Article Figures & Data
Figures
- Figure 1.
Generation of Siva conditional knockout mice. A, targeting scheme for generating Siva conditional knockout mice. The Siva-targeting vector contains a positive selection marker [Puromycin cassette (Puro)] flanked by loxP sites (triangles) and a negative selection marker [diphtheria toxin (dTA)]. The four exons comprising the Siva locus (gray boxes) are flanked by loxP and Lox-Puro-Lox sites on the 5′ and 3′ ends, respectively. The Puro cassette was removed in vivo by limited Cre expression, leaving a single 3′ loxP site. Upon subsequent Cre recombinase expression, the Siva locus gets excised, resulting in a Siva-null allele. These recombination events were detected by Southern blot analysis using Xmn1/EcoR1 restriction digests and subsequent probing with a 5′ or 3′ fragment external to the targeting vector. This leads to generation of fragments of different sizes in all cases, as shown. B1, BamH1; E1, EcoR1; Xm1, Xmn1; Xh1, Xho1. B, Southern blot analyses of mouse embryonic stem (ES) cells targeted at the Siva locus. Analyses of a wild-type (Siva+/+) and a targeted (Sivafl/+) ES cell clone are shown. DNA was digested with Xmn1/EcoR1. Left, upon probing with the 3′ probe, the 11-kb band indicates the wild-type allele and the 8.7-kb band indicates the targeted conditional allele. Right, upon probing with the 5′ probe, the 8.5-kb band indicates the wild-type allele and the 4.5-kb band indicates the targeted conditional allele. C, PCR analysis of recombined allele. MEFs generated from E13.5 Sivafl/− embryos (where fl denotes the conditional knockout allele) were infected either with adenovirus-expressing Cre (Ad-Cre) to excise the Siva floxed allele or with empty adenovirus (Ad-Emp) as a control. Primers spanning the 5′ loxP site (F1 and R1) or the remaining loxP site after excision of the Siva locus (F1 and R2) were used. The absence of the floxed allele following Ad-Cre infection verifies the ability of Cre to fully excise the Siva locus.
- Figure 2.
Siva deficiency inhibits lung tumorigenesis. A, schematic of the timeline for tumor study. Six- to 8-week-old mice were infected with Ad-Cre via intratracheal injection, and lungs were analyzed 18 weeks later. B, representative photomicrographs of lungs from KrasLSL-G12D;Siva+/+ (left, n = 12) and KrasLSL-G12D;Sivafl/− (right, n = 17) mice 18 weeks after Ad-Cre infection by intratracheal injection. C, box plot depicting the median number and quartiles of hyperplasias and tumors (adenomas and adenocarcinomas) per total lung area in KrasLSL-G12D;Siva+/+ and KrasLSL-G12D;Sivafl/− mice. Dots represent outlier data points. Hyperplasias: **, P = 0.008; tumors: **, P = 0.0058 by the Wilcoxon rank sum test between KrasLSL-G12D;Siva+/+ and KrasLSL-G12D;Sivafl/− mice. D, box plot depicting median tumor burden and quartiles calculated as tumor area per total lung area for hyperplasias and tumors (adenomas and adenocarcinomas). Dots represent outlier data points. Hyperplasia: P = 0.066; tumors: ***, P = 0.003 by Wilcoxon rank-sum test between KrasLSL-G12D;Siva+/+ and KrasLSL-G12D;Sivafl/− mice. E, representative photomicrographs of hyperplasias and tumors (adenomas and adenocarcinomas) taken at ×100 and ×400 magnification.
- Figure 3.
Siva knockdown inhibits cellular proliferation and transformation in mouse NSCLC cells. A, top, Western blot analysis of SIVA in LSZ4 and LSZ2 NSCLC cell lines. Two independent shRNAs targeting Siva were used to knock down Siva. shRNA targeting GFP was used as a negative control. ACTIN serves as a loading control. Bottom, quantification of SIVA protein levels following expression of two Siva shRNAs compared with expression of shGFP, after normalization to ACTIN. B, left, average percentage of BrdUrd incorporation upon Siva knockdown in LSZ4 and LSZ2 cells compared with control knockdown with shGFP. Error bars, ±SD. P values by the Student t test comparing each shRNA to shGFP: LSZ4: **, P = 0.006, 0.002; LSZ2: **, P = 0.003, 0.007. Right, representative images of BrdUrd immunofluorescence. Blue, DAPI; red, BrdUrd. C, Left, average number of colonies in low plating assay upon Siva knockdown in LSZ4 and LSZ2 cells compared with control knockdown with shGFP. Error bars, ±SD. P values by the Student t test: LSZ4: ***, P = 0.0002; **, P = 0.0025; LSZ2: ***, P = 0.0004, 0.0001. Right, representative images of colonies in low plating assay stained with crystal violet. D, Top, average number of colonies in soft-agar assay upon Siva knockdown in LSZ4 cells relative to control knockdown with shLacZ. Error bars, ±SD. *, P value by the Student t test: 0.047. Bottom, representative images of colonies in soft-agar assay stained with Giemsa. DAPI, 4,6-diamidino-2-phenylindole.
- Figure 4.
SIVA knockdown inhibits cellular proliferation and transformation in human NSCLC cells. A, SIVA expression in A549 cells, as assessed by quantitative RT-PCR normalized to β-Actin, upon expression of shRNA targeting SIVA or shRNA targeting GFP. B, representative cellular proliferation assay in A549 cells over 7 days following SIVA or control GFP shRNA transduction. The experiment was repeated in duplicate with two independent shRNAs to SIVA each time. C, average percentage of BrdUrd incorporation in SIVA knockdown cells. Error bars, ±SD. P value by the Student t test: 0.058. D, left, average number of colonies in low plating assay following SIVA or control GFP shRNA transduction. Error bars, ±SD. Right, representative images of colonies in low plating assay stained with crystal violet. *, P value by the Student t test: 0.05. E, left, average number of colonies in soft-agar assay upon knockdown of SIVA relative to control cells expressing shGFP. Error bars, ±SD. **, P value by the Student t test: 0.0013. Right, representative images of soft-agar assay stained with Giemsa. F, survival curve from human NSCLC patients generated using KMplot.com and gene expression from the SIVA1 probe 203489_at. SIVA expression levels: low, black; high, gray.
- Figure 5.
Phenotypes induced by Siva knockdown are p53-independent. A, Western blot analysis of p53 levels upon Siva knockdown in LSZ4 cells. Western blot analysis for SIVA shows knockdown with shSiva relative to shGFP. ACTIN serves as a loading control. B, qRT-PCR analysis of p53 target gene expression, after normalization to β-Actin, in NSCLC cells transduced with shSiva or shGFP. Error bars, ±SD. n = 2. C, Western blot analysis of p53 and SIVA upon knockdown of Trp53 and/or Siva in LSZ4 cells. ACTIN was used as a loading control. D, average of percentage of BrdUrd incorporation in LSZ4 cells upon knockdown of Trp53 and/or Siva. shGFP and shLuc served as controls. Error bars, ±SD. n = 3. E, phase-contrast images of LSZ4 cells upon knockdown of Trp53 and/or Siva. F, left, images of colony formation in soft-agar assays performed in Trp53-null NSCLC cells with knockdown of Siva. Right, average number of colonies formed relative to shLacZ-transduced cells in soft-agar assays. Error bars, ±SD; n = 3. **, P = 0.01.
- Figure 6.
SIVA loss decreases metabolic function of NSCLC cells. A, heatmap of gene expression based on hierarchical clustering of microarray data from LSZ2 and LSZ4 cells with control knockdown using two control hairpins (shGFP and shLacZ; red) or knockdown of Siva using two independent hairpins (shSiva1 and shSiva2; blue). Genes identified for the heatmap were significantly modulated (P value less than or equal to 0.05) and had a fold change equal to or greater than 1.5. Yellow signifies downregulated genes, and blue signifies upregulated genes. P value for each gene is denoted on the right-hand side with either red (high) or blue (low) bars. B, GO term analysis of genes with altered expression upon Siva shRNA transduction into LSZ2 and LSZ4 cells relative to cells with control shRNA transduction. The P value for each category is shown on the right side. GO term analysis was performed using GeneSpring-GX software (Agilent). C, OCR and ECAR in LSZ4 cells upon Siva knockdown or in shGFP-transduced control cells. Oligo, oligomycin [ATP synthase inhibitor (electron transport chain inhibitor)]; FCCP (uncoupler); anti, antimycin (proton gradient disrupter). *, P value by the Student t test: 0.024. D, mitochondrial DNA content assessed by quantitative PCR for mitochondrial-encoded genes upon knockdown of Siva in LSZ4 cells and in shGFP-transduced control cells. Normalized to β2-microglobulin. *, P < 0.05; **, P < 0.01; ***, P < 0.005. E, top, Western blot analysis of autophagy related protein LC3-II in LSZ4 cells upon shSiva or shGFP control transduction. ACTIN used as a loading control. Bottom, quantification of LC3-II levels upon Siva knockdown without (top) and with Bafilomycin A1 (Baf; bottom), relative to ACTIN. P value by the Student t test: *, 0.05; **, 0.007. F, average percentage of BrdUrd incorporation in LSZ4 cells with control (shLacZ) or Siva (shSiva) knockdown in the absence (untreated) or presence of chloroquine. Cells were incubated with 100 nmol/L chloroquine for 18 hours prior to BrdUrd pulse. Graph represents average ±SD of three experiments. *, P < 0.05; **, P < 0.01; ns, not significant by the Student t test. n = 3.
- Figure 7.
SIVA loss decreases metabolic function of NSCLC cells. A, gene set enrichment analysis (GSEA) of genes with altered expression upon Siva shRNA transduction into LSZ2 and LSZ4 cells relative to cells with control shRNA transduction. The FDR P value and normalized enrichment score (NES) for each category are shown on the right side. The GSEA signature for decreased mTOR signaling is highlighted in gray. B, select GSEA profiles (left) enrichment of genes upregulated upon mTOR inhibition in the presence of AKT upregulation. FDR q-value: 0.039 (right) enrichment of genes downregulated upon mTOR activation. FDR q-value: 0.069. C, Western blot analysis of mTOR signaling targets in LSZ4 cells upon shSiva or shGFP control transduction. ACTIN serves as a loading control. D, average percentage of BrdUrd incorporation in LSZ4 cells treated with 50 nmol/L Torin1 or 50 nmol/L rapamycin for 18 hours. Error bars, ±SD. P value by the Student t test: *, Torin1: 0.048; **, rapamycin: 0.009. E, Western blot analysis of mTOR upstream signaling components in LSZ4 cells upon shSiva or shGFP control transduction. ACTIN serves as a loading control. F, average percentage of BrdUrd incorporation in LSZ4 cells with control (shLacZ) or Siva (shSiva) knockdown and without (siControl) or with TSC2 (siTsc2) knockdown. Error bars, ±SD; *, P < 0.05; **, P < 0.01 by the Student t test. G, model depicting the role of SIVA in promoting tumorigenesis through activation of mTOR signaling, which can itself affect metabolism, proliferation, tumorigenesis, and autophagy. SIVA may also have mTOR-independent effects on these processes.
Additional Files
Supplementary Data
Files in this Data Supplement:
- Supplementary Figure Legends - Supplementary Figure Legends
- Supplementary Figure 1 - <i>Siva</i> heterozygous mice display reduced tumor number and tumor burden.
- Supplementary Figure 2 - <i>Siva</i> loss does not affect proliferation in non-lung cancer cell lines.
- Supplementary Figure 3 - <i>Siva</i> knockdown does not induce apoptosis.
- Supplementary Figure 4 - <i>Siva</i> loss does not affect NFkappaB signaling.
- Supplementary Table 1 - qPCR Primer List.
Volume 5, Issue 6
