Fig 2: H6PD knockdown reduces cancer cell migration and increases cell adhesion. A) SUM159, MCF7, and MDA-MB-453 cells were transfected with mock or H6PD siRNAs, and cell migration was assessed by a migration assay. Data represent the number of migrated cells counted after crystal violet staining in 10 independent visual fields (left) or as a percentage of migrated cells after H6PD knockdown, compared with mock-transfected cells (right). Graphs depict the means from at least 3 independent experiments ± sd in SUM159 cells. mRNA analysis of the indicated genes 48 h after H6PD knockdown in SUM159 cells. B) SUM159 cells were transfected with mock or H6PD siRNAs and reseeded after 72 h on fibronectin-coated plates or were left untreated. Adhesion was evaluated spectrometrically after crystal violet staining. Absorbance data were normalized to the values of mock-transfected cells on fibronectin-coated wells. C) qPCR analysis of mRNA levels for the depicted genes, 48 or 72 h after siRNA transfection in SUM159 cells. D) SUM159 cells were transfected with H6PD siRNA or mock siRNA and, 72 h later, were subjected to immunofluorescence with an antibody against a-tubulin. E) Western blot analysis of a-tubulin levels in SUM159 cells 72 h after knockdown of H6PD compared to mock-transfected cells. Ns, nonsignificant. *P < 0.05, **P < 0.01, ***P < 0.001 vs. mock siRNA.

Fig 3: Silencing of H6PD leads to an increase in the ER redox status and mitochondrial superoxide levels. A) SUM159 cells were transfected with mock or H6PD siRNAs, and redox changes were observed 48 and 72 h after treatment, using the ER-targeted ratiometric probe roGFP1-iEER. The OxD value was determined from the ratios of fluorescence emission intensities after excitation at 405 and 488 nm. Means ± sd were calculated from 3 independent experiments. Representative graphs of the ratio of fluorescence intensities at baseline, after full oxidation and full reduction of the sensor are shown from 2 H6PD- and 2 mock-siRNA–transfected cells at each time point. B) H6PD was down-regulated in SUM159 cells, and 48 or 72 h later, superoxide levels were monitored using the red indicator under normal conditions or after addition of ethanol as stressor. The measurements were performed in real time by using high-throughput imaging, and the values represent the mean of 3 independent experiments. Baseline and stressed levels were documented in each experiment and are shown separately for better comprehension of scale. For simplicity, only the statistical significance of the latest time point is depicted in every graph. Ns, nonsignificant. **P < 0.01, ***P < 0.001 vs. mock siRNA.

Fig 4: Effect of H6PD knockdown on ER folding proteins. H6PD or mock-transfected SUM159 cells were analyzed by Western blot, as in Fig. 3, for estimation of levels of the heat shock family proteins Grp94 and -78 (A), the PDI family proteins PDI, ERp44, and ERp72 (B) and the chaperones calreticulin and calnexin (C). Ns, nonsignificant. *P < 0.05 vs. mock siRNA.

Fig 5: Alterations in UPR signaling after H6PD silencing. SUM159 cells were mock or H6PD siRNA transfected. A, B) Western blot analysis was performed at 72 h after transfection for semiquantitative analysis of protein levels of PERK, eIF2a, peIF2a, and ATF4 (A) and ATF6, sXBP1, and CHOP (B). A representative Western blot image is shown (left) in addition to the relative protein levels measured from 3 independent experiments by densitometric analysis of the bands after normalization to actin loading control levels (right). C) mRNA expression of ATF4, ATF6, and CHOP 72 h after H6PD down-regulation compared to mock-transfected cells, as assessed through qPCR. Values were normalized to the levels of the gene prolyl cis-trans isomerase A. Ns, nonsignificant. *P < 0.05, **P < 0.01, ***P < 0.001 vs. mock siRNA.