Journal: Molecular cell

Article Title: SLC19A1 is an importer of the immunotransmitter cGAMP

doi: 10.1016/j.molcel.2019.05.006

Figure Lengend Snippet: (A) Chemical structures of the known SLC19A1 substrates folinic acid and methotrexate, as compared to cGAMP.

Article Snippet: Recombinant DNA For creation of pcDNA3-FLAG-HA-SLC19A1, the DNA sequence encoding human SLC19A1 was amplified from a U937 cell cDNA library using SLC19A1 FWD and SLC19A1 REV primers ( Supplemental Table 1 ) and insert into the XbaI-BamHI sites of pcDNA3-FLAG-HA (Addgene).

Techniques:

Journal: Molecular cell

Article Title: SLC19A1 is an importer of the immunotransmitter cGAMP

doi: 10.1016/j.molcel.2019.05.006

Figure Lengend Snippet: (A) Effect of AICAR on extracellular cGAMP signaling. U937 WT and SLC19A1−/− cells were pretreated with 1 mM AICAR for 20 min followed by treatment with 100 μM 2’3’-cGAMP for 2 h (n = 4 biological replicates).

Article Snippet: Recombinant DNA For creation of pcDNA3-FLAG-HA-SLC19A1, the DNA sequence encoding human SLC19A1 was amplified from a U937 cell cDNA library using SLC19A1 FWD and SLC19A1 REV primers ( Supplemental Table 1 ) and insert into the XbaI-BamHI sites of pcDNA3-FLAG-HA (Addgene).

Techniques:

Journal: Molecular cell

Article Title: SLC19A1 is an importer of the immunotransmitter cGAMP

doi: 10.1016/j.molcel.2019.05.006

Figure Lengend Snippet: (A) Role of SLC19A1 in THP-1 cells. THP-1 WT and SLC19Ar−/− cells were pretreated with 1 mM SSZ or 500 uM MTX for 15 min, and then treated with 100 μM cGAMP for 2 h (n = 3 (SSZ) or 2 (MTX) biological replicates).

Article Snippet: Recombinant DNA For creation of pcDNA3-FLAG-HA-SLC19A1, the DNA sequence encoding human SLC19A1 was amplified from a U937 cell cDNA library using SLC19A1 FWD and SLC19A1 REV primers ( Supplemental Table 1 ) and insert into the XbaI-BamHI sites of pcDNA3-FLAG-HA (Addgene).

Techniques:

Journal: bioRxiv

Article Title: SLC19A1 is a cyclic dinucleotide transporter

doi: 10.1101/539767

Figure Lengend Snippet: SLC19A1 is critical for CDN-induced reporter expression. a,b, mRNA expression levels of (a) SLC19A1 or (b) IRF3 in THP-1 cells expressing a CRISPRi vector and a control non-targeting gRNA or gRNAs targeting IRF3 or SLC19A1 (two gRNAs each). Error bars represent ± SE of at two biological replicates. Statistical analysis was performed to compare each cell line to the control using a one-way ANOVA followed by Dunnetts’s post-test. *** P ≤0.001; **** P ≤ 0.0001; n.s. not significant.

Article Snippet: Membranes were probed in 5% Bovine Serum Albumin (Fisher) in 1 X TBS-T with anti-SLC19A1 Picoband antibody (Boster Bio).

Techniques: Expressing, Plasmid Preparation

Journal: bioRxiv

Article Title: SLC19A1 is a cyclic dinucleotide transporter

doi: 10.1101/539767

Figure Lengend Snippet: SLC19A1 is required for CDN-induced reporter expression. a, dCas9-KRAB-expressing THP-1 cells transduced with non-targeting gRNA (control), IRF3-1 gRNA or SLC19A1-1 gRNA were exposed to 2’3’-RR CDA (1.67 µg/ml) or 2’3’-cGAMP (10 µg/ml). 20h later, tdTomato expression was analyzed by flow cytometry. Representative dot plots of three independent experiments are shown. b, THP-1 cells expressing the indicated CRISPRi gRNAs or non-targeting gRNA (control), were stimulated with indicated 2’3’-RR CDA (1.67 µg/ml), 2’3’-cGAMP (10 µg/ml), 3’3’ CDA (20 µg/ml) or hIFN-β (100 ng/ml). After 18-22h, tdTomato expression was quantified as in (a). Combined results of three independent experiments are shown. c, Control THP-1 cells and SLC19A1-1 gRNA expressing THP-1 cells transduced with SLC19A1 (SLC. tr.) were exposed to 2’3’-RR CDA (1.67 µg/ml), 2’3’-cGAMP (15 µg/ml) or hIFN-β (100 ng/ml). After 18-22h, tdTomato reporter expression was quantified. Combined results of three independent experiments are shown. d, Control THP-1 cells (7 clonal lines) and SLC19A1 -/-cells (9 clonal lines) were exposed to 2’3’-RR CDA (2.22 µg/ml), 2’3’-cGAMP (10 µg/ml), and tdTomato reporter expression was analyzed by flow cytometry 20h after stimulation. e, Various cell lines expressing a control vector or an SLC19A1 expression vector were stimulated with 2’3’-RR CDA (1.67 µg/ml) or 2’3’-cGAMP (10 µg/ml). After 20h, reporter expression was quantified by flow cytometry. f, Various cell lines expressing a control vector or an SLC19A1 expression vector were stimulated with hIFN-β (100 ng/ml) or murine IFN-β (100 ng/ml) in the case of RAW cells. After 20h, reporter expression was quantified by flow cytometry. g, THP-1 cells were incubated with increasing concentrations of 2’3’-RR CDA, 2’3’-cGAMP or hIFN-β in the presence of the SLC19A1 inhibitor sulfasalazine or DMSO as vehicle control. After 18h, tdTomato reporter expression was analyzed by flow cytometry. For each concentration of sulfasalazine, reporter expression in treated cells was compared to reporter expression in cells treated with the same amount of vehicle (DMSO). In panels b and c, e, f, and g, error bars represent ± SE of three biological replicates. Statistical analysis was performed using one-way ANOVA followed by Tukey’s post-test (b and c), unpaired two-tailed Student’s t tests for (d), two-way ANOVA followed by uncorrected Fisher’s LSD tests (e and f), and two-way ANOVA followed by Tukey’s post-tests to compare the significance between the CDNs and hIFN-β in (g). * P ≤ 0.05; ** P ≤ 0.01;*** P ≤0.001; **** P ≤ 0.0001; n.s. not significant

Article Snippet: Membranes were probed in 5% Bovine Serum Albumin (Fisher) in 1 X TBS-T with anti-SLC19A1 Picoband antibody (Boster Bio).

Techniques: Expressing, Transduction, Flow Cytometry, Plasmid Preparation, Incubation, Concentration Assay, Two Tailed Test

Journal: bioRxiv

Article Title: SLC19A1 is a cyclic dinucleotide transporter

doi: 10.1101/539767

Figure Lengend Snippet: SLC19A1 is critical for STING activation by CDNs. a, b, Induction of CXCL10 (a) or CCL5 (b) mRNA in control (non-targeting gRNA) THP-1 cells or THP-1 cells expressing the indicated CRISPRi gRNAs after 5h stimulation with 5 µg/ml 2’3’-RR CDA. c, Immunoblot analysis of (phospho-) protein expression in control THP-1 cells or THP-1 cells expressing the indicated CRISPRi gRNAs. Cells were stimulated for 2h with 10 µg/ml 2’3’-RR CDA or left unstimulated. TransferrinR.: Transferrin receptor; p-TBK1: TKB1 phosphorylated at position Ser172; p-IRF3: IRF3 phosphorylated at position Ser296; p-STING: STING phosphorylated at position Ser366. Immunoblots are representative of two independent experiments with similar results. d, Control THP-1 cells or SLC19A1-depleted THP-1 cells were transfected with 3 εg interferon-stimulatory DNA (ISD) for 3h and the induction of IFNB mRNA was measured by RT-qPCR. In panels a, and b: error bars represent ± SE of at least three biological replicates, In panel d: error bar represents ± SE of two biological replicates. Statistical analysis was performed using a one-way ANOVA followed by Dunnett’s post-test for the comparison of the CDN-stimulated IRF3, SLC19A1 , and STING -depleted cell lines to the control CDN-stimulated cells in (a) and an unpaired two-tailed Student’s t test for (d). **** P ≤ 0.0001; n.s. not significant.

Article Snippet: Membranes were probed in 5% Bovine Serum Albumin (Fisher) in 1 X TBS-T with anti-SLC19A1 Picoband antibody (Boster Bio).

Techniques: Activation Assay, Expressing, Western Blot, Transfection, Quantitative RT-PCR, Two Tailed Test

Journal: bioRxiv

Article Title: SLC19A1 is a cyclic dinucleotide transporter

doi: 10.1101/539767

Figure Lengend Snippet: SLC19A1 transports CDNs into cells. a, [ 32 P] 2’3’-cGAMP uptake by THP-1 monocytes transduced with empty vector (control) or SLC19A1 expression vector (left panel), or transduced with a non-targeting control CRISPRi gRNA or SLC19A1 CRISPRi gRNA (right panel). b, [ 32 P] 2’3’-cGAMP uptake by THP-1 monocytes in the presence of 100 µM competing, unlabeled cyclic di-nucleotides. c, d , Competitive inhibition of [ 32 P] 2’3’-cGAMP uptake by THP-1 cells in the presence of varying concentrations of competing, unlabeled 2’3’-cGAMP (IC 50 = 1.89 ± 0.11 µM) or Folic Acid (IC 50 = 4.79 ± 0.08 µM). e, Inhibition of [ 32 P] 2’3’-cGAMP uptake by THP-1 cells in the presence of varying concentrations of sulfasalazine (IC 50 = 2.06 ± 0.17 µM). f, [ 32 P] 2’3’-cGAMP uptake by primary (1°) peritoneal leukocytes (PL) or splenocytes in the presence of excess folic acid or Sulfasalazine (SSZ). g, Binding of SLC19A1 to 2’3’ cGAMP. Coomassie staining and Western blot analysis of pulldowns with 2’3’ cGAMP-Sepharose (+) or control ethanoloamine-Sepharose (−) beads. The beads were incubated with recombinant C-terminal domain of mSTING (mSTING-CTD) or with recombinant hSLC19A1 before precipitation and analysis. h, 2’3’cGAMP competes binding of SLC19A1 to 2’3’ cGAMP-Sepharose beads. Soluble 2’3’-cGAMP (250 µM) was added (+) or not (−) to the mixtures of 2’3’ cGAMP-Sepharose and hSLC19A1, before precipitation and Western blot analysis. Data are representative of three independent experiments with similar results. Data are representative of three independent experiments with similar results. In all panels, error bars represent ± SE of biological replicates. Red dashed lines represent the 95% confidence interval for the non-linear regression. Statistical analysis was performed using an unpaired two-tailed Student’s t-test (a) or one-way ANOVA (b and f) followed by Tukey’s post-test. ***P ≤ 0.001; ****P ≤0.0001

Article Snippet: Membranes were probed in 5% Bovine Serum Albumin (Fisher) in 1 X TBS-T with anti-SLC19A1 Picoband antibody (Boster Bio).

Techniques: Transduction, Plasmid Preparation, Expressing, Inhibition, Binding Assay, Staining, Western Blot, Incubation, Recombinant, Two Tailed Test

Journal: bioRxiv

Article Title: SLC19A1 is a cyclic dinucleotide transporter

doi: 10.1101/539767

Figure Lengend Snippet: CDN uptake in C1R and U937 cells. a, Time course of [ 32 P] 2’3’ cGAMP uptake by CIR cells transduced (tr.) with empty vector or SLC19A1 . b, Time course of [ 32 P] 2’3’ cGAMP uptake by U937 monocytes in the presence of excess folic acid or sulfasalazine (SSZ). In all panels, error bars represent ± SD of biological replicates. Dashed lines represent the 95% confidence interval for the non-linear regression. Statistical analysis was performed using an unpaired two-tailed Student’s t-test (a) or one-way ANOVA (b) followed by Tukey’s post-test. ***P ≤ 0.001; ****P ≤ 0.0001

Article Snippet: Membranes were probed in 5% Bovine Serum Albumin (Fisher) in 1 X TBS-T with anti-SLC19A1 Picoband antibody (Boster Bio).

Techniques: Plasmid Preparation, Two Tailed Test

Journal: bioRxiv

Article Title: SLC19A1 is a cyclic dinucleotide transporter

doi: 10.1101/539767

Figure Lengend Snippet: SLC19A1 interacts with 2’3’ cGAMP. a, Sodium dodecyl sulfate (SDS)-PAGE analysis followed by Coomassie Blue staining of mSTING-C-Terminal Domain (CTD) pull-downs with 2’3 cGAMP (+) or control (−) Sepharose. b, SDS-PAGE analysis followed by Coomassie Blue staining of His-tagged hSLC19A1 pull-downs with 2’3’ cGAMP (+) or control (−) Sepharose as well as the input material following Ni-NTA affinity purification (right panel). c, SDS-PAGE analysis followed by Western blot analysis of His-tagged hSLC19A1 pull-downs with 2’3’ cGAMP (+) or control (−) Sepharose as well as the input material following Ni-NTA affinity purification. The two panels were run on the same gel but separated for comparison to the panels in B. d, SDS-PAGE analysis followed by Western blot analysis of 8xHis-tagged hSLC19A1 affinity purification (AP) with 2’3’ cGAMP Sepharose in the absence (−) and presence (+) of free, unbound 2’3’ cGAMP (250 µM).

Article Snippet: Membranes were probed in 5% Bovine Serum Albumin (Fisher) in 1 X TBS-T with anti-SLC19A1 Picoband antibody (Boster Bio).

Techniques: SDS Page, Staining, Affinity Purification, Western Blot

Journal: bioRxiv

Article Title: SLC19A1 is a cyclic dinucleotide transporter

doi: 10.1101/539767

Figure Lengend Snippet: RNA-Seq data of STING and SLC19A1 mRNA expression in 934 human cancer cell lines available at the Cancer Cell Line Encyclopedia. Expression is presented as transcripts per kilobase million (TPM). Data is downloaded from the European Bioinformatics Institute Gene expression Atlas (URL: https://www.ebi.ac.uk/gxa/home ). The data set included three of the cell lines we examined, as shown.

Article Snippet: Membranes were probed in 5% Bovine Serum Albumin (Fisher) in 1 X TBS-T with anti-SLC19A1 Picoband antibody (Boster Bio).

Techniques: RNA Sequencing Assay, Expressing

Journal: bioRxiv

Article Title: SLC19A1 is a cyclic dinucleotide transporter

doi: 10.1101/539767

Figure Lengend Snippet: SLC46A1 or SLC46A3 depletions, in combination with SLC19A1 depletion have no additional effect on stimulation by CDNs. THP-1 cells were transduced with non-targeting control CRISPRi gRNAs or SLC19A1 -targeting CRISPRi gRNA in combination with a second control CRISPRi gRNA or SLC46A1 -targeting CRISPRi gRNA in (a) or SLC46A3 -targeting gRNA in (b). Cells were stimulated with 2’3’-RR CDA (1.67 µg/ml), 2’3’-cGAMP (10 µg/ml), or hIFN-β (100 ng/ml). tdTomato reporter expression was measured by flow cytometry 18-22h after stimulation. Combined data of three independent experiments. Statistical analysis was performed using a one-way ANOVA followed by a Tukey’s post-test, comparing only the effects of depleting SLC46A1 (a) or SLC46A3 (b). Error bars represent ± SE of independent replicates. * P ≤ 0.05; ** P ≤ 0.01; n.s. not significant.

Article Snippet: Membranes were probed in 5% Bovine Serum Albumin (Fisher) in 1 X TBS-T with anti-SLC19A1 Picoband antibody (Boster Bio).

Techniques: Transduction, Expressing, Flow Cytometry

Journal: Cancer Science

Article Title: Thymidylate synthase inhibitor raltitrexed can induce high levels of DNA damage in MYCN ‐amplified neuroblastoma cells

doi: 10.1111/cas.14485

Figure Lengend Snippet: Protein expression levels of reduced folate carrier (RFC), thymidylate synthase (TS), and dihydrofolate reductase (DHFR) and their correlation with N‐Myc expression. A, Cell lysates were subjected to western blot analysis with anti‐N‐Myc, anti‐RFC, anti‐TS, anti‐DHFR, and anti‐β‐actin Abs. B, Protein expression levels of RFC, TS, and DHFR (normalized against β‐actin) in 6 MYCN ‐amplified and 4 MYCN nonamplified (Nonamp) neuroblastoma cell lines. C, Flow cytometric analysis of fluorescein methotrexate (F‐MTX) incorporation in 2 MYCN ‐amplified (IMR‐32 and KELLY) and 2 MYCN nonamplified (SH‐SY5Y and SK‐N‐FI) cells. Cells were incubated with 0, 1, 3, and 10 μmol/L F‐MTX for 2 h and washed with fresh culture medium without F‐MTX for 30 min. A total of 20 000 cells were analyzed by flow cytometer

Article Snippet: The following Abs were used: N‐Myc (D4B2Y) rabbit mAb (#51705; Cell Signaling Technology), reduced folate carrier/SLC19A1 Ab (NBP1‐59904; Novus Biologicals), thymidylate synthase (D5B3) XP rabbit mAb (#9045; Cell Signaling Technology), DHFR rabbit polyclonal Ab (15194‐1‐AP; Proteintech), monoclonal anti‐β‐actin clone AC‐15 (A5441; Sigma‐Aldrich), cleaved PARP (Asp214) (D64E10) XP rabbit mAb (#5625; Cell Signaling Technology), cleaved caspase‐3 (Asp175) (5A1E) rabbit mAb (#9664; Cell Signaling Technology), anti‐phospho‐histone H2A.X (Ser139) clone JBW301 (#05‐636; Merck), phospho‐Chk1/2 Ab sampler kit (#9931; Cell Signaling Technology), RPA32/RPA2 (4E4) rat mAb (#2208; Cell Signaling Technology), monoclonal mouse anti‐Human p53 protein clone DO‐7 (M7001; Agilent), p21 Waf1/Cip1 (12D1) rabbit mAb (#2947; Cell Signaling Technology), anti‐rabbit IgG, HRP‐linked antibody (#7074; Cell Signaling Technology), anti‐mouse IgG, HRP‐linked antibody (#7076; Cell Signaling Technology), and anti‐rat IgG, HRP‐linked antibody (#7077; Cell Signaling Technology).

Techniques: Expressing, Western Blot, Amplification, Incubation, Flow Cytometry