is a Novel Cytokine-induced Phospho-regulatory Site That Is Constitutively Activated in Primary Hematopoietic Malignancies *

doi: 10.1074/jbc.M111.319756

Figure Lengend Snippet: Phosphorylation of Stat5b Ser-193 displays rapid kinetics and is inducible by multiple cytokines. A, a phospho-specific polyclonal Stat5b Ser(P)-193 antibody was generated and tested by dot blot analysis using increasing amounts of Stat5b Ser-193 (CLAQLSPQERL), Ser(P)-193 (CLAQL(pS)PQERL), Ser-731 (KDQAPSPAVCP), and Ser(P)-731 (KDQAP(pS)PAVCP) peptides spotted onto PVDF membrane (where pS indicates phospho-serine). IB, immunoblotting. B, YT cells stimulated with IL-2 for 15 min were fluorescently labeled using α-phospho-Tyr (α-pY) Stat5 (Cy2, green), α-Ser(P)-193 (α-pS193) Stat5b (Cy3, red), and DAPI (blue) and visualized by confocal microscopy. The overlay (bottom panel) shows co-localization of phospho-Tyr Stat5 and Ser(P)-193 Stat5b. For peptide competition analysis, the polyclonal Ser(P)-193 Stat5b antibody was preblocked with increasing amounts of non-phospho-peptide (lanes a–c) or phospho-peptide (lanes d–f) for 1 h at 4 °C followed by staining of YT cells treated with IL-2 for 15 min. C, YT cells were stimulated without (−) (lane a) or with IL-2 (+) from 0 to 60 min (lanes b–e) and fixed with cold methanol. Cells were then stained with α-phospho-Tyr Stat5 (Cy2, green), α-Ser(P)-193 Stat5b (Cy3, red), and DAPI (blue) and visualized by confocal microscopy. The overlay (bottom panel) shows co-localization of phospho-Tyr Stat5 and Ser(P)-193 Stat5b. Insets show a higher magnification view of α-Ser(P)-193 Stat5b (Cy3, red)-stained YT cells treated with IL-2 for the indicated time points. D, quiescent PHA-activated human PBMCs were stimulated with medium (−) (lane a) or IL-2 (lane b), IL-7 (lane c), IL-9 (lane d), or IL-15 (lane e) for 15 min and fixed with cold methanol. Confocal images using α-phospho-Tyr Stat5 (Cy2, green), α-Ser(P)-193 Stat5b (Cy3, red), and DAPI (blue) and overlay images are shown. Immunofluorescent images were captured using PASCAL software on a Zeiss LSM 510 Meta confocal microscope at 63× magnification. Representative data from three independent experiments are shown.

Article Snippet: Human Stat5b cDNA (OriGene) was PCR-amplified and subcloned into the pCMV-Tag2 (Stratagene).

Techniques: Generated, Dot Blot, Western Blot, Labeling, Confocal Microscopy, Staining, Software, Microscopy

Journal: The Journal of Biological Chemistry

Article Title: Signal Transducer and Activator of Transcription 5b (Stat5b) Serine 193 Is a Novel Cytokine-induced Phospho-regulatory Site That Is Constitutively Activated in Primary Hematopoietic Malignancies *

doi: 10.1074/jbc.M111.319756

Figure Lengend Snippet: Stat5b Ser-193 undergoes phosphorylation in an mTOR-dependent manner. A, YT cells were left untreated (lanes a and b) or pretreated with increasing concentrations of rapamycin (1–100 nm) (lanes c–f) for 1 h followed by stimulation with IL-2 (lanes b–f) for 15 min. α-pY, α-phospho-Tyr; α-pS193, α-Ser(P)-193. B, YT cells were left untreated (lanes a and b) or pretreated with 10–200 nm PP242 hydrate for 1 h followed by stimulation with IL-2 for 15 min (lanes b–f) as indicated. The cells were fixed and stained with α-phospho-Tyr Stat5 (Cy2, green), α-Ser(P)-193 Stat5b (Cy3, red), and DAPI (blue) and visualized by confocal microscopy. The overlay (bottom panel) shows co-localization of phospho-Tyr Stat5 and Ser(P)-193 Stat5b. Immunofluorescent images were captured using PASCAL software on a Zeiss LSM 510 Meta confocal microscope at 63× magnification. Representative data from three independent experiments are shown.

Article Snippet: Human Stat5b cDNA (OriGene) was PCR-amplified and subcloned into the pCMV-Tag2 (Stratagene).

Techniques: Staining, Confocal Microscopy, Software, Microscopy

Journal: The Journal of Biological Chemistry

Article Title: Signal Transducer and Activator of Transcription 5b (Stat5b) Serine 193 Is a Novel Cytokine-induced Phospho-regulatory Site That Is Constitutively Activated in Primary Hematopoietic Malignancies *

doi: 10.1074/jbc.M111.319756

Figure Lengend Snippet: PP2A, but not PP1, negatively regulates Stat5b Ser-193 phosphorylation in YT cells. A, cells were pretreated without (lanes a and b) or with DMSO (lanes c and d) or 50 nm CA (lanes e and f) for 1 h before stimulation without (lanes a, c, and e) or with IL-2 (lanes b, d, and f) for 15 min at 37 °C. The cells were fixed and stained with α-phospho-Tyr Stat5 (Cy2, green), α-Ser(P)-193 (α-pS193) Stat5b (Cy3, red), and DAPI (blue) and visualized by confocal microscopy. The overlay (bottom panel) shows co-localization of phospho-Tyr Stat5 and Ser(P)-193 Stat5b. NT, not treated. B, YT cells were left untreated (lanes a and b) or pretreated with 150 nm OA (lanes c and d), 25 nm FOS (lanes e and f), or 1 μm TAU (lanes g and h) for 1 h before stimulation without (−) or with (+) IL-2 for 15 min at 37 °C. The cells were fixed and analyzed using α-Ser(P)-193 Stat5b and DAPI as described above. Immunofluorescent images were captured using PASCAL software on a Zeiss LSM 510 Meta confocal microscope at 63× magnification. Representative data from three independent experiments are shown.

Article Snippet: Human Stat5b cDNA (OriGene) was PCR-amplified and subcloned into the pCMV-Tag2 (Stratagene).

Techniques: Staining, Confocal Microscopy, Software, Microscopy

Journal: The Journal of Biological Chemistry

Article Title: Signal Transducer and Activator of Transcription 5b (Stat5b) Serine 193 Is a Novel Cytokine-induced Phospho-regulatory Site That Is Constitutively Activated in Primary Hematopoietic Malignancies *

doi: 10.1074/jbc.M111.319756

Figure Lengend Snippet: Stat5b Ser-193 phosphorylation is required for its maximal DNA binding and transcriptional activity. A, HEK293 cells were transfected with IL-2Rβ, IL-2Rγ, Jak3, and Stat5b (WT, S193A, or S193E) and incubated for 48 h. The cells were then stimulated without (−) or with IL-2 (+) for 15 min. Stat5b WT (lanes a and b), Stat5b S193A (lanes c and d), and Stat5b S193E (lanes e and f) were blotted with α-phospho-Tyr (α-pY) Stat5 (upper panel) or total Stat5 (lower panel). WB, Western blot. B, nuclear extracts (5 μg) isolated from transfected HEK293 described in A were incubated with a 32P-radiolabeled oligonucleotide probe corresponding to the Stat5 binding site in the β-casein gene promoter. The extracts indicated were co-incubated with N-terminal directed α-Stat5 (lane h) or normal rabbit IgG (Cntrl) (lane i). The bracket indicates the location of free probe, and the arrows indicate the location of non-supershifted and supershifted Stat5b-DNA complexes. Representative data from two independent experiments are shown. C, HEK293 cells transfected as described in A were treated without (−) or with (+) IL-2 for 6 h. Control cells were transfected with Stat5b alone (lanes a and b). At 48 h after transfection, the cells were lysed, and luciferase activities were measured and normalized to β-galactosidase activity. Statistical significance was determined using analysis of variance (*, p < 0.05). Representative data from three independent experiments are shown. Error bars indicate S.D.

Article Snippet: Human Stat5b cDNA (OriGene) was PCR-amplified and subcloned into the pCMV-Tag2 (Stratagene).

Techniques: Binding Assay, Activity Assay, Transfection, Incubation, Western Blot, Isolation, Luciferase

Journal: The Journal of Biological Chemistry

Article Title: Signal Transducer and Activator of Transcription 5b (Stat5b) Serine 193 Is a Novel Cytokine-induced Phospho-regulatory Site That Is Constitutively Activated in Primary Hematopoietic Malignancies *

doi: 10.1074/jbc.M111.319756

Figure Lengend Snippet: Stat5b Ser-193 is constitutively phosphorylated in HTLV-1-transformed tumor T-cell lines and primary leukemia and lymphoma patient tumor cells. A, immunofluorescent confocal microscopy was utilized to detect phospho-Tyr Stat5 (α-pY Stat5) and Ser(P)-193 Stat5b (α-pS193 Stat5b) in normal PHA-activated quiescent human PBMCs stimulated with IL-2 or without for 15 min (lanes a and b), non-PHA-activated PBMCs cells (lane c), MT-2 (lane d), HUT-102 (lane e), and HUT-78 (lane f). B, immunofluorescent confocal microscopy was utilized to detect phospho-Tyr Stat5 and Ser(P)-193 Stat5b in primary tumor cells isolated from patients diagnosed with ALL (lane d), AML (lane e), and B-cell lymphoma (lane f). Normal PHA-activated quiescent human PBMCs stimulated with IL-2 or without IL-2 for 15 min (lanes a and b) and non-PHA-activated PBMCs cells (lane c) served as controls. Immunofluorescent images were captured using PASCAL software on a Zeiss LSM 510 Meta confocal microscope at 63× magnification. Representative data from three independent experiments are shown.

Article Snippet: Human Stat5b cDNA (OriGene) was PCR-amplified and subcloned into the pCMV-Tag2 (Stratagene).

Techniques: Transformation Assay, Confocal Microscopy, Isolation, Software, Microscopy

Journal: Communications Chemistry

Article Title: A Hybrid compound H93 treats prostate cancer by directly binding UHRF1 and promoting protein dimerization

doi: 10.1038/s42004-025-01744-3

Figure Lengend Snippet: a DU145 (Left) or VCaP (Right) cells were treated with indicated concentrations of H93 for 48 h, and the total DNA methylation level was measured by 5-methylcytosine (5mC) DNA dot-blot assays. Experiments were performed in triplicate. b DU145 (Left) or VCaP (Right) cells were treated with 0 (black dots), 10 (black squares) or 20 μM (black triangles) of H93 for 48 h, and the total DNA methylation level was measured by 5mC ELISA assays. The data are represented as the mean ± SD, n = 3 independent experiments. c Quantitated gene expression changes of representative tumor suppressor genes in DU145 (Upper) and VCaP (Lower) cells treated with 0 (empty black circles), 2.5 (black squares), 5 (black triangles), 10 (black rhombus) or 20 μM (black dots) of H93. n = 3 independent experiments. d–f DU145 subcutaneous xenografts were induced in immune-deficient nude mice, and then treated with vehicle (20 ml kg −1 day -1 ) or H93 (50 or 100 mg kg −1 day −1 ) by oral administration. n = 8 per group. The tumor nodes were harvested 28 days after treatment. d The tumor volumes (Left) and weights (Right) were compared with vehicle 20 ml kg −1 day −1 group depicted as black dots, H93 50 mg kg −1 day −1 group as black squares, H93 100 mg kg −1 day −1 group as black triangles. n = 8 animals. e The body weights of nude mice were continuously monitored and vehicle 20 ml kg −1 day −1 group was depicted as black line, H93 50 mg kg −1 day −1 group as red line, H93 100 mg kg −1 day −1 group as glaucous line. n = 8 animals. f Representative images of Hematoxylin-Eosin (HE), Ki67 immunochemistry (IHC) and transferase-mediated deoxyuridine triphosphate-biotin nick end labeling (TUNEL) staining in DU145 xenografts subjected to different concentrations of H93 (Left), and the quantitative data of Ki67 (Middle) and TUNEL (Right) staining, with vehicle 20 ml kg −1 day −1 group depicted as black dots, H93 50 mg kg −1 day −1 group as black squares, H93 100 mg kg −1 day −1 group as black triangles, and n = 40 views. The length of scale bars is 200 μm (×100) or 50 μm (×400). * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001. The data are shown as mean ± SD.

Article Snippet: Colorimetric TUNEL Apoptosis Assay Kit (Cat#C1098) was purchased from Beyotime Biotech.

Techniques: DNA Methylation Assay, Dot Blot, Enzyme-linked Immunosorbent Assay, Gene Expression, End Labeling, TUNEL Assay, Staining

Journal: bioRxiv

Article Title: RNA methylation influences TDP43 binding and disease pathogenesis in models of amyotrophic lateral sclerosis and frontotemporal dementia

doi: 10.1101/2022.04.03.486880

Figure Lengend Snippet: Density of UG nucleotide sequences 100bp upstream and downstream of m6A modifications identified by cross-linking induced mutation sites (CIMS; A ) or cross-linking induced truncation sites (CITS; B ) in relation to random sequences (red line). Grey shading represents 95% confidence regions. ( C ) Schematic of HaloTag immunoprecipitation and dot blot procedure. ( D ) Dot blot for total RNA (detected by methylene blue) or m6A-modified RNA (detected by anti-m6A antibody) isolated by immunoaffinity purification of HaloTag-labeled proteins in HEK293T cells overexpressing HaloTag, TDP43-HaloTag or YTHDF2-HaloTag from 3 biological replicates. ( E ) Diagram illustrating insertion of the HaloTag open reading frame into the endogenous TARDBP locus immediately 5’ to the TDP43 start codon, resulting in a fusion of HaloTag to the N-terminus of TDP43. ( F ) Halo-TDP43 HEK293T cells labeled live with JF646 Halo dye (red), then fixed, permeabilized, and immunostained with anti-TDP43 antibody (green) prior to imaging. DAPI (blue) marks the nucleus of each cell. Scale bar = 10µm. ( G ) Dot blot for total RNA (detected by methylene blue) or m6A-modified RNA (detected by anti-m6A antibody) isolated by immunoaffinity purification of endogenous HaloTag-TDP43 or exogenous HaloTag. Additional replicates shown in Sup. Fig. 1.

Article Snippet: Coverslips were then incubated overnight with blocking buffer + rabbit anti-TDP43 antibody (Proteintech, #10782-2-AP) at 1:500 to stain for TDP43.

Techniques: Mutagenesis, Immunoprecipitation, Dot Blot, Modification, Isolation, Immunoaffinity Purification, Labeling, Imaging

Journal: bioRxiv

Article Title: RNA methylation influences TDP43 binding and disease pathogenesis in models of amyotrophic lateral sclerosis and frontotemporal dementia

doi: 10.1101/2022.04.03.486880

Figure Lengend Snippet: ( A ) HaloTag-TDP43 immunoprecipitation was followed by DART-seq to delineate m6A sites within TDP43 target RNAs. HaloTag-TDP43 HEK293T cells were transfected with APOBEC1-YTH or APOBEC1-YTHmut and crosslinked before immunoaffinity purification of HaloTag-labeled proteins. Immunoprecipitated RNAs were then sequenced and C-T transitions were identified in the context of DRACH motifs (red shaded box, D=A/G/T, R=A/G, H=A/C/T). Absolute counts ( B ) and relative frequency ( C ) of base pair transitions observed by RNA-seq in each condition. Shaded boxes represent transition types expected from APOBEC1 activity. ( D ) Example m6A sites identified by DART-seq in RPL10A . C-T transitions are highlighted in red, and DRACH motifs in pink. Green arrow, transcription start site; red hexagon, transcription stop site; thick blue bars, coding exons; thin blue bars, untranslated region. ( E ) Absolute count and relative distribution ( F ) of DART-seq reads in cells expressing APOBEC1-YTH and APOBEC1-YTHmut. UTR, untranslated region; CDS, coding sequence. ( G ) Scatter plot of TDP43 targets, determined by fold enrichment in precipitated RNA from HaloTag-TDP43 cells (expressing APOBEC1-YTH and APOBEC10YTHmut) compared to cells transfected with HaloTag. Red dots signify transcripts showing > 2-fold enrichment in both APOBEC1-YTH and APOBEC1-YTHmut expressing cells. TARDBP , yellow dot, identified as high confidence target. ( H ) Stacked bar graph showing percentage of m6A modified RNA in TDP43 targets (red) and non-targets (black). ( I ) Cumulative distribution of RNA methylation in TDP43 targets (red) and non-targets (black). p = 1.87×10 −55 by Kolmogorov Smirnov test. ( J ) Euler diagram depicting overlap between TDP43 targets identified in this study, and those identified by TDP43 cross linking and immunoprecipitation followed by RNA-sequencing (CLIP-seq) in HEK293T cells (Hallegger et al ., 2021) . **p=1.5×10 −117 , hypergeometric test. ( K ) Pie charts demonstrating the percentage of methylated RNA among TDP43 targets (pink) and non-targets (grey). **p<1×10 −5 chi-square test.

Article Snippet: Coverslips were then incubated overnight with blocking buffer + rabbit anti-TDP43 antibody (Proteintech, #10782-2-AP) at 1:500 to stain for TDP43.

Techniques: Immunoprecipitation, Transfection, Immunoaffinity Purification, Labeling, RNA Sequencing, Activity Assay, Expressing, Sequencing, Modification, Methylation

Journal: bioRxiv

Article Title: RNA methylation influences TDP43 binding and disease pathogenesis in models of amyotrophic lateral sclerosis and frontotemporal dementia

doi: 10.1101/2022.04.03.486880

Figure Lengend Snippet: ( A ) TARDBP gene map, illustrating TDP43 binding region (TBR), the location of the DRACH motif (pink square), and the C-T transition (red box) identified by DART-seq within this domain, representing an m6A site. ( B ) Schematic of the TARDBP minigene reporter, consisting of the mCherry ORF upstream of TARDBP exon 6 and 3.4 Kb of the TARDBP 3’ UTR. The A residue adjacent to the detected C-T transition via DART-seq in the WT reporter (mCherry-TBR) was mutated to a G, precluding methylation the mutant reporter (mCherry-mTBR). Red, methylated residue; blue line, DRACH motif; dagger, C-T transition from DART-seq. ( C ) HaloTag-TDP43 was isolated by immunoaffinity purification from HaloTag-TDP43 HEK293T cells expressing mCherry-TBR or mCherry-mTBR, and reporter RNA detected in elution fractions by qRT-PCR. ( D ) Outline of TDP43 autoregulation assay. Excess TDP43 binds to the reporter, triggering reporter splicing, destabilization, and reduced mCherry fluorescence. ( E ) Primary rodent neurons were transfected with WT (mCherry-TBR) or mutant (mCherry-mTBR) reporters, together with EGFP or TDP43-EGFP. After 7d, mCherry expression was assessed by fluorescence microscopy. Scale bar= 20 µm. Normalized RFP (mCherry) intensity in primary neurons expressing WT mCherry-TBR reporter ( F ) or mutant mCherry-mTBR ( G ) reporter together with EGFP or TDP43(WT)-EGFP. Cherry-TBR+GFP n= 160, Cherry-TBR+TDP43(WT)-GFP n= 58, Cherry-mTBR+GFP n= 105, Cherry-mTBR+TDP43(WT)-GFP n= 44. Data in C plotted as mean ± SD, collected from 3 biological replicates. ns= not significant, *p< 0.05, **p< 0.01; one-way ANOVA with Tukey’s test. Data in F and G plotted as mean ± SD, color coded by biological replicate. ns = not significant, *p < 0.05; Welch’s t-test.

Article Snippet: Coverslips were then incubated overnight with blocking buffer + rabbit anti-TDP43 antibody (Proteintech, #10782-2-AP) at 1:500 to stain for TDP43.

Techniques: Binding Assay, Residue, Methylation, Mutagenesis, Isolation, Immunoaffinity Purification, Expressing, Quantitative RT-PCR, Fluorescence, Transfection, Microscopy

Journal: bioRxiv

Article Title: RNA methylation influences TDP43 binding and disease pathogenesis in models of amyotrophic lateral sclerosis and frontotemporal dementia

doi: 10.1101/2022.04.03.486880

Figure Lengend Snippet: ( A ) Genome-wide analysis of RNA methylation via epitranscriptomic array. RNA was extracted from control (n= 3) and sporadic ALS (sALS) patient (n= 4) spinal cord samples, prior to m6A RNA immunoprecipitation. The resulting samples were separated into methylated and non-methylated RNA, then labeled with distinct fluorescent dyes (red and green stars) prior to hybridization, allowing relative quantification of methylation at each annotated locus. ( B ) Principal component analysis (PCA) plot comparing methylation levels of control (grey) and ALS (red) patient samples. ( C ) Hierarchical clustering of mRNA methylation profiles from control and ALS mRNA samples. ( D ) Volcano plot depicting fold change in mRNA methylation levels in ALS compared to control spinal cord. ( E ) Hierarchical clustering of lncRNA methylation profiles from control ALS lncRNA samples. ( F ) Volcano plot showing fold change in lncRNA methylation levels in ALS compared to control spinal cord. In D and F , grey horizontal vertical lines represent p= 0.05 and fold change (FC)= 2. ( G ) Euler diagram demonstrating overlap (n= 322, p= 5.09×10 −119 , hypergeometric test) among TDP43 substrates and methylated transcripts identified in HEK293T cells, in additional to hypermethylated transcripts determined via m6A array in sALS spinal cord. Comparisons were limited to the subset of transcripts expressed in both HEK293T cells and human spinal cord (nTPM>2). ( H ) Based on comparisons with the GEO transcription factor loss-of-function database via Enrichr , there was strong enrichment for TDP43-regulated genes not only among the set of 2034 transcripts hypermethylated in sALS spinal cord, but also among the 322 TDP43 targets that were also hypermethylated in sALS (A1 in G ). Combined score = (log 10 p * Z-score). ( I ) Immunohistochemical staining for m6A in control and sALS spinal cord sections. Scale bars= 50 µm. ( J ) Quantification of m6A antibody reactivity in spinal cord neurons from control (n= 110 neurons) and sALS (n= 277 neurons) sections. Plot shows mean +/- SD, color coded by patient. ****p< 0.0001 via Mann-Whitney test.

Article Snippet: Coverslips were then incubated overnight with blocking buffer + rabbit anti-TDP43 antibody (Proteintech, #10782-2-AP) at 1:500 to stain for TDP43.

Techniques: Genome Wide, Methylation, Control, RNA Immunoprecipitation, Labeling, Hybridization, Quantitative Proteomics, Immunohistochemical staining, Staining, MANN-WHITNEY

Journal: bioRxiv

Article Title: RNA methylation influences TDP43 binding and disease pathogenesis in models of amyotrophic lateral sclerosis and frontotemporal dementia

doi: 10.1101/2022.04.03.486880

Figure Lengend Snippet: ( A ) Representative images of rodent primary neurons transfected with plasmids expressing Cas9-2A-EGFP and sgRNA targeting the neuronal protein NeuN or negative control (LacZ). 5d after transfection, neurons were fixed and immunostained for NeuN (red). White dashed circles indicate nucleus stained with Hoechst (blue). ( B ) NeuN antibody reactivity measured in EGFP-positive neurons expressing sgLacZ (n= 565) or sgNeuN (n= 654), ****p < 0.0001 by Mann-Whitney. ( C ) Schematic depicting m6A writers (green), erasers (red), and readers (orange) targeted by CRISPR/Cas9. ( D ) Primary neurons expressing EGFP and TDP43-mApple were assessed at regular 24h intervals by fluorescence microscopy, and their survival assessed by automated image analysis. Individual neurons are assigned unique identifiers (yellow number) and tracked until their time of death (red), indicated by cellular dissolution, blebbing, or neurite retraction. Scale bar= 20µm. ( E ) Cumulative hazard plot depicting risk of death for neurons expressing TDP43(WT) + non-targeting (NT) (red line), mApple + NT (grey line), or TDP43(WT) + Atxn2 sgRNA (purple line). †p<2.0 ×10 −16 , Hazard ratio (HR)= 3.45; ***p= 5.81 ×10 −4 , HR= 0.80). ( F ) Forest plot showing HR for TDP43-overexpressing neurons upon knockdown of m6A writers (green), erasers (dark red), and readers (orange), in comparison to nontargeting (NT) control. Dashed line indicates HR= 1, representing the survival of the reference condition, neurons expressing TDP43-mApple and NT sgRNA. Values >1 indicate increased toxicity, whereas values <1 denote relative protection. Error bars represent 95% CI. ( G ) Alkbh5 knockout significantly increases TDP43 associated toxicity. †p=3.11 ×10 −5 , HR= 1.59; ***p= 2.65×10 −11 , HR= 2.03. ( H ) Ythdf2 knockout significantly extends survival in TDP43-expressing neurons. ***p <2.0 ×10 −16 , HR= 1.69; †p= 6.2 ×10 −6 , HR= 0.71. ( I ) YTHDF2 overexpression is toxic to neurons. ***p= 3.07×10 −5 , HR= 1.30. ( J ) METTL3/14 overexpression enhances TDP43-dependent toxicity in neurons. †p = 5.53 ×10 −4 , HR= 1.32; ***p =4.16 ×10 −6 , HR= 1.31. p values in E, G-J determined via Cox proportional hazards analysis, with a minimum 3 of biological replicates.

Article Snippet: Coverslips were then incubated overnight with blocking buffer + rabbit anti-TDP43 antibody (Proteintech, #10782-2-AP) at 1:500 to stain for TDP43.

Techniques: Transfection, Expressing, Negative Control, Staining, MANN-WHITNEY, CRISPR, Fluorescence, Microscopy, Dissolution, Knockdown, Comparison, Control, Knock-Out, Over Expression

Journal: bioRxiv

Article Title: RNA methylation influences TDP43 binding and disease pathogenesis in models of amyotrophic lateral sclerosis and frontotemporal dementia

doi: 10.1101/2022.04.03.486880

Figure Lengend Snippet: ( A ) Immunostaining of YTHDF2 in control and sALS patient spinal cord samples. Scale bar= 50 µm. ( B ) Quantification of YTHDF2 immunoreactivity in spinal cord neurons from control (n= 117 neurons) and sALS (n= 193 neurons) samples. Plot shows mean +/- SD, color coded by sample. ****p< 0.0001 via Mann-Whitney test. ( C ) Strategy used to create isogenic iPSCs expressing native TDP43(WT)-Dendra2 or TDP43(M337V)-Dendra2. ( D ) Representative images of untransduced (grey) and transduced (green) iNeurons expressing shRNA against YTHDF2 (shYTHDF2) and a GFP reporter. Time of death (red circles) for each cell is used to determine cumulative risk of death, plotted in ( E ) and ( F ). Scale bar= 20µm. shRNA-mediated knockdown of YTHDF2 significantly extended the survival of TDP43(M337V)-Dendra2 iNeurons ( E ; †p= 8.42×10 −12 , HR= 6.25; ***p= 4.82×10 −9 , HR=0.32; #p= 0.08, HR= 1.84) as well as mutant C9ORF72 iNeurons ( F , †p= 1.42×10 −11 , HR= 2.85; ***p= 1.42×10 −16 , HR= 0.32). ns= not significant. Values in ( E , F ) calculated by Cox proportional hazards analysis, with a minimum 3 biological replicates.

Article Snippet: Coverslips were then incubated overnight with blocking buffer + rabbit anti-TDP43 antibody (Proteintech, #10782-2-AP) at 1:500 to stain for TDP43.

Techniques: Immunostaining, Control, MANN-WHITNEY, Expressing, shRNA, Knockdown, Mutagenesis

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Identification and characterization of topoisomerase III beta poisons.

doi: 10.1073/pnas.2218483120

Figure Lengend Snippet: Fig. 1. Comparative Cellular Cytotoxicity Screen to identify TOP3B poisons. (A) A pair of isogenic HCT116 and HCT116-TOP3B-KO cells were constructed to express GFP and mCherry, respectively. Both cell lines were mixed at 1:1 ratio, seeded together, and allowed to attach for 48 h prior to treatment with drugs at indicated concentrations for an additional 72 h. On each cell culture plate, one column of dimethyl sulfoxide (DMSO)-treated samples served as control. At the end of drug treatment, the entire cell culture plate was imaged in multiple fluorescent channels to calculate the relative viability rates of the wild-type and TOP3B-KO cells, normalized to the DMSO-treated control wells on the same plate. The 4 potential outcomes are represented from left to right in the middle of the figure: 1) If TOP3B-KO cells show selective resistance, the compound potentially targets TOP3B; 2) if a compound is selectively toxic to TOP3B-KO cells, the compound potentially interferes with parallel pathways of TOP3B, so that in KO cells, inhibiting the parallel pathway leads to adverse effects; 3) if a compound shows no effect (same results as DMSO-treated samples), it does not interfere with any pathways related to TOP3B and is discarded; and 4) if a compound shows high toxicity to both HCT116 and TOP3B-KO cells, it is retested at lower concentrations. (B) Representative fluorescent microscopy images of a DMSO control and drug-treated samples are shown. After deconvoluting into individual fluorescent channels, the drug-treated sample was compared to the DMSO control sample in each fluorescent channel. The GFP channel conveys the effect of the drug on the HCT116 cells while the mCherry channel conveys the effect of the drug on TOP3B-KO cells. Each sample is normalized to the DMSO-treated control samples on the same plate.

Article Snippet: Human TOP3B- Myc- Flag cDNA open reading frame (ORF) was purchased from OriGene (RC223204) and transfected in HEK293 cells using Lipofectamine 3000 Reagent (ThermoFisher Scientific) according to the manufacturer’s protocol for 48 h before drug treatments.

Techniques: Construct, Cell Culture, Control, Microscopy

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Identification and characterization of topoisomerase III beta poisons.

doi: 10.1073/pnas.2218483120

Figure Lengend Snippet: Fig. 3. Modified RADAR assay to demonstrate drug-induced cellular TOP3Bccs. (A) Schematic procedures of the modified RADAR assay. (B) Chemical structures of six hit compounds selected from the screen using modified RADAR assays. (C) HEK293 cells transiently transfected with Flag-tagged TOP3B (TOP3B OE) were treated with the indicated compounds at 100 µM for 1 h prior to analysis by modified RADAR. Anti-Flag antibody was used to probe the level of TOP3Bccs. The same samples probed with anti-dsDNA antibodies served as loading controls. The six compounds showed a spectrum of potency in inducing TOP3Bccs.

Article Snippet: Human TOP3B- Myc- Flag cDNA open reading frame (ORF) was purchased from OriGene (RC223204) and transfected in HEK293 cells using Lipofectamine 3000 Reagent (ThermoFisher Scientific) according to the manufacturer’s protocol for 48 h before drug treatments.

Techniques: Modification, Transfection

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Identification and characterization of topoisomerase III beta poisons.

doi: 10.1073/pnas.2218483120

Figure Lengend Snippet: Fig. 4. Band shift assays demonstrating the induction of DNA- and RNA-TOP3Bccs by NSC690634 and NSC96932 with recombinant TOP3B. (A) Simplified scheme of the band shift assay (11). A single-strand DNA or RNA oligo substrate (containing a short hairpin section) labeled on the 3′-end with a fluorophore is incubated with recombinant human TOP3B. A small population of the TOP3B forms TOP3Bccs with the DNA or RNA substrate. The resulting molecular weight of TOP3Bccs with the attached fluorophore is significantly higher than that of free DNA and RNA substrates, regardless of the positions of TOP3B binding sites. (B) The DNA-TOP3Bcc (Upper) and RNA-TOP3Bcc (Lower) are resolved from the free DNA and RNA on sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and directly visualized by the fluorophores attached to the oligo constructs. The formation of DNA- and RNA-TOP3Bccs (100 to 180 kDa) is TOP3B- dependent and the TOP3Bccs were titrated with each of the six lead compounds to reach equilibrium. The effect of each compound is compared to the DMSO- treated control samples.

Article Snippet: Human TOP3B- Myc- Flag cDNA open reading frame (ORF) was purchased from OriGene (RC223204) and transfected in HEK293 cells using Lipofectamine 3000 Reagent (ThermoFisher Scientific) according to the manufacturer’s protocol for 48 h before drug treatments.

Techniques: Electrophoretic Mobility Shift Assay, Recombinant, Labeling, Incubation, Molecular Weight, Binding Assay, Polyacrylamide Gel Electrophoresis, SDS Page, Construct, Control

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Identification and characterization of topoisomerase III beta poisons.

doi: 10.1073/pnas.2218483120

Figure Lengend Snippet: Fig. 5. NSC690634 and NSC96932 induce endogenous TOP3Bccs in different cell lines. (A) Representative modified RADAR assays showing that NSC690634 (100 µM) induces endogenous TOP3Bccs in a time-dependent manner in the indicated human cell lines, immunodetection with anti-TOP3B antibodies. (B) Modified RADAR assay showing that NSC690634 (4 h) induces endogenous TOP3Bccs in a dose-dependent manner in three human cell lines, immunodetection with anti-TOP3B antibodies. (C) Same as Fig. 5A, but with NSC96932. (D) Same as Fig. 5B, but with NSC96932 (1 h). (E) HEK293 cells transiently over-expressing TOP3B were treated with NSC690634 or NSC96932 (100 µM for 1 h) prior to analysis by in vivo complex of enzyme (ICE) bioassays. Ultracentrifugation of cell lysates on a CsCl gradient enabled separation of DNA and RNA species. The separated fractions were quantified and 1.5 µg of DNA or RNA were blotted on a slot blot for independent detection of DNA- and RNA-TOP3Bccs through immunodetection probed with anti-Flag antibodies. The upper row contained 1.5 µg of nontreated control DNA or RNA samples. Representative blot of 3 independent experiments is shown. (F) Genomic DNA of HCT116 and HCT116-TOP3B-KO cells with or without treatment with NSC690634 (100 µM, 4 h) was collected and blotted on nitrocellulose membrane before immunoprobing by S9.6 antibody (7). RNase H-treated samples serve as negative controls.

Article Snippet: Human TOP3B- Myc- Flag cDNA open reading frame (ORF) was purchased from OriGene (RC223204) and transfected in HEK293 cells using Lipofectamine 3000 Reagent (ThermoFisher Scientific) according to the manufacturer’s protocol for 48 h before drug treatments.

Techniques: Modification, Immunodetection, Expressing, In Vivo, Dot Blot, Control, Membrane

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Identification and characterization of topoisomerase III beta poisons.

doi: 10.1073/pnas.2218483120

Figure Lengend Snippet: Fig. 6. Structure–activity analysis of bisacridine analogs of NSC690634 demonstrates that linker length is critical for inducing TOP3Bccs. (A) Chemical structures of a series of bisacridines. The length of the carbon linker between the two acridine moieties increases from left to right. (B) HEK293 cells transiently over-expressing TOP3B (TOP3B OE) were treated with DMSO (control) or with the indicated bisacridines (100 µM for 1 h) prior to analysis by modified RADAR and probed with anti-Flag antibody. The same samples probed with anti-dsDNA antibody served as loading controls. Only NSC690634 showed a strong induction of TOP3Bccs while all the other structurally related compounds with different linker lengths failed to induce cellular TOP3Bccs. (C) In vitro band shift assays demonstrate that linker length is critical for inducing TOP3Bccs by the bisacridine NSC690634. Compounds without linkers did not trap TOP3B, whereas NSC690634 (3-carbon linker) strongly enhanced TOP3Bccs with RNA. Bisacridine compounds with longer carbon linkers not only failed to induce TOP3Bcc but they also destabilized TOP3Bccs with both DNA and RNA.

Article Snippet: Human TOP3B- Myc- Flag cDNA open reading frame (ORF) was purchased from OriGene (RC223204) and transfected in HEK293 cells using Lipofectamine 3000 Reagent (ThermoFisher Scientific) according to the manufacturer’s protocol for 48 h before drug treatments.

Techniques: Activity Assay, Expressing, Control, Modification, In Vitro, Electrophoretic Mobility Shift Assay

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Identification and characterization of topoisomerase III beta poisons.

doi: 10.1073/pnas.2218483120

Figure Lengend Snippet: Fig. 7. Structure activity analysis of thiacyanine analogs of NSC96932 demonstrates that the linker length is critical for inducing TOP3Bccs. (A) Chemical structures of a series of thiacyanine analogs. The length of the carbon linker between the two indole ring moieties increases from Left to Right. (B) HEK293 cells transiently over-expressing TOP3B (TOP3B OE) were treated with the indicated thiacyanine analogs (100 µM for 1 h) prior to analysis by modified RADAR and probed with anti-Flag antibody. The same samples probed with anti-dsDNA antibody served as loading controls. Only the thiacyanine with 3-carbon linkers (NSC96932, NSC356711, and NSC93472) induced endogenous TOP3Bccs while the compounds with longer carbon-linker lengths failed to induce cellular TOP3Bccs.

Article Snippet: Human TOP3B- Myc- Flag cDNA open reading frame (ORF) was purchased from OriGene (RC223204) and transfected in HEK293 cells using Lipofectamine 3000 Reagent (ThermoFisher Scientific) according to the manufacturer’s protocol for 48 h before drug treatments.

Techniques: Activity Assay, Expressing, Modification

Journal: Nature communications

Article Title: Replication-associated formation and repair of human topoisomerase IIIα cleavage complexes.

doi: 10.1038/s41467-023-37498-6

Figure Lengend Snippet: Fig. 2 | TOP3Accs are predominantly formed in replicative cells. a Outline of the experimental protocol for transfection, cell synchronization, followed by RADAR assay in U2OS cells with ectopic expression of TOP3A-R364W. Cells at different phases of cell cycle were harvested at the indicated times and protein-DNA adducts were probed by RADAR assay. b Representative slot blot for TOP3Accs detection by RADAR assay from cells harvested as indicated in panel A. TOP3Accs were detected with anti-TOP3A antibody. c U2OS cells expressing FLAG-tagged TOP3A-R364W after 48 h transfection were pre-extracted, fixed and analyzed by confocal micro- scopy. Representative images showing TOP3A co‐localization with CDC45, RPA1 and RPA2. TOP3A foci were detected using anti-FLAG antibody. Scale bars: 10 μm. d Upper panel: workflow of the iPOND experiments; click reactions were performed at the end of the 15 min EdU pulse or following thymidine chase. Lower left panels: lysates form mock-transfected (NT), TOP3A-WT- and TOP3A-R364W-transfected cells were immunoblotted with the indicated antibodies. H3 was used as a loading

Article Snippet: 10μg of each RADAR assay sample was digested with 250 units benzonase nuclease (EMD Millipore, 100 units/μl) in the presence of 5mM CaCl2, followed by SDS-PAGE electrophoresis for immunodetection of total TOP3Accs and ubiquitinated and SUMOylated TOP3Accs by probing with rabbit anti-TOP3A (dilution 1:1000, Proteintech, Rosemont, IL, CAT#: 14525-1-AP), mouse anti-ubiquitin antibody (dilution 1:500, Cell Signaling Technology, Cat# 3936), rabbit anti-SUMO-1 (dilution 1:1000, Cell Signaling Technology, Cat# 4940) and rabbit anti-SUMO2/3 antibody (dilution 1:1000, Cell Signaling Technology, Cat# 4971), respectively.

Techniques: Transfection, Expressing, Dot Blot

Journal: Nature communications

Article Title: Replication-associated formation and repair of human topoisomerase IIIα cleavage complexes.

doi: 10.1038/s41467-023-37498-6

Figure Lengend Snippet: Fig. 3 | Trapped TOP3Accs alter DNA replication. a Schematic representation of the DNA combing assay protocol and representative single molecule images. Nascent DNA was labeled with CldU (30 min) followed by IdU (30 min) in U2OS cells transfected with TOP3A-WT and TOP3A- R338W. Scale bars: 100 μm. b Histogram showing reduced replication fork speed in U2OS cells expressing R364W-TOP3A. c Representative images of a symmetric and an asymmetric replicon observed in DNA combing. Scale bars: 100 μm. d Self-trapping TOP3A-R364W induces asym- metric replication forks in U2OS cells. The values of x and y axis represent repli- cation speed (kb/min) of the right fork and left fork, respectively. The percentages of asymmetric forks from the indicated cells are indicated in red. n = number of forks analyzed. Asymmetric forks were classified as those forks when the difference between length of left forks and right forks emerging from the same origin was greater than 30%. e Histogram showing the percentages of unidirectional (grey), asymmetrical (green) and symmetrical (pink) forks in the indicated cell lines.

Article Snippet: 10μg of each RADAR assay sample was digested with 250 units benzonase nuclease (EMD Millipore, 100 units/μl) in the presence of 5mM CaCl2, followed by SDS-PAGE electrophoresis for immunodetection of total TOP3Accs and ubiquitinated and SUMOylated TOP3Accs by probing with rabbit anti-TOP3A (dilution 1:1000, Proteintech, Rosemont, IL, CAT#: 14525-1-AP), mouse anti-ubiquitin antibody (dilution 1:500, Cell Signaling Technology, Cat# 3936), rabbit anti-SUMO-1 (dilution 1:1000, Cell Signaling Technology, Cat# 4940) and rabbit anti-SUMO2/3 antibody (dilution 1:1000, Cell Signaling Technology, Cat# 4971), respectively.

Techniques: Labeling, Transfection, Expressing

Journal: Nature communications

Article Title: Replication-associated formation and repair of human topoisomerase IIIα cleavage complexes.

doi: 10.1038/s41467-023-37498-6

Figure Lengend Snippet: Fig. 7 | Proposed model for parallel pathways resolving stalled cellular TOP3Accs. a–c Differential roles of SPRTN, MRE11 and TDP2 for TOP3Accs repair as a function of cell cycle. U2OS cells were transfected with either siControl or siSPRTN (a),siMRE11 (b),siTDP2 (c) followed by co-transfection with TOP3A-R364W. Cells (in S and G2/M phase) were collected after synchronization by double-thymidine block. RADAR assays were performed with anti-TOP3A antibody. Representative slot-blot images are shown. Quantitation from three independent RADAR assays is shown to the bottom of slot blot images. Error bar indicates mean ± SE. Statistical significance was determined by two-way ANOVA with Sidak’s multiple comparisons test. ****Adjusted p-value ≤0.0001(siControl vs siSPRTN: S-phase, siControl vs siTDP2: S-phase, siControl vs siMRE11: G2/M-phase), **Adjusted p-value = 0.0016

Article Snippet: 10μg of each RADAR assay sample was digested with 250 units benzonase nuclease (EMD Millipore, 100 units/μl) in the presence of 5mM CaCl2, followed by SDS-PAGE electrophoresis for immunodetection of total TOP3Accs and ubiquitinated and SUMOylated TOP3Accs by probing with rabbit anti-TOP3A (dilution 1:1000, Proteintech, Rosemont, IL, CAT#: 14525-1-AP), mouse anti-ubiquitin antibody (dilution 1:500, Cell Signaling Technology, Cat# 3936), rabbit anti-SUMO-1 (dilution 1:1000, Cell Signaling Technology, Cat# 4940) and rabbit anti-SUMO2/3 antibody (dilution 1:1000, Cell Signaling Technology, Cat# 4971), respectively.

Techniques: Transfection, Cotransfection, Blocking Assay, Dot Blot, Quantitation Assay

Journal: Molecular cell

Article Title: RNA methylation influences TDP43 binding and disease pathogenesis in models of amyotrophic lateral sclerosis and frontotemporal dementia.

doi: 10.1016/j.molcel.2022.12.019

Figure Lengend Snippet: Figure 1. TDP43 recognizes m6A-modified RNA (A and B) UG density 100 bp up- and downstream of m6A modifications identified by cross-linking induced mutation sites (CIMSs) (A) or cross-linking induced truncation sites (CITSs) (B) in relation to random sequences (red line). Gray shading, 95% CI. (C) Schematic of HaloTag immunoprecipitation and dot blot procedure. (D) Dot blot for total RNA (detected by methylene blue) or m6A-modified RNA (detected by anti-m6A antibody) following HaloTag immunoaffinity purification in HEK293T cells overexpressing HaloTag, TDP43-HaloTag or YTHDF2-HaloTag from 3 biological replicates. (E) Diagram illustrating insertion of HaloTag downstream of the TARDBP start codon (green line), encoding HaloTag-TDP43. (F) Halo-TDP43 HEK293T cells labeled live with JF646 dye (red), then fixed, permeabilized, and immunostained for TDP43 (green) prior to imaging. DAPI (blue) marks the nucleus. Scale bars, 10 mm. (G) Dot blot for total and m6A RNA isolated by immunoaffinity purification of endogenous HaloTag-TDP43 or overexpressed HaloTag. Empty space between lanes was clipped in (D) and (E). Additional replicates shown in Figure S1.

Article Snippet: Coverslips were then incubated overnight with blocking buffer + rabbit anti-TDP43 antibody (Proteintech, #10782-2-AP) at 1:500 to stain for TDP43.

Techniques: Mutagenesis, Immunoprecipitation, Dot Blot, Labeling, Imaging, Isolation

Journal: Molecular cell

Article Title: RNA methylation influences TDP43 binding and disease pathogenesis in models of amyotrophic lateral sclerosis and frontotemporal dementia.

doi: 10.1016/j.molcel.2022.12.019

Figure Lengend Snippet: Figure 3. m6A modifications influence TDP43 binding and autoregulation (A) TARDBP gene map, illustrating TDP43 binding region (TBR), the location of the DRACH motif (pink square), and the C-T transition (red box) identified by DART- seq within this domain, representing an m6A site.

Article Snippet: Coverslips were then incubated overnight with blocking buffer + rabbit anti-TDP43 antibody (Proteintech, #10782-2-AP) at 1:500 to stain for TDP43.

Techniques: Binding Assay

Journal: Molecular cell

Article Title: RNA methylation influences TDP43 binding and disease pathogenesis in models of amyotrophic lateral sclerosis and frontotemporal dementia.

doi: 10.1016/j.molcel.2022.12.019

Figure Lengend Snippet: Figure 6. YTHDF2 reduction extends neuronal survival in human neuron disease models (A) YTHDF2 immunostaining in control and sALS spinal cord. Scale bars, 50 mm. (B) YTHDF2 immunoreactivity in spinal neurons from control (n = 117 neurons) and sALS (n = 193 neurons) samples. Plot shows mean ± SD, color coded by sample. ****p < 0.0001 via Mann-Whitney test. (C) Strategy used to create isogenic iPSCs expressing native TDP43(WT)-Dendra2 or TDP43(M337V)-Dendra2. (D–F) (D) Representative images of untransduced (gray) and transduced (green) iNeurons expressing shRNA against YTHDF2 (shYTHDF2). Time of death (red circles) for each cell is used to determine cumulative risk of death, plotted in (E) and (F). Scale bars, 20 mm. YTHDF2 knockdown significantly extended the survival of TDP43(M337V)-Dendra2 iNeurons (E; yp = 8.42 3 1012, HR = 6.25; ***p = 4.82 3 109, HR = 0.32; #p = 0.08, HR = 1.84) as well as mutant C9ORF72 iNeurons (F, yp = 1.42 3 1011, HR = 2.85; ***p = 1.42 3 1016, HR = 0.32). ns, not significant. Values in (E) and (F) calculated by Cox proportional hazards analysis, with a minimum 3 biological replicates.

Article Snippet: Coverslips were then incubated overnight with blocking buffer + rabbit anti-TDP43 antibody (Proteintech, #10782-2-AP) at 1:500 to stain for TDP43.

Techniques: Immunostaining, Control, MANN-WHITNEY, Expressing, shRNA, Knockdown, Mutagenesis