Fig 1: Heregulin treatment of LNCaP cells disrupts AR-SLIRP association through a non-Erk dependent pathway. (A) Heregulin induces loss of AR-SLIRP interaction. LNCaP cells were treated with heregulin (10 ng/ml), EGF (100 ng/ml), Gas6 (100 ng/ml), interleukin-6 (10 ng/ml) or bombesin (1 nM) for 60 min before harvesting. Protein extracts were immunoprecipitated with the AR antibody and immunoblotted with the indicated antibodies. Whole cell lysates were immunoblotted with the indicated antibodies. (B) Dasatinib but not U0126 inhibits heregulin-induced AR-SLIRP disassociation. LNCaP cells were pretreated with dasatinib (10 nM), U0126 (10 μM) or vehicles for 2 hrs before treatment with heregulin (10 ng/ml) for 60 min. Protein extracts were immunoprecipitated with the AR antibody and immunoblotted with the indicated antibodies. (C) Known AR tyrosine phosphorylation sites do not contribute to Ack1-induced AR-SLIRP dissociation. 293 T cells were transfected with the expression vectors encoding AR wild-type (wt), Y267F, Y363F, or Y534F (1 μg of each), along with constitutively active Ack1 (1 μg) or empty vector (1 μg). After 24 hrs, protein extracts were immunoprecipitated with the AR antibody and immunoblotted with the antibodies against SLIRP or AR. (D) Lack of the ligand-binding domain of AR impairs AR-SLIRP interaction. 293 T cells were transfected with the expression vectors encoding FLAG-tagged full-length (FL) AR (2 μg), truncated (T) AR (amino acids 1–660) (2 μg), or empty vector (2 μg). After 24 hrs, protein extracts were immunoprecipitated with the FLAG antibody and immunoblotted with the antibodies against SLIRP or AR. Immunoblotting of whole cell extracts was performed as indicated.
Fig 2: Ack1 and androgen ligand disrupt the interaction between AR and SLIRP. (A) Ack1 activation abrogates association between AR and SLIRP in 293 T cells. 293 T cells were transfected with the expression vectors encoding AR (1 μg), along with constitutively active Ack1 (ca) (1 μg), wild-type Ack1 (wt) (1 μg), kinase-dead Ack1 (kd) (1 μg), or empty vector (1 μg). After 24 hrs, protein extracts were immunoprecipitated with AR antibody and immunoblotted with antibodies against SLIRP, AR, or Ack1. Whole cell lysates (WCL) were immunoblotted with the indicated antibodies. (B) DHT treatment abrogates interaction of AR and SLIRP in 293 T cells. 293 T cells were transfected as indicated and treated with DHT (10 nM). After 24 hrs, immunoprecipitation and immunoblotting were performed using the indicated antibodies. (C) DHT treatment and Ack1 activation disrupt interaction between AR and SLIRP in LNCaP cells. For left two lanes, LNCaP cells were treated with DHT (10 nM) or vehicle for 24 hrs. For right two lanes, LNCaP cells were transfected with the expression vectors encoding constitutively active Ack1 (ca), or empty vector for 24 hrs. Protein extracts were immunoprecipitated with AR antibody and immunoblotted with antibodies against SLIRP or AR. Whole cell lysates were immunoblotted with the indicated antibodies. (D) Immunoprecipitation of SLIRP demonstrates that Ack1 and DHT disrupt interaction between AR and SLIRP. 293 T cells were transfected with the expression vectors encoding AR (1 μg) and FLAG-tagged SLIRP (1 μg), along with constitutively active Ack1 (1 μg) or empty vector (1 μg). Cells were treated with DHT (10 nM) or vehicle. After 24 hrs, protein extracts were immunoprecipitated with FLAG antibody and immunoblotted with the antibody against SLIRP. Whole cell lysates were immunoblotted with the indicated antibodies.
Fig 3: Whole transcriptome analysis identifies three groups of AR target genes regulated by SLIRP. LNCaP cells were transfected with nonsense control siRNA or SLIRP-siRNA and then treated with vehicle or 1 nm DHT for another 24 hrs. RNA isolated from 4 replicates of each treatment condition and subjected to high throughput sequencing. (A) The heat map of unsupervised hierarchical cluster analysis is shown. The highlighted expression level of SLIRP show the effect of SLIRP siRNA. (B) Clustering analysis using the signature of 176 androgen-regulated genes identified three classes of gene expression patterns that respond differentially to loss of SLIRP. Loss of SLIRP increased androgen-induced expression in C2 genes whereas loss of SLIRP led to decreased androgen-induced expression in C1 genes. Loss of SLIRP had little or no effect on C3 genes.
Fig 4: Antiviral signaling is amplified via the MDA5-mtRNA-SLIRP positive feedback loop.(A) Cytosolic mtRNA expression in poly I:C transfected cells with or without SLIRP (n=4). (B, C) The effect of BAK1 downregulation on the ISG (B) and mtRNA (C) induction by poly I:C. (D) Analysis of mtRNA expression upon poly I:C transfection with or without MDA5. (E) The mtRNA expression upon IFN-β treatment. (F) Western blot analysis for SLIRP localization upon poly I:C with or without MDA5. JNK is used as a marker for the cytosol fraction while AIF is used as a marker for the membrane fraction. (G, H) The expression of MDA5 mRNA (G) and protein (H) expression after poly I:C transfection in cells deficient in SLIRP. (I) The effect of SLIRP downregulation on the IRF3 phosphorylation upon poly I:C. (J) A model for the positive feedback amplification of antiviral signaling consists of mtRNA induction, cytosolic release, and MDA5 activation. Unless mentioned, three independent experiments were carried out, and error bars denote s.e.m. All of the statistical significances were calculated using one-tail Student’s t-tests; * p ≤ 0.05, ** p ≤ 0.01, and *** p ≤ 0.001.
Fig 5: Stabilization by SLIRP results in mtRNA upregulation during antiviral response.(A, B) Analysis of total (A) and strand-specific (B) mtRNA expression upon introduction of dsRNAs with different lengths in HCT116 cells. (C) smRNA-FISH analysis for the individual strand of ND5 mtRNAs in poly I:C transfected cells. Nuclei were stained using DAPI. Quantified signal intensities calculated by taking an average of over 30 images are presented below. Scale bar, 25 μm. (D) Heatmap of log2 fold changes for the two strands of mtRNAs upon poly I:C transfection. The three columns represent biological replicates. (E) Analysis of mtDNA copy number upon poly I:C transfection in HCT116 cells. (F) Normalized total mtRNA levels upon poly I:C transfection 48 h after IMT1 treatment. Data are normalized only to those of DEPC-transfected cells in order to analyze the degree of mtRNA induction by poly I:C. (G) Heatmap of the degree of mtRNA induction upon poly I:C transfection in cells transfected with the indicated siRNAs. (H, I) Analysis of total (H) and strand-specific (I) mtRNAs in control and SLIRP knocked-down HCT116 cells. Three independent experiments were carried out, and error bars denote s.e.m. All of the statistical significances were calculated using one-tail Student’s t-tests; * p ≤ 0.05, ** p ≤ 0.01, and *** p ≤ 0.001.
Supplier Page from Abcam for Anti-SLIRP antibody