Journal: Cancers

Article Title: Inhibition of the Transcription Factor PU.1 Suppresses Tumor Growth in Mice by Promoting the Recruitment of Cytotoxic Lymphocytes Through the CXCL9-CXCR3 Axis

doi: 10.3390/cancers17162684

Figure Lengend Snippet: DB enhances the expression of CXCL9 mRNA in tumor-associated macrophages (TAMs). ( A ) Volcano plot showing differentially expressed genes between TAMs isolated from DB- or vehicle-treated melanoma tumors. Red dots indicate genes with an adj. p < 0.01. ( B ) DB-affected genes (FC > 1.5, adj. p < 0.05) were analyzed for GO terms using the Metascape online program. The top 20 GO and hallmark terms are presented. ( C ) Bubble heatmap showing five cytokines displaying increased expression in DB-treated TAMs compared to vehicle-treated TAMs. ( D ) CXCL9 mRNA levels in TAMs isolated from DB- or vehicle-treated tumors were determined by real-time PCR. ( E ) TAM depletion by clodronate abolished the effect of DB on CXCL9 mRNA expression. Shown are the mRNA levels of CXCL9 (relative to GAPDH) in tumors under the specified treatments. Statistical significance was determined by one-way ANOVA followed by Tukey’s multiple comparisons test ( n = 3–4).

Article Snippet: InVivoMAb, rat IgG2a isotype control, anti-mouse CXCR3 (CD183), anti-mouse CXCL9 (MIG), and polyclonal Armenian hamster IgG isotype control antibodies were purchased from Bio X Cell (Lebanon, NH, USA).

Techniques: Expressing, Isolation, Real-time Polymerase Chain Reaction

Journal: Cancers

Article Title: Inhibition of the Transcription Factor PU.1 Suppresses Tumor Growth in Mice by Promoting the Recruitment of Cytotoxic Lymphocytes Through the CXCL9-CXCR3 Axis

doi: 10.3390/cancers17162684

Figure Lengend Snippet: Inhibition of PU.1 by DB and siRNAs enhances CXCL9 transcription in BMDMs . ( A , B ) Macrophage colony-stimulating factor-derived BMDMs were exposed to the drug vehicle or DB (500 nM) for 18–20 h and then activated by LPS (100 ng/mL, 5 h ( A ) or 18 h ( B )). ( A ) RNAs from BMDMs were isolated and mRNA expression of CXCL9, IL-1β, and PU.1 was analyzed by RT-qPCR. ( B ) Production of IL-1β and CXCL9 was measured from the cell culture media using the Luminex platform. Statistical significance was determined by one-way ANOVA followed by Tukey’s multiple comparisons test ( n = 3; * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001). ( C ) Similarly, BMDMs treated with control or PU.1-targeting siRNAs were activated by LPS for 5 h. Top left panel: PU.1 mRNA levels (relative to GAPDH) from the same cells, determined by RT-qPCR. Top right panels: Western blot of total cell lysates of BMDMs transfected with PU.1-specific siRNA or random control siRNA (siCNT). β-actin was included as a loading control. Bottom panels: Expression levels of CXCL9 and IL-1β in BMDMs transfected with PU.1-specific or control siRNA, with or without LPS treatment. * adj. p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001, one-way ANOVA test followed by Tukey’s multiple comparisons test ( n = 3).

Article Snippet: InVivoMAb, rat IgG2a isotype control, anti-mouse CXCR3 (CD183), anti-mouse CXCL9 (MIG), and polyclonal Armenian hamster IgG isotype control antibodies were purchased from Bio X Cell (Lebanon, NH, USA).

Techniques: Inhibition, Derivative Assay, Isolation, Expressing, Quantitative RT-PCR, Cell Culture, Luminex, Control, Western Blot, Transfection

Journal: Cancers

Article Title: Inhibition of the Transcription Factor PU.1 Suppresses Tumor Growth in Mice by Promoting the Recruitment of Cytotoxic Lymphocytes Through the CXCL9-CXCR3 Axis

doi: 10.3390/cancers17162684

Figure Lengend Snippet: CXCL9- and CXCR3-neutralizing antibodies abolish the anti-tumor effect of DB by reducing TILs . ( A ) Growth curves of B16-OVA melanoma treated with DB or vehicle (every 2 days) in combination with neutralizing antibodies for CXCL9 (αCXCL9, every 3 days), CXCR3 (αCXCR3, every 4 days), control IgG (every 4 days). ( B ) Relative frequencies of immune cells (CD45 + ), CD4 or CD8 T cells (CD45 + /CD3 + , CD4 + or CD8 + ), and NK cells (CD45 + /CD3 − /NK1.1 + ). Macrophages (CD45 + /CD11b + /F4/80 + ) were included for comparison. ( C , D ) Immunohistochemical staining of CD8 and Granzyme B (GrzB) in tumors using the specified treatments. Tumor tissues were stained with CD8 − and GrzB-specific antibodies, followed by incubation with HRP-conjugated secondary antibodies. HRP signals were detected using the DAB substrate kit, as described in Materials and Methods. The corresponding quantification data are presented in the right panels of ( C , D ); CD8 immunohistochemistry (IHC) is quantified as cells per field of view (cells/FOV), and Granzyme B (GrzB) IHC is quantified by the optical density of DAB staining. Statistical significance was assessed using a one-way ANOVA followed by Tukey’s multiple comparisons test. p < 0.05. Sample sizes: n = 5–6 for panel ( A ); n = 3–4 for other panels.

Article Snippet: InVivoMAb, rat IgG2a isotype control, anti-mouse CXCR3 (CD183), anti-mouse CXCL9 (MIG), and polyclonal Armenian hamster IgG isotype control antibodies were purchased from Bio X Cell (Lebanon, NH, USA).

Techniques: Control, Comparison, Immunohistochemical staining, Staining, Incubation, Immunohistochemistry

Journal: Cancers

Article Title: Inhibition of the Transcription Factor PU.1 Suppresses Tumor Growth in Mice by Promoting the Recruitment of Cytotoxic Lymphocytes Through the CXCL9-CXCR3 Axis

doi: 10.3390/cancers17162684

Figure Lengend Snippet: The CXCL9-CXCR3 axis is responsible for DB-induced transcriptional changes in tumors. B16−OVA tumors under the same treatments described in A were harvested on day 13 for mRNA sequencing. Transcript counts were analyzed by GSEA and were visualized using the Cytoscape program. Enriched GO nodes (dots) and leading edge (grouped in circles with key functional annotation) are presented in pair-wise comparisons (up-regulation in red, down-regulation in blue), including DB + IgG vs. Vehicle + IgG ( A ), Vehicle + αCXCR3 vs. Vehicle + IgG ( B ), DB + αCXCR3 vs. DB + IgG ( C ), DB + αCXCR3 vs. Vehicle + IgG ( D ).

Article Snippet: InVivoMAb, rat IgG2a isotype control, anti-mouse CXCR3 (CD183), anti-mouse CXCL9 (MIG), and polyclonal Armenian hamster IgG isotype control antibodies were purchased from Bio X Cell (Lebanon, NH, USA).

Techniques: Sequencing, Functional Assay

Journal: Molecular Biology of the Cell

Article Title: SRSF1 regulates the assembly of pre-mRNA processing factors in nuclear speckles

doi: 10.1091/mbc.E12-03-0206

Figure Lengend Snippet: SRSF1-depleted cells show reduced cellular transcription. (A, a) LacI-mCherry–localized gene locus in the DOX-treated control and SRSF1, SRSF2, and PRP6 siRNA–transfected CLTon cells. (A, b and c) RT-PCR and immunoblot analyses show reduced reporter RNA (β-globin) and protein (GFP) levels in the SR protein–depleted (SRSF1 and SRSF2) cells. GAPDH RNA and MEK protein are used as loading controls. (B) Localization of MS2BP–YFP as an indicator of active transcription at the gene locus (+DOX) in control (a–a′′) and SRSF1-depleted (b–b′′) CLTon cells. (c). Localization of YFP-rTa (c′, d′, e′, f′), YFP-RNA pol II (g′, h′), and YFP-CdK9 (i′, j′, k′, l′) at the gene locus (+DOX) in control and SRSF1-depleted CLTon cells. Note the absence of MS2-BP-YFP (b–b′′), RNA pol II (h–h′′Cdk9 (l–l′′), and YFP-SRSF2 (o–o′′), and the presence of YFP-rTa (f–f′′) at the DOX-induced gene locus of SRSF1-depleted cells. The YFP-SRSF2 in the SRSF1-depleted cells (o′, o′′) are imaged with less exposure time compared with control siRNA–treated cells for the better clarity of nuclear speckles. DNA is counterstained with DAPI. Scale bars, 5 μm.

Article Snippet: Antibodies used in the present study include the following: B′′-U2-snRNP (mouse immunoglobulin G [mIgG]; IF, 1:100; Western blot [WB], 1:250), SF3a60 (rabbit IgG [rIgG]; IF, 1:200; WB, 1:2000; Kramer et al. , 1994 ), SRSF1 (mIgG, mAb103; IF, 1:300; Hanamura et al. , 1998 ), SRSF1 (mIgG, mAb96; WB, 1:1000; Hanamura et al. , 1998 ), SRSF2 (mIgG; WB, 1:20; Cavaloc et al. , 1999 ), SRSF2 (SC35, mIgG; IF, 1:300; ), SRSF3 (rIgG; WB, 1:1000; Invitrogen), U170K (mIgG; IF, 1:150; S. Hoch, Agouron Institute, La Jolla, CA), 3C5 (mIgM; IF, 1:100; ), U2AF-65 (rIgG; WB, 1:1000), U2AF-35 (rIgG; WB, 1:1000), SON (rIgG; IF, 1:5000; Sharma et al. , 2010 ), Y12 (anti-Sm, mIgG; IF, 1:20; Abcam, Cambridge, MA), RNA polymerase II (H5 antibody, mIgM; IF, 1:100), RNA pol II (H14 antibody, mIgM; IF, 1:100), Cdk9 (rIgG; IF, 1:100; Santa Cruz Biotechnology, Santa Cruz, CA), MEK (rIgG; WB, 1:2000), T7 (mIgG; IF, 1:1000, WB, 1:5000; Novus Biologicals, Littleton, CO), α-tubulin (mIgG; WB, 1:5000; Sigma-Aldrich), GFP (mIgG; WB, 1:500; Covance, Berkeley, CA), mRFP (WB, 1:500; Chemicon International, Temecula, CA), and HA tag (clone 12CA5, mIgG; WB, 1:500).

Techniques: Control, Transfection, Reverse Transcription Polymerase Chain Reaction, Western Blot

Journal: Molecular Biology of the Cell

Article Title: SRSF1 regulates the assembly of pre-mRNA processing factors in nuclear speckles

doi: 10.1091/mbc.E12-03-0206

Figure Lengend Snippet: Immobilization of SRSF1 on chromatin leads to association with nuclear speckle or de novo formation of nuclear speckle. (A) CLTon cells are cotransfected with CFP-LacI vector (a′), CFP-LacI-SRSF1 (b′, f′), or YFP-LacI-SRSF1 (c′, d′, e′) and YFP-SRSF2 (a′′, b′′), CFP-SRSF3 (c′′), CFP-U170K (d′′), CFP-UAP56 (e′′), and YFP-SON (f′′). (B, a–d) RNA-FISH shows the nuclear distribution of endogenous MALAT1 (a′′, b′′), poly(A)+ RNA (c′′), and U2snRNA (d′′) in CLTon cells that are transfected with CFP-LacI vector (a′), CFP-LacI-SRSF1 (b′, d′), or YFP-LacI-SRSF1 (c′). Immunolocalization of RNA pol II (H14 antibody; e′′) and YFP-Cdk9 localization (f′′) in CLTon cells, which are transfected with CFP-LacI-SRSF1 (e′, f′). Note that CFP-LacI-SRSF1 fails to recruit YFP-SON and RNA pol II to the de novo–formed speckles. A and B, a–f, represent the stably integrated LacI locus (blue) in the CLTon cells. Scale bars, 5 μm.

Article Snippet: Antibodies used in the present study include the following: B′′-U2-snRNP (mouse immunoglobulin G [mIgG]; IF, 1:100; Western blot [WB], 1:250), SF3a60 (rabbit IgG [rIgG]; IF, 1:200; WB, 1:2000; Kramer et al. , 1994 ), SRSF1 (mIgG, mAb103; IF, 1:300; Hanamura et al. , 1998 ), SRSF1 (mIgG, mAb96; WB, 1:1000; Hanamura et al. , 1998 ), SRSF2 (mIgG; WB, 1:20; Cavaloc et al. , 1999 ), SRSF2 (SC35, mIgG; IF, 1:300; ), SRSF3 (rIgG; WB, 1:1000; Invitrogen), U170K (mIgG; IF, 1:150; S. Hoch, Agouron Institute, La Jolla, CA), 3C5 (mIgM; IF, 1:100; ), U2AF-65 (rIgG; WB, 1:1000), U2AF-35 (rIgG; WB, 1:1000), SON (rIgG; IF, 1:5000; Sharma et al. , 2010 ), Y12 (anti-Sm, mIgG; IF, 1:20; Abcam, Cambridge, MA), RNA polymerase II (H5 antibody, mIgM; IF, 1:100), RNA pol II (H14 antibody, mIgM; IF, 1:100), Cdk9 (rIgG; IF, 1:100; Santa Cruz Biotechnology, Santa Cruz, CA), MEK (rIgG; WB, 1:2000), T7 (mIgG; IF, 1:1000, WB, 1:5000; Novus Biologicals, Littleton, CO), α-tubulin (mIgG; WB, 1:5000; Sigma-Aldrich), GFP (mIgG; WB, 1:500; Covance, Berkeley, CA), mRFP (WB, 1:500; Chemicon International, Temecula, CA), and HA tag (clone 12CA5, mIgG; WB, 1:500).

Techniques: Plasmid Preparation, Transfection, Stable Transfection