Journal: Cancer Research

Article Title: Blocking Platelet-Derived Growth Factor-D/Platelet-Derived Growth Factor Receptor β Signaling Inhibits Human Renal Cell Carcinoma Progression in an Orthotopic Mouse Model

doi: 10.1158/0008-5472.can-04-4313

Figure Lengend Snippet: Figure 1. PDGF-D is overexpressed in human renal cell carcinoma samples. Normal kidney tissue in the tissue microarray stains with anti–PDGF-D antibody in collecting tubes (A-D). In clear cell type (E and G), granular cell type (F), and granular and clear cell type (H) tumor tissues, PDGF-D stains strongly and homogenously. The arrow indicates positive staining. I, isotype control staining of the tumor tissues. Antigoat immunoglobulin stains negatively of the tumor tissues. Bar, 50 Am.

Article Snippet: Tissue microarray slides bearing 50 human renal cell carcinoma tumor sections and nine normal human kidney sections were purchased from Imgenex (San Diego, CA).

Techniques: Microarray, Staining, Control

Journal: Oncogene

Article Title: ISCA2 inhibition decreases HIF and induces ferroptosis in clear cell renal carcinoma

doi: 10.1038/s41388-022-02460-1

Figure Lengend Snippet: A Schematic showing sample preparation workflow for DARTS assay. Lysates from ACHN cells that were exposed to 1% O 2 for 16 h were divided into 4 equal samples and subjected to indicated treatments. B (i) Mass spectrometry data from DARTS assay indicating intensity of ISCA2 fragments in cell lysates treated with Pronase together with #1 or DMSO (Con); (ii) Western blot showing effects of control or Pronase treatment in ACHN cell lysates treated with indicated concentrations of #1. Densitometric analysis of ISCA2 band intensities normalized to actin is shown above blots. B , C Thermal shift assays showing effects of incubation of recombinant ISCA2 (4 μg) with ( B ) DMSO (green) or 200 μM #1 (magenta), or ( C ) DMSO (green) or 100 µM #25 (red). Quantitation of ISCA2 melting temperatures are shown inset (mean ± SEM). D Effects of transfections with non-targeting (Con) or ISCA2 (A2) siRNA on indicated proteins in hypoxic ACHN cells. Knockdown was performed using two consecutive siRNA transfections over 72 h each. E – G Iron content of 786-0 cells treated with ( E ) #1, ( F ) #25, for 24 h; or ( G ) transfected with siCon or siISCA2 (6 days) as determined using ICP-MS.

Article Snippet: TMAs and tissue were stained for ISCA2 (HPA030492 Atlas antibodies, Bromma, Sweden) using conditions optimized using normal kidney according to the manufacturer’s protocols using the BenchMark Ultra automated slide stainer (Ventana Medical Systems, Roche, Oro Valley, AZ).

Techniques: Sample Prep, Mass Spectrometry, Western Blot, Control, Incubation, Recombinant, Quantitation Assay, Transfection, Knockdown

Journal: Oncogene

Article Title: ISCA2 inhibition decreases HIF and induces ferroptosis in clear cell renal carcinoma

doi: 10.1038/s41388-022-02460-1

Figure Lengend Snippet: A Western blots showing effect of 6-day siRNA knockdown of ISCA1 (A1), ISCA2 (A2), IBA57 (A57) or non-targeting siRNA (Con) in ACHN cells in 20% O 2 or after 6 h at 1% O 2 ± the proteasome inhibitor MG132 (5 μM and 1 μM for 6 h in 20% and 1% O 2 respectively). Densitometric analysis of ISCA2 band intensities normalized to GAPDH is shown in S A. B Effect of 2- and 4-day knockdown of ISCA2 using a second ISCA2 siRNA construct (A#2) in ACHN cells in 1% O 2 . Densitometric analysis of ISCA2 band intensities normalized to actin is shown above blots. C Effect of 6-day knockdown with indicated siRNAs in RCC4 cells. D Effect of transfection of indicated siRNAs on the transcription of HIF1A and EPAS1 normalized to B2M in ACHN cells. E Effect of ISCA2 siRNA on HIF-1/2α translation in RCC4 cells. Cells were transfected with ISCA2 siRNA for 3 days, then labelled with puromycin. Puromycin incorporation was determined by immunoprecipitation of HIF-1/2α followed by western blot using an antibody against puromycin. F Effect of 5-day siRNA knockdowns on metals accumulation in ACHN cells detected using ICP-MS. Approximately equal cell numbers were submitted for analysis. All data are representative or averages of at least two independent experiments shown as mean ± SEM.

Article Snippet: TMAs and tissue were stained for ISCA2 (HPA030492 Atlas antibodies, Bromma, Sweden) using conditions optimized using normal kidney according to the manufacturer’s protocols using the BenchMark Ultra automated slide stainer (Ventana Medical Systems, Roche, Oro Valley, AZ).

Techniques: Western Blot, Knockdown, Construct, Transfection, Immunoprecipitation

Journal: Oncogene

Article Title: ISCA2 inhibition decreases HIF and induces ferroptosis in clear cell renal carcinoma

doi: 10.1038/s41388-022-02460-1

Figure Lengend Snippet: A Effect of co-treatment of ISCA2 siRNA and DMSO or liproxstatin (2.5 μM) on cell viability determined using resazurin in ACHN cells. siRNA transfection was performed for a total of 5 days with liproxstatin (or DMSO) added for the last 24 h. B , C Cell viability assays (resazurin) of 786-0 cells treated with ( B ) #1 or ( C ) #25, ± DFO (100 μM), liproxstatin (1 μM) or ZVAD-FMK (20 μM); or ( H ) #25 ± DFO (100 μM), NAC (1 mM), liproxstatin (1 μM) or ZVAD-FMK (20 μM). Treatments were performed for 24 h. Average IC 50 values (µM) are shown in brackets. D Impact of #25 treatment (48 h) on BODIPY 581/591 fluorescence detected by flow cytometry in 786-0 cells. E Impact of 48 hours’ treatment with #25, PT2385 (PT) or 6 hours’ treatment with RSL3 on malondialdehyde (MDA), an indicator of lipid peroxidation in RCC10 cells. F Transmission electron microscopy of RCC10 cells treated with DMSO or #25 for 24 h (2 representative micrographs of each condition). DMSO-treated cells show normal mitochondria whereas #25-treated cells show damaged mitochondria including lost or irregular cristae (white arrows) and irregular matrix with voids (yellow arrows).

Article Snippet: TMAs and tissue were stained for ISCA2 (HPA030492 Atlas antibodies, Bromma, Sweden) using conditions optimized using normal kidney according to the manufacturer’s protocols using the BenchMark Ultra automated slide stainer (Ventana Medical Systems, Roche, Oro Valley, AZ).

Techniques: Transfection, Fluorescence, Flow Cytometry, Transmission Assay, Electron Microscopy

Journal: Oncogene

Article Title: ISCA2 inhibition decreases HIF and induces ferroptosis in clear cell renal carcinoma

doi: 10.1038/s41388-022-02460-1

Figure Lengend Snippet: A ISCA2 levels in pVHL mutant ccRCC cells ± stable pVHL reconstitution. B Resazurin cell viability assay of 786-0 parental or 786-0 cells with pVHL reconstitution transfected with indicated siRNAs (24 h post-transfection). C , D Representative plot showing viability (resazurin) of RCC4 and RCC4 + VHL cells ( C ), and 786-0 and 786-0 +VHL cells ( D ), after treatment with #25 for 24 h. IC 50 values (μM) are shown in brackets. E Western blots showing levels of endogenous (Endo) and overexpressed FLAG-ISCA2 in stable clones expressing empty vector (Vec) or ISCA2 from 786-0 and RCC10 cells (2 ISCA2 clones each). F , G Representative plots showing viability of RCC10 ( F ), or 786-0 ( G ) cells stably expressing empty vector (Vec) or ISCA2 treated with #25 for 24 h. IC 50 values (μM) are shown in brackets. H Representative plots showing viability of RCC10 cells stably expressing empty vector (Vec) or ISCA2 treated with Erastin for 24 h. IC 50 values (μM) are shown in brackets.

Article Snippet: TMAs and tissue were stained for ISCA2 (HPA030492 Atlas antibodies, Bromma, Sweden) using conditions optimized using normal kidney according to the manufacturer’s protocols using the BenchMark Ultra automated slide stainer (Ventana Medical Systems, Roche, Oro Valley, AZ).

Techniques: Mutagenesis, Viability Assay, Transfection, Western Blot, Clone Assay, Expressing, Plasmid Preparation, Stable Transfection

Journal: Oncogene

Article Title: ISCA2 inhibition decreases HIF and induces ferroptosis in clear cell renal carcinoma

doi: 10.1038/s41388-022-02460-1

Figure Lengend Snippet: A Immunohistochemistry showing representative expression of ISCA2 in normal kidney (top) and in ccRCC (bottom). B Quantitation of ISCA2 H-Score in paired uninvolved and ccRCC cores from 19 patients. **** p < 0.0001 determined via Mann Whitney test. C , D Kaplan–Meier curves showing associations of ( C ) ISCA2 protein, or ( D ) ISCA2 transcript levels above (high) or below (low) the median with overall survival using a tumor microarray (TMA; 94 cases) or data from TCGA-KIRC (522 cases) respectively.

Article Snippet: TMAs and tissue were stained for ISCA2 (HPA030492 Atlas antibodies, Bromma, Sweden) using conditions optimized using normal kidney according to the manufacturer’s protocols using the BenchMark Ultra automated slide stainer (Ventana Medical Systems, Roche, Oro Valley, AZ).

Techniques: Immunohistochemistry, Expressing, Quantitation Assay, MANN-WHITNEY, Microarray

Journal: Oncogene

Article Title: ISCA2 inhibition decreases HIF and induces ferroptosis in clear cell renal carcinoma

doi: 10.1038/s41388-022-02460-1

Figure Lengend Snippet: A , B Effect of treatment of 786-0 subcutaneous xenografts with vehicle or indicated doses of compound #25 (8 mice/group) administered orally once daily, on tumor volume ( A ), or mouse body weights ( B ). ** p < 0.01; *** p < 0.001; **** p < 0.0001 from Students’ T -tests of vehicle versus 60 mg/kg treated group. p was not significant (NS) for 30 mg/kg treated group versus vehicle. C Effect of treatment of RENCA subcutaneous xenografts in syngeneic Balb/c mice with vehicle or 60 mg/kg #25 (8 mice/group) once daily. D Western blots showing HIF-1α and GAPDH with densitometric values normalized to GAPDH below. E MDA content of RENCA tumors from mice treated with vehicle or 60 mg/kg #25. F Proposed model for effects of ISCA2 blockade. Inhibition of ISCA2 using either small molecules or siRNA results in perceived iron deprivation, which induces upregulation of IRP2, TFRC and downregulation of FTH1, that together promote iron/metals accumulation that triggers cell death via ferroptosis, and also drives the later downregulation of IRP1, IRP2 and TFRC. This perceived iron deprivation also drives a shift to the IRE-binding form of IRP1 which inhibits HIF-2α translation. ISCA2 inhibition may also cause other mitochondrial/extramitochondrial defects dependent or independent of perceived iron deprivation that inhibits the translation of HIF-1α through unknown mechanisms. Thus, ISCA2 inhibition depletes both HIF-1α and HIF-2α and promotes cell death via ferroptosis.

Article Snippet: TMAs and tissue were stained for ISCA2 (HPA030492 Atlas antibodies, Bromma, Sweden) using conditions optimized using normal kidney according to the manufacturer’s protocols using the BenchMark Ultra automated slide stainer (Ventana Medical Systems, Roche, Oro Valley, AZ).

Techniques: Western Blot, Inhibition, Binding Assay