Journal: Aging (Albany NY)

Article Title: Overexpression of steroid sulfotransferase genes is associated with worsened prognosis and with immune exclusion in clear cell-renal cell carcinoma

doi: 10.18632/aging.102392

Figure Lengend Snippet: Overexpression of steroid SULT genes is associated with worsened prognosis in ccRCC. Reproduced from the TCGA KIRC dataset with cBioPortal platform, shown are ( A ) Percentage of ccRCC case with overexpression of steroid sulfotransferase (SULT) genes amongst 534 cases; ( B – C ) Progression-free and overall survival (OS) between ccRCC patients with steroid SULT genes upregulated and unchanged. Reproduced from TCGA KIRC dataset with Human Protein Atlas platform, shown are ( D ) OS of ccRCC patients with high or low mRNA level of SULT1A1, SULT1E1, SULT2A2 and SULT2B1 at automatically defined cutoffs. Reproduced from TCGA KIRC dataset with GEPIA and UALCAN platform, shown are ( E–H ) Contrast in gene expressions of steroid SULT genes between normal and cancer tissue, respectively (****P < 0.0001).

Article Snippet: The human SULT1A1, SULT1E1, SULT2A1 and SULT2B1 cDNA clones were obtained from Origene and a lentivirus-based vector was constructed as previously reported.

Techniques: Over Expression

Journal: Nature chemical biology

Article Title: Correlating chemical sensitivity and basal gene expression reveals mechanism of action

doi: 10.1038/nchembio.1986

Figure Lengend Snippet: ( a ) Expression–sensitivity correlations for the compound austocystin D and CYP2J2 expression. Reactive functionalities are depicted with a red arrow. ( b ) Cytotoxicity of austocystin D and a selective CYP2J2 inhibitor (CYP2J2i) across renal CCLs with different expression levels of CYP2J2 , and effects of co-treatment of austocystin D with CYP2J2i. Cell viability values are normalized to vehicle-only (DMSO) treatment, with each point representing the mean of n = 2 independent experiments with 2 technical replicates each. ( c ) Cytotoxicity of austocystin D in MCF7-ER-Snail-1 6SA cells either induced to undergo epithelial-to-mesenchymal transition (blue) or vehicle-treated (black). Each point is the mean of n = 2 technical replicates. Two independent inductions are shown. ( d ) Expression–sensitivity correlations for the compound RITA and SULT1A1 expression. ( e ) Protein expression of SULT1A1 and sensitivity to RITA of six renal CCLs. CCLs described in ref. as capable (*) or incapable (‡) of metabolizing RITA into a cytotoxic form are indicated. Each point is mean ± s.d. for n = 3 independent experiments. For uncropped gel image, see . ( f ) Sulfotransferase enzyme activity of varying amounts of recombinant SULT1A1 at a fixed concentration of RITA in the presence of the sulfate donor 3′-phosphoadenosine-5′-phosphosulfate. Each point is mean ± s.d. for n = 3 independent experiments.

Article Snippet: Recombinant human SULT1A1 (R&D Systems) activity in the presence of 0.2 mM 3′-phosphoadenosine-5′-phosphosulfate and the indicated concentrations of RITA was measured by a phosphatase-coupled assay using the Universal Sulfotransferase Activity Kit (R&D Systems) according to the manufacturer’s instructions.

Techniques: Expressing, Activity Assay, Recombinant, Concentration Assay

Journal: Virology Journal

Article Title: Cytosolic sulfotransferase 1A1 regulates HIV-1 minus-strand DNA elongation in primary human monocyte-derived macrophages

doi: 10.1186/s12985-016-0491-9

Figure Lengend Snippet: SULT1A1 is highly expressed in primary human monocyte-derived macrophages (MDMs). a The cellular sulfonation pathway. The first step of the cellular sulfonation pathway involves import through a sulfate transporter of a sulfate ion that is then used as a substrate by either 3′-phosphoadenosine-5′phosphosulfate (PAPS) synthetase enzymes PAPSS1 or PAPSS2. These proteins catalyze two enzymatic steps to generate PAPS, the high-energy universal sulfonate-donor from sulfate and two molcules of ATP. PAPS can be transported across the Golgi membrane and used by the Golgi sulfotransferases to generate sulfonated proteins, glycoproteins, glycoproteins, glycolipids, and proteoglycans. Alternatively, PAPS can be used by cytosolic sulfotransferases (SULTS) to sulfonate small molecules such as hormones, neurotransmitters, and xenobiotics. b Human CD4+ T cells and CD14+ monocytes were isolated from donor PBMCs by magnetic bead isolation. Resting CD4+ T cells were lysed directly after separation, and the remaining CD4+ T cells were activated using CD3/CD28 beads for three days. Monocytes were cultured for 7 days in the presence of 20 ng/ml M-CSF, were lysed, subjected to gel electrophoresis, and immunoblotting was performed to detect SULT1A1 or the loading control Ku86 protein

Article Snippet: Primary antibodies used in the study include mouse anti-human SULT1A1 mAb (clone 638708, R&D Systems) used at 1:2000 dilution, rabbit anti-HIV-1 Vif (clone A319, NIH AIDS Reagent program) used at 1:500, rabbit anti-HIV-1 Vpu (clone ab81532, Abcam) used at 1:1000 dilution, mouse anti-human Ku-86 mAb (clone B-1, Santa Cruz Biotechnology) used at 1:500 dilution, and anti-human mouse GAPDH (clone 6C5, Abcam) used at 1:5000.

Techniques: Derivative Assay, Membrane, Isolation, Cell Culture, Nucleic Acid Electrophoresis, Western Blot, Control

Journal: Virology Journal

Article Title: Cytosolic sulfotransferase 1A1 regulates HIV-1 minus-strand DNA elongation in primary human monocyte-derived macrophages

doi: 10.1186/s12985-016-0491-9

Figure Lengend Snippet: SULT1A1 knockdown is associated with decreased viral gene expression following infection of MDMs with VSV-G pseudotyped HIV-1 and SIV vectors. a Schematic showing experimental timeline. Briefly, CD14+ monocytes were isolated from human donor PBMCs using positive selection with magnetic beads. Monocytes were differentiated into MDMs and on day 7 were electroporated with siRNA and plated at 1.5 × 10 4 MDMs per well in a 48 well plate. After 96 h, protein knockdown was confirmed by immunoblot and cells were infected with 100 μl (corresponding to 164 ng p24 HIV-1 and 234 ng p27 SIV viral vectors) of the indicated virus. Luciferase and cell viability measurements were determined at 24 h post-infection. b Representative immunoblot showing SULT1A1 knockdown 96 h post transfection with SULT1A1 siRNAs (1–3) and AllStars Negative siRNA Control (ASN), Qiagen. SULT1A1 expression is compared to endogenous Ku86 used as a loading control. Results from one representative donor are shown. SULT1A1 expression was generally decreased by 70–80 % with siRNA treatment compared to the control ASN siRNA (as shown in Fig. 2c, middle panel). c HIV-1 luciferase reporter expression ( left panel ), SULT1A1 protein expression ( middle panel ), and cell viability of MDMs ( right panel ) were measured 24 h after infection with the VSV-G pseudotyped NL43-Luc HIV-1 vector. Results shown are from 6 donors assayed twice. All values were compared to ASN control. d SIV-1 luciferase reporter expression (left panel), SULT1A1 protein expression ( middle panel ), and cell viability ( right panel ) for MDMs 24 h post infection with VSV-G-pseudotyped SIVagm-Luc. Mean and SD shown, *** p < 0.0005 ** p < 0.005 * p < 0.05 one sample t test. Results shown are from 6 donors assayed twice. Samples with <60 % SULT1A1 knockdown and/or <65 % cell viability were not used in the analysis. All values were compared to ASN control

Article Snippet: Primary antibodies used in the study include mouse anti-human SULT1A1 mAb (clone 638708, R&D Systems) used at 1:2000 dilution, rabbit anti-HIV-1 Vif (clone A319, NIH AIDS Reagent program) used at 1:500, rabbit anti-HIV-1 Vpu (clone ab81532, Abcam) used at 1:1000 dilution, mouse anti-human Ku-86 mAb (clone B-1, Santa Cruz Biotechnology) used at 1:500 dilution, and anti-human mouse GAPDH (clone 6C5, Abcam) used at 1:5000.

Techniques: Knockdown, Gene Expression, Infection, Isolation, Selection, Magnetic Beads, Western Blot, Virus, Luciferase, Transfection, Control, Expressing, Plasmid Preparation

Journal: Virology Journal

Article Title: Cytosolic sulfotransferase 1A1 regulates HIV-1 minus-strand DNA elongation in primary human monocyte-derived macrophages

doi: 10.1186/s12985-016-0491-9

Figure Lengend Snippet: SULT1A1 knockdown is associated with decreased viral gene expression following infection of MDMs with a replication-competent HIV-1 vector. a The same method was used as in Fig. , however 100 μl (corresponding to 66.3 ng p24) HIV-1 Env + JM1186-Rluc virus was added to the cells at day 11 and luciferase and cell viability was assayed at 72 h post-infection. b HIV-1 luciferase reporter expression, SULT1A1 protein expression, and cell viability of MDMs 24 h post infection with HIV-1 Env + JM1186-RLuc.. Mean and SD shown, *** p < 0.0005 ** p < 0.005 * p < 0.05 one sample t test. Results shown are from 6 donors assayed twice. Samples with <60 % SULT1A1 knockdown and/or <65 % cell viability were not used in the analysis. All values were compared to ASN control

Article Snippet: Primary antibodies used in the study include mouse anti-human SULT1A1 mAb (clone 638708, R&D Systems) used at 1:2000 dilution, rabbit anti-HIV-1 Vif (clone A319, NIH AIDS Reagent program) used at 1:500, rabbit anti-HIV-1 Vpu (clone ab81532, Abcam) used at 1:1000 dilution, mouse anti-human Ku-86 mAb (clone B-1, Santa Cruz Biotechnology) used at 1:500 dilution, and anti-human mouse GAPDH (clone 6C5, Abcam) used at 1:5000.

Techniques: Knockdown, Gene Expression, Infection, Plasmid Preparation, Virus, Luciferase, Expressing, Control

Journal: Virology Journal

Article Title: Cytosolic sulfotransferase 1A1 regulates HIV-1 minus-strand DNA elongation in primary human monocyte-derived macrophages

doi: 10.1186/s12985-016-0491-9

Figure Lengend Snippet: SULT1A1 regulates HIV-1 reverse transcription. a MDMs were treated with control siRNA or SULT1A1-specific siRNA and subesequently challenged with VSV-G pseudotyped NL43-Luc HIV-1 vector. DNA was isolated 24 h post-infection and qPCR was performed using primers that detect early RT DNA products or late RT DNA products compared to the cellular PBGD gene as an endogenous control. The levels of early and late RT products were normalized to the ASN siRNA control. Results shown are from MDMs derived from 6 donors tested twice. b MDMs were transfected with SULT1A1 siRNA 2 or ASN siRNA and total cellular RNA was isolated 24 h post infection with VSV-G pseudotyped NL43-Luc HIV-1 vector. qPCR using primers specific for HIV-1 multiply spliced mRNA (MS RNA, forward and reverse primers span the first and second exons of Tat/Rev, respectively) was performed, and relative MS RNA (normalized to GAPDH) was then normalized to ASN siRNA control. Results shown are from MDMs derived from 6 donors tested twice. c Representative immunoblot showing HIV-1 Vpu and Vif protein levels compared to endogenous Ku86 or GAPDH loading control, respectively, from protein lysate collected 48 h post infection with VSV-G pseudotyped NL43-Luc HIV-1 vector pre-treated with either ASN siRNA control or SULT1A1 siRNA 2. d Quantitative immunoblot analysis using Image Studio software of Vpu and Vif as shown in Fig. 4c. Mean and SD shown, *** p < 0.0005 ** p < 0.005 * p < 0.05 one sample t test. Results shown are from 6 donors assayed twice. Samples with <60 % SULT1A1 knockdown and/or <65 % cell viability were not used in the analysis. All values were compared to ASN control

Article Snippet: Primary antibodies used in the study include mouse anti-human SULT1A1 mAb (clone 638708, R&D Systems) used at 1:2000 dilution, rabbit anti-HIV-1 Vif (clone A319, NIH AIDS Reagent program) used at 1:500, rabbit anti-HIV-1 Vpu (clone ab81532, Abcam) used at 1:1000 dilution, mouse anti-human Ku-86 mAb (clone B-1, Santa Cruz Biotechnology) used at 1:500 dilution, and anti-human mouse GAPDH (clone 6C5, Abcam) used at 1:5000.

Techniques: Reverse Transcription, Control, Plasmid Preparation, Isolation, Infection, Derivative Assay, Transfection, Western Blot, Software, Knockdown

Journal: Virology Journal

Article Title: Cytosolic sulfotransferase 1A1 regulates HIV-1 minus-strand DNA elongation in primary human monocyte-derived macrophages

doi: 10.1186/s12985-016-0491-9

Figure Lengend Snippet: SULT1A1 influences the kinetics of minus-strand DNA elongation. a The step during HIV-1 reverse transcription that SULT1A1 regulates was investigated by quantitative real-time PCR analysis with the indicated primer sets. The stages of reverse transcription are shown: 1. Viral genomic RNA; 2. Minus-strand DNA initiation; 3. Minus-strand DNA transfer; 4. Minus-strand DNA elongation and plus-strand DNA initiation; 5 and 6. Plus-strand DNA transfer; 7. Plus-strand DNA elongation. b MDMs were treated with control or SULT1A1-specific siRNA and challenged with VSV-G pseudotyped NL43-Luc HIV-1 vector. Total cellular DNA was isolated at 8, 16, or 24 h post infection. Quantitative real-time PCR was performed to measure the relative abundance of DNA products corresponding to specific steps during the process of reverse transcription using primers indicated in Fig. 5a. Results shown are from MDMs derived from 6 donors assayed once. Mean and SD shown, *** p < 0.0005 ** p < 0.005, one sample t test

Article Snippet: Primary antibodies used in the study include mouse anti-human SULT1A1 mAb (clone 638708, R&D Systems) used at 1:2000 dilution, rabbit anti-HIV-1 Vif (clone A319, NIH AIDS Reagent program) used at 1:500, rabbit anti-HIV-1 Vpu (clone ab81532, Abcam) used at 1:1000 dilution, mouse anti-human Ku-86 mAb (clone B-1, Santa Cruz Biotechnology) used at 1:500 dilution, and anti-human mouse GAPDH (clone 6C5, Abcam) used at 1:5000.

Techniques: Reverse Transcription, Real-time Polymerase Chain Reaction, Control, Plasmid Preparation, Isolation, Infection, Derivative Assay

Journal: Molecular cancer therapeutics

Article Title: Decreased DNA damage and improved p53 specificity of RITA analogs

doi: 10.1158/1535-7163.MCT-22-0119

Figure Lengend Snippet: (A) Chemical structures of RITA, NSC777196, NSC782846. (B) Correlation of SULT1A1 mRNA level with sensitivity to RITA, NSC777196 and NSC782846 in NCI-60 cell line database. (C) Upper panels, SULT1A1 protein level detected by Western blot in seven cancer cell lines, including breast carcinoma T47D, MDA-MB-231; skin cancer A431; osteosarcoma U2OS, SJSA; melanoma A375; and lung carcinoma H1299. β-Actin was used as loading control. Lower panels, NSC777196 efficiently suppressed the growth of high SULT1A1-expressing cells (U2OS, T47D and A431, red lines) while having negligible effect in cells with low SULT1A1 (A375, MDA-MB-231, SJSA, H1299, black lines). The antitumor effect of NSC782846 was less dependent on the level of SULT1A1 expression. Shown are the results obtained from five biological replicates. (D) Cell viability of WT and SULT1A1−/− MCF7 cells after 24 hours treatment with RITA, NSC777196 and NSC782846 (n=5, * p<0.05, ** p<0.01, *** p<0.001).

Article Snippet: A375 and SJSA cells, stably expressing SULT1A1 cDNA (OriGene, #RC201601L1), were generated by lentivirus transduction as described previously ( 16 ).

Techniques: Western Blot, Expressing

Journal: Molecular cancer therapeutics

Article Title: Decreased DNA damage and improved p53 specificity of RITA analogs

doi: 10.1158/1535-7163.MCT-22-0119

Figure Lengend Snippet: (A) mRNA level of representative oncogenes after 16 hours treatment of MCF7 SULT1A1−/− cells with 10μM NSC777196 or 10μM NSC782846 measured by qPCR (n=3 * p<0.05, ** p<0.01, ***p<0.001). (B) Downregulation of selected oncogenes upon treatment of MCF7 SULT1A1−/− cells by 10μM NSC777196 and 10μM NSC782846 as detected by Western blotting. (C) Protein levels of selected oncogenes in MCF7 SULT1A1−/− in which p53 was depleted by 2 different shRNAs treated with 10μM NSC777196 or 10μM NSC782846 for 24 hours. Scrambled shRNA and GFP shRNA were used as controls. (D) Apoptotic cells were detected by Annexin V-PI double staining using FACS after treatment of MCF7 SULT1A1 −/− cells with 10μM NSC777196 or 10μM NSC782846 for 16 hours. Representative pictures are shown in left panel. The total number of cells in the Q2 and Q3 quadrant was regarded as apoptotic cells. Percentages of apoptotic cells are shown in the bar graph (right panel; n=3 * p<0.05).

Article Snippet: A375 and SJSA cells, stably expressing SULT1A1 cDNA (OriGene, #RC201601L1), were generated by lentivirus transduction as described previously ( 16 ).

Techniques: Western Blot, shRNA, Double Staining

Journal: Molecular cancer therapeutics

Article Title: Decreased DNA damage and improved p53 specificity of RITA analogs

doi: 10.1158/1535-7163.MCT-22-0119

Figure Lengend Snippet: (A) DNA damage marker γH2AX and p53 detected by Western blotting upon 24-hour treatment of MCF7 SULT1A1−/− cells with 1μM RITA, or 10μM and 25μM of NSC777196 or NSC782846, respectively. (B) DNA damage marker γH2AX detected by immunostaining upon treatment by RITA (1μM), NSC777196 (10μM) and NSC782846 (10μM) in MCF7 SULT1A1 −/− cells. (C) RNA synthesis monitored by EU incorporation in MCF7 SULT1A1−/− cells treated with RITA (1μM), NSC777196 (10μM), NSC782846 (10μM), and ActD (2.5 mM), then incubated for 1 hour with 1 mM EU. EU was labeled by click chemistry and visualized by fluorescent microscopy. (D) Protein levels of total RNA Pol II and phospho-Ser2 RNA Pol II in MCF7 SULT1A1−/− cells treated with RITA (1μM), NSC777196 (10μM), NSC782846 (10μM) for 24 hours. (E) The levels of representative oncogenes and RNA Pol II upon treatment with low dose of NSC777196 and NSC782846 in MCF7 WT cells as detected by Western blotting. (F) RNA synthesis monitored by EU incorporation in MCF7 WT cells treated with NSC777196 (0.25μM), NSC782846 (1μM), then incubated for 1 hour with 1 mM EU. EU was labeled by click chemistry and visualized by fluorescent microscopy.

Article Snippet: A375 and SJSA cells, stably expressing SULT1A1 cDNA (OriGene, #RC201601L1), were generated by lentivirus transduction as described previously ( 16 ).

Techniques: Marker, Western Blot, Immunostaining, Incubation, Labeling, Microscopy