Journal: Nature Communications

Article Title: Trifunctional sphingomyelin derivatives enable nanoscale resolution of sphingomyelin turnover in physiological and infection processes via expansion microscopy

doi: 10.1038/s41467-024-51874-w

Figure Lengend Snippet: a , b TFSMs are enriched within the Golgi and mitochondria. HeLa cells were treated with TFSM 1 for 2 h ( a ) or 24 h ( b ). Samples were fixed and clicked to BODIPY-FL-DBCO (backbone) or Atto647N-azide (headgroup). Golgi and mitochondria were visualized by an anti-Golgi matrix protein 130 (GM130) and anti-Peroxiredoxin (Prx3) antibody, respectively. n = 1. Scale bars: 25 µm ( a ) or 5 µm ( b ). c , d FRET measurement of TFSM conversion. HeLa or human umbilical vein endothelial cells (HuVEC) were incubated with TFSM 1, TFSM 2, or BODIPY-FL-C 12 -SM for 2 h. Then, the compounds were removed, and cells were either treated with bSMase or left untreated for 3 h. Samples treated with TFSMs were fixed, clicked with BODIPY-FL-DBCO (backbone) and AlexaFluor TM 546 (AF)546-azide (headgroup) and FRET efficiency was determined by acceptor bleaching. Cells treated with BODIPY-FL-C 12 -SM were detached, and lipids were extracted by CHCl 3 :MeOH. The proportion of unmetabolized SM was determined by thin-layer chromatography. Scale bars: 25 µm. n = 3. Statistics: Two-way ANOVA and Šídák’s multiple comparisons ( d ). Bars represent means ± SD. n corresponds to biological replicates. Source data and detailed statistics are provided as a Source Data file.

Article Snippet: 7163670), 1:50 anti-Prx3 antibody (OriGene, Cat. No. TA322472) or 4 μg/ml anti-chlamydial HSP60 antibody (SantaCruz, Cat. No. sc-57840, Lot.

Techniques: Incubation, Thin Layer Chromatography

Journal: Redox Biology

Article Title: ALDH1L2 induces resistance to chemotherapy in small cell lung cancer by inhibiting ferroptosis

doi: 10.1016/j.redox.2026.104098

Figure Lengend Snippet: ALDH1L2 promotes SCLC chemoresistance by negatively regulating the hyperoxidized PRDX3 and PRDX3 dimer content in the plasma membrane. (A) Immunofluorescence detection of the colocalization of ALDH1L2 and PRDX3 in SCLC cells. The cell nuclei are labeled with blue fluorescence, the ALDH1L2 protein is labeled with green fluorescence, and the PRDX3 protein is labeled with red fluorescence. Scale bar: 20 μm. (B) Coimmunoprecipitation assays revealed the interaction of the ALDH1L2 protein with the PRDX3 protein in chemoresistant SCLC cells. (C) Coimmunoprecipitation assays revealed the interaction of the TRX2 protein with the ALDH1L2 protein and the PRDX3 protein in chemoresistant SCLC cells. (D-E) The reaction of PRDX3 with hydroperoxides was analyzed by immunoblotting. Bar plots show the results of the semiquantitative analysis of the gray values of the protein bands by ImageJ software. (F–H) Analysis of the effect of ALDH1L2 knockdown on the content of hyperoxidized PRDXs protein in each fraction of H69AR and H446DDP cells. The results under no treatment, cisplatin treatment (10 μg/mL for 24 h) and erastin treatment (20 μM for 72 h) are shown in the graph. Bar plots show the results of the semiquantitative analysis of the gray values of the protein bands by ImageJ software. (I) Coimmunoprecipitation assays revealed that hyperoxidized PRDXs protein could be immunoprecipitated by anti-PRDX3 antibody. (J-N) Analysis of the effect of ALDH1L2 knockdown on the content of oxidized PRDX3 dimers in each fraction of H69AR and H446DDP cells. The results under no treatment, cisplatin treatment (10 μg/mL for 24 h) and erastin treatment (20 μM for 72 h) are shown in the graph. LE, long exposure; SE, short exposure. Bar plots show the results of the semiquantitative analysis of the gray values of the protein bands by ImageJ software. The data are expressed as mean ± standard deviation (n = 3). ns, not significant; ∗, p < 0.05; ∗∗, p < 0.01; ∗∗∗, p < 0.001; ∗∗∗∗, p < 0.0001.

Article Snippet: The purified PRDX3 protein was obtained from MedChemExpress (HY– P71147 , MCE, USA).

Techniques: Clinical Proteomics, Membrane, Immunofluorescence, Labeling, Fluorescence, Western Blot, Software, Knockdown, Immunoprecipitation, Standard Deviation

Journal: Redox Biology

Article Title: ALDH1L2 induces resistance to chemotherapy in small cell lung cancer by inhibiting ferroptosis

doi: 10.1016/j.redox.2026.104098

Figure Lengend Snippet: PRDX3 contributes to the ALDH1L2-mediated chemoresistance program in SCLC. (A-D) RT-qPCR was used to verify the knockdown and overexpression efficiency of PRDX3 in SCLC cells at the mRNA level. (E-L) Immunoblotting was performed to validate the knockdown and overexpression efficiency of PRDX3 in SCLC cells at the protein level. Bar plots show the results of the semiquantitative analysis of the gray values of the protein bands by ImageJ software. (M − P) CCK-8 assay to determine the role of PRDX3 in the regulation of chemoresistance elicited by ALDH1L2 in SCLC cells. IC50, half maximal inhibitory concentration. The data are expressed as mean ± standard deviation (n = 3). ns, not significant; ∗, p < 0.05; ∗∗, p < 0.01; ∗∗∗, p < 0.001; ∗∗∗∗, p < 0.0001.

Article Snippet: The purified PRDX3 protein was obtained from MedChemExpress (HY– P71147 , MCE, USA).

Techniques: Quantitative RT-PCR, Knockdown, Over Expression, Western Blot, Software, CCK-8 Assay, Concentration Assay, Standard Deviation

Journal: Redox Biology

Article Title: ALDH1L2 induces resistance to chemotherapy in small cell lung cancer by inhibiting ferroptosis

doi: 10.1016/j.redox.2026.104098

Figure Lengend Snippet: The PRDX3 inhibitor thiostrepton synergizes with chemotherapy to suppress tumor growth in SCLC. (A) CCK-8 assay to determine the IC50 values of thiostrepton in human normal bronchial epithelial BEAS-2B cells and SCLC cells (n = 3). (B–C) Verification of the inhibitory effect of thiostrepton on PRDX3 in SCLC cells by immunoblotting. Bar plots show the results of the semiquantitative analysis of the gray values of the protein bands by ImageJ software (n = 3). (D) The growth of tumors in the orthotopic SCLC model was recorded with the live animal imaging system (n = 5). (E) Line chart demonstrating the quantitative bioluminescence intensities for each group. (F) Growth curves of xenografted tumors derived from H69AR cells under different treatments are shown (n = 5). The tumor volume was calculated as follows: (longest diameter) × (shortest diameter) 2 /2. (G) Xenografted tumors derived from H69AR cells under different treatments were removed and photographed after the mice were euthanized. (H) Weights of xenografted tumors derived from H69AR cells under different treatments are shown. IC50, half maximal inhibitory concentration. The data are expressed as mean ± standard deviation. ns, not significant; ∗, p < 0.05; ∗∗, p < 0.01; ∗∗∗, p < 0.001; ∗∗∗∗, p < 0.0001.

Article Snippet: The purified PRDX3 protein was obtained from MedChemExpress (HY– P71147 , MCE, USA).

Techniques: CCK-8 Assay, Western Blot, Software, Imaging, Derivative Assay, Concentration Assay, Standard Deviation

Journal: Human molecular genetics

Article Title: Protein misfolding and clearance in the pathogenesis of a new infantile onset ataxia caused by mutations in PRDX3.

doi: 10.1093/hmg/ddac146

Figure Lengend Snippet: Figure 1. Brain MRI and pedigree of the proband. (A–F) Consecutive brain MRI of the patient from onset of ataxia at 19 months to 6.5 years of age. (A–E) Midsagittal T1-weighted images showing vermian atrophy over time. (F–K) Coronal FLAIR images revealing atrophy of cerebellar hemispheres and early appearance of cerebellar cortical hyperintensities (red arrow head). (L) Quantitative analysis using the midsagittal vermis relative diameter (MVRD). The vermis diameter/total posterior cranial fossa diameter ratio (%) was used to represent the proportion of both values. (M) Pedigree of the proband. The arrow indicates the proband MD-174. Mutation segregation PRDX3 c.489C > G (p.D163E) is below each symbol.

Article Snippet: The PRDX3 cDNA (NM_006793) was tagged with MycDDK in C-ter and cloned in pCMV6 (Origene Technologies, Inc., Rockville, MD, USA).

Techniques: Mutagenesis

Journal: Human molecular genetics

Article Title: Protein misfolding and clearance in the pathogenesis of a new infantile onset ataxia caused by mutations in PRDX3.

doi: 10.1093/hmg/ddac146

Figure Lengend Snippet: Figure 3. PRDX3 p.D163E expression in primary neurons. (A) Representative pictures of primary cortical neurons expressing at D5 either WT-PRDX3 or PRDX3 p.D163E. Boxed area shows the regions magnified and indicated as proximal and distal (solid and dashed lines, respectively). Arrowheads and red lines show the GFP positive regions that colocalized with PRDX3 protein in the neurites. Scale bar = 10 μm. (B) Schematic representation of the vector for the co-expression of PRDX3 and GFP linked by an internal ribosome entry site (IRES). (C) Upper graphs show either the ratio of the intensity of GFP and PRDX3 labeling. n = 20 control or mutant neurons. ∗P < 0.05, two-way ANOVA test. Average ± SEM. Lower graph shows the colocalization expressed as Manders’ Overlap Coefficient of PRDX3 over GFP labeling, overlapping areas are lined in red in (A). n ≥15 control or mutant neurons. ∗P < 0.05, two- way ANOVA test. Mean ± SEM. (D) Representative pictures of primary neurons at D5 expressing either PRDX3 WT or p.D163E mutant co-labeled with MitoTracker® Red. Full arrowheads indicate regions of co-localization. Scale bar = 10 μm. (E) Representative neurons at D12 expressing either PRDX3 WT or PRDX3 p.D163E mutant labeled with GFP. The box indicates the area of the neurite magnified or the right panels. Arrowheads indicate axonal swellings. Scale bar = 10 μm. (F) Quantification of neurite integrity based on the neuronal morphology. Ctrl, n = 50; PRDX3 WT, n = 109; PRDX3 mut, n = 109. ∗P < 0.05; Fisher’s exact test.

Article Snippet: The PRDX3 cDNA (NM_006793) was tagged with MycDDK in C-ter and cloned in pCMV6 (Origene Technologies, Inc., Rockville, MD, USA).

Techniques: Expressing, Plasmid Preparation, Labeling, Control, Mutagenesis

Journal: Human molecular genetics

Article Title: Protein misfolding and clearance in the pathogenesis of a new infantile onset ataxia caused by mutations in PRDX3.

doi: 10.1093/hmg/ddac146

Figure Lengend Snippet: Figure 4. Mitochondrial morphology in HeLa cells overexpressing WT-PRDX3 and mutated forms. (A) Distribution of cell percentage according to mitochondrial morphology and obtained percentages for WT and each mutation. (B–E) Quantitative analysis of mitochondrial network morphology by the ImageJ™Mito-Morphology macro. Quantification of the number of mitochondria (B), and the elongation and interconnectivity indices (C, D). (E, F) Quantitative analysis of mitochondrial network morphology by the ImageJ™plugin MiNA. Mean Branch Length is the average of the lengths of all lines used to represent the mitochondrial structures, and the footprint is the total area in the cell occupied by mitochondrial structures. Only statically significant comparatives are shown. Error bars represent SEM. ∗P < 0.05; ∗∗P < 0.01. One-way ANOVA and Tukey post hoc test.

Article Snippet: The PRDX3 cDNA (NM_006793) was tagged with MycDDK in C-ter and cloned in pCMV6 (Origene Technologies, Inc., Rockville, MD, USA).

Techniques: Mutagenesis

Journal: Human molecular genetics

Article Title: Protein misfolding and clearance in the pathogenesis of a new infantile onset ataxia caused by mutations in PRDX3.

doi: 10.1093/hmg/ddac146

Figure Lengend Snippet: Figure 5. Electron microscopy in transfected HeLa cells expressing PRDX3 p.D163E. HeLa cells were transiently transfected with plasmid DNA encoding PRDX3 p.D163E fused to Myc-DDK tag, immunostained and further processed for transmission electron microscopy (TEM) analysis. (A) Correlative light and electron microscopy (CLEM) to identify transfected cells and highlight the localization of the mutated protein (green fluorescence). (B) Enlarged picture of the square marked in (A), showing an overlay of p.D163E fluorescence signal and perinuclear clumped mitochondria at the ultrastructural level, observed in more detail in lower panels (C, D). Two additional analyzed transfected cells (E) and representative TEM high-magnification images (F– H) of the boxed regions in (E) displaying aberrant mitochondrial morphology and lipid droplets (LDs) absent in control cells. (I) Control non-transfected HeLa cells and successive higher resolution views (J–L) of the selected region in (I).

Article Snippet: The PRDX3 cDNA (NM_006793) was tagged with MycDDK in C-ter and cloned in pCMV6 (Origene Technologies, Inc., Rockville, MD, USA).

Techniques: Electron Microscopy, Transfection, Expressing, Plasmid Preparation, Transmission Assay, Fluorescence, Control

Journal: Human molecular genetics

Article Title: Protein misfolding and clearance in the pathogenesis of a new infantile onset ataxia caused by mutations in PRDX3.

doi: 10.1093/hmg/ddac146

Figure Lengend Snippet: Figure 6. PRDX3 p.D163E expression in fibroblasts and structural analysis. (A, B) PRDX3 expression in fibroblasts cultures derived from a healthy control and the patient (MD-174). (A) Immunofluorescence showing the subcellular location of WT-PRDX3 and PRDX3 p.D163E (green), the mitochondrial network with MitoTracker® Red and DAPI (blue). Scale bar = 50 μm. (B) WB analysis of the WT and mutated PRDX3 expression in both soluble and insoluble fractions. α-Tubulin was used to normalize the detected protein levels. (C) Filament organization of human PRDX3 observed in the crystal structure (PDB ID 5JCG). The subunits of each one of the stacked dodecamers are shown in different colors with different intensities for adjacent subunits. (D) Structural model of human ring-like PRDX3 dodecamer. The two subunits of each dimer are colored in light or dark blue, respectively. Localization of D163 is mapped with red spheres. (E) Detail of the intersubunit interface, where D163 is located. The side chain of D163 as well as that for the catalytic cysteine 47 is shown with stick representation and labeled.

Article Snippet: The PRDX3 cDNA (NM_006793) was tagged with MycDDK in C-ter and cloned in pCMV6 (Origene Technologies, Inc., Rockville, MD, USA).

Techniques: Expressing, Derivative Assay, Control, Immunofluorescence, Labeling

Journal: Human molecular genetics

Article Title: Protein misfolding and clearance in the pathogenesis of a new infantile onset ataxia caused by mutations in PRDX3.

doi: 10.1093/hmg/ddac146

Figure Lengend Snippet: Figure 7. Overexpression of mutated forms of PRDX3 in HeLa cells. (A) WB analysis of PRDX3 expression in transfected Hela cells with plasmids expressing WT PRDX3 and mutants. α-Tubulin was used as loading control. (B) Densitometric quantification of proteins detected by WB. Error bars represent SEM. ∗P < 0.05, ∗∗P < 0.01, one-way ANOVA followed by Dunnett’s test compared to WT. (C) Protein levels were normalized to calculate relative presence in both fractions for WT and mutated PRDX3 proteins. (D) WB analysis from transfected HeLa cells treated with MG-132 (proteasome inhibitor) or bafilomycin (autophagic inhibitor). The autophagic substrate p62 was used as a bafilomycin control and α-tubulin as loading control.

Article Snippet: The PRDX3 cDNA (NM_006793) was tagged with MycDDK in C-ter and cloned in pCMV6 (Origene Technologies, Inc., Rockville, MD, USA).

Techniques: Over Expression, Expressing, Transfection, Control

Journal: International journal of cancer

Article Title: Downregulation of the c-MYC target gene, peroxiredoxin III, contributes to arsenic trioxide-induced apoptosis in acute promyelocytic leukemia.

doi: 10.1002/ijc.24341

Figure Lengend Snippet: FIGURE 5 – Overexpression of Prx-III in U937 cells inhibits ATO- induced apoptosis. (a) Untransfected (UT) U-937 cells (Lane 1). U- 937 cells (1 3 106) were stably transfected by electroporation with 50 ng of control vector (Lane 2, pcDNA3.1/neo1) or with 50 ng of pcDNA3.1-Neo-Prdx3 vector (Lanes 3 and 4) as explained in the ‘‘Material and methods’’ section. Serial dilutions were performed to obtain individual clones with moderate (U937-14.1, Lane 3) or high (U937-13.13, Lane 4) levels of Prx III expression compared with non- transfected (U937-UT, Lane 1) U-937 cells. (b) Apoptosis was meas- ured by flow cytometry after staining with Annexin V-FITC/PI. Val- ues represent the percentage of apoptotic cells relative to the total cells. Values represent means 6 SD of triplicates.

Article Snippet: Full length human Prdx3 (OriGene, Rockville, MD) was cloned into the pcDNA3.1/neo1 mammalian expression vector (Invitrogen) using the EcoRI and NotI sites to generate the pcDNA3.1Neo- Prdx3 plasmid.

Techniques: Over Expression, Stable Transfection, Transfection, Electroporation, Control, Plasmid Preparation, Clone Assay, Expressing, Cytometry, Staining

Journal: International journal of cancer

Article Title: Downregulation of the c-MYC target gene, peroxiredoxin III, contributes to arsenic trioxide-induced apoptosis in acute promyelocytic leukemia.

doi: 10.1002/ijc.24341

Figure Lengend Snippet: FIGURE 7 – ATO treatment of NB4 cells reduces the levels of c-MYC binding to its canonical E-box in the Prdx3 gene. (a) Representative eth- idium bromide-stained agarose gels of PCR products: NB4 cells (2 3 106) were treated with 2 lM ATO for 4 and 24 hr. Samples were processed as described under ‘‘Material and methods’’. Equal volumes of soluble chromatin were used for IP with c-MYC, polymerase-II or IgG antibodies. Lane 1 corresponds to the IP with the c-MYC antibody and further PCR amplification with primers A, which encompass the c-MYC canonical recognition site in the Prdx3 gene. Lane 2 corresponds to the PCR product of the total input DNA amplified with primers A. Lane 3 corresponds to the IP with the c-MYC antibody and further PCR amplification with the primers B, which amplified a negative distal site of the Prdx3 gene. Lane 4 corresponds to the PCR product of the total input DNA amplified with the primers B. Lane 5 corresponds to the PCR products after IP with the Pol-II antibody followed by amplification with primers to GAPDH. Lane 6 corresponds to the IP with the IgG antibody followed by PCR amplification with primers A. (b) Densitometric analysis of the intensity of bands of IP/c-MYC (Lane 1) relative to input DNA (Lane 2) and of IP/c-MYC (Lane 1) relative to GAPDH (Lane 5). Percentages were calculated in relation to the untreated cells. (c) U-937 cells (1 3 106) were cotransfected with either a reporter plasmid containing the full-length (T-pGL4) Prdx3 promoter or a reporter plasmid (P-pGL4) containing only one Prdx3 canonical recognition site to c-MYC, together with a plasmid containing the human c-MYC (c-MYC-pCMV6-XL5) or with an empty vector (pCMV6-XL5). RLU were calculated as described in the ‘‘Material and methods’’. Each value represents means and 6 SD of three independent experiments.

Article Snippet: Full length human Prdx3 (OriGene, Rockville, MD) was cloned into the pcDNA3.1/neo1 mammalian expression vector (Invitrogen) using the EcoRI and NotI sites to generate the pcDNA3.1Neo- Prdx3 plasmid.

Techniques: Binding Assay, Staining, Plasmid Preparation

Journal: bioRxiv

Article Title: Oxidants are dispensable for HIF1α stability in hypoxia

doi: 10.1101/2020.05.05.079681

Figure Lengend Snippet: (A) Experimental approach employed to examine changes in the protein levels of endogenous antioxidant enzymes in hypoxia. (B-I) Immunoblots of catalase (peroxisome), GPX1 and GPX4 (cytosol and mitochondria) or MnSOD and PRDX3 (mitochondria) in (B-E) SH-SY5Y oDD Luc. cells that stably overexpressed ODD (HIF O xygen D ependent D omain-luciferase fusion) or (F-I) PCNs exposed to normoxia or hypoxia for 8h. The protein levels of catalase, GPX4 and GPX1 were normalized to actin, while those of MnSOD and PRDX3 were normalized to the mitochondrial protein, citrate synthase. A monoclonal antibody to luciferase (indicated as anti-ODD-Luc. in the figure) was used to detect changes in ODD-luciferase protein levels in SH-SY5Y ODD-Luc. cells. (J-K) Pancreatic islets were exposed to hypoxia for 2h and then were lysed and processed for mRNA expression analysis of HIF1α target genes, Kdm6b and Bnip3. The densitometric data and gene expression data were pooled from three independent experiments an presented as mean ± SD. The densitometric data and gene expression data were statistically analysed using Student’s t test (B-K). (n.s.) indicates non-significant difference, (**) indicates p<0.01 and (***) indicates p<0.001 with respect to respective normoxia controls. (L) Hypoxia leads to a large decrease in H 2 O 2 levels (top), increased NAD(P)H (middle), and decreased insulin secretion (bottom). Glucose stimulation by 20 mM glucose was added as reference, and oxygen levels were changed using an artificial gas equilibration device placed inline in the flow system. All three experiments were carried out separately, but using the same flow culture system.

Article Snippet: Protein extracts were prepared using 1% Triton buffer containing protease inhibitor, separated by SDS-PAGE, transferred onto nitrocellulose membranes and probed with antibodies against GFP (Cell Signaling Technology; 2555), MnSOD (Sigma-Aldrich; HPA001814), catalase (Sigma-Aldrich; C0979), GPX1 (Cell Signaling Technology; 3286S and Novus Biologicals; NBP1-33620), GPX4 (LSBio; LS B1596), PRDX3 (Novus Biologicals; NBP2-19777), Luciferase (Santa Cruz Biotechnology; sc-74548), HIF1α (Novus Biologicals; NB100-479) and citrate synthase (Cell Signaling Technology; 14309S).

Techniques: Western Blot, Stable Transfection, Luciferase, Expressing, Gene Expression

Journal: bioRxiv

Article Title: Oxidants are dispensable for HIF1α stability in hypoxia

doi: 10.1101/2020.05.05.079681

Figure Lengend Snippet: (A, B) Relative changes in Hiflα mRNA in SH-SY5Y cells in response to forced expression of various antioxidant enzymes. Data were pooled from three independent experiments in the form of mean ± SD. One way ANOVA with Dunnett’s post-test was used for comparing cells expressing catalase or GPX1 with respect to cells expressing GFP and Student’s t test was used for comparing cells expressing PRDX3 with respect to GFP. (n.s.) indicates non-significant difference with respect to respective GFP controls under normoxia while (ns) and (#) indicate non­significant difference and the statistical difference of p<0.05, respectively, with respect to GFP control in hypoxia. (C, D, E) Changes in half-life of HIF1α in SH-SY5Y ODD-Luc cells expressing catalase or GPX1 or PRDX3 with respect to that of respective GFP controls in hypoxia. ODD Half-life was assessed by performing a pulse chase experiment by adding 35mM cycloheximide at every twenty minutes for a total of four hours using luciferase activity assay in SH-SY5Y cells expressing these antioxidants pre-treated either with or without 10 mM MG132. Data were pooled from three independent experiments in the form of mean ± SD. Two way ANOVA with Bonferroni’s post-test was used for statistical analysis. (*), (**), and (***) indicate statistical differences of p<0.05, p<0.01 and p<0.001 with respect to respective GFP controls in hypoxia. (°°°), (♦♦♦) and (#) represent statistical differences of p<0.001 w.r.t CAT, p<0.001 w.r.t GPX1 and p<0.05 w.r.t PRDX3, respectively.

Article Snippet: Protein extracts were prepared using 1% Triton buffer containing protease inhibitor, separated by SDS-PAGE, transferred onto nitrocellulose membranes and probed with antibodies against GFP (Cell Signaling Technology; 2555), MnSOD (Sigma-Aldrich; HPA001814), catalase (Sigma-Aldrich; C0979), GPX1 (Cell Signaling Technology; 3286S and Novus Biologicals; NBP1-33620), GPX4 (LSBio; LS B1596), PRDX3 (Novus Biologicals; NBP2-19777), Luciferase (Santa Cruz Biotechnology; sc-74548), HIF1α (Novus Biologicals; NB100-479) and citrate synthase (Cell Signaling Technology; 14309S).

Techniques: Expressing, Control, Pulse Chase, Luciferase, Activity Assay

Journal: bioRxiv

Article Title: Oxidants are dispensable for HIF1α stability in hypoxia

doi: 10.1101/2020.05.05.079681

Figure Lengend Snippet: Relative changes in mRNA levels of HlFla target genes (A, B) Enolase2 and (C, D) Bnip3 in SH-SY5Y cells expressing various antioxidant enzymes as compared to that of respective GFP controls. Data were pooled from three independent experiments in the form of mean ± SD. One way ANOVA with Dunnett’s post-test was used for comparing the statistical difference between cells expressing MnSOD, catalase or GPX1 with respect to that of GFP control and Student’s t test was used to compare the statistical difference between cells expressing PRDX3 with that of GFP control. (n.s.) indicates non-significant difference with respect to respective GFP controls under normoxia only while (ns), (#) and (###) indicate non-significant difference, and the statistical differences of p<0.05, and p<0.001, respectively, with respect to GFP control in hypoxia. (E, F) Correlation between relative changes in either Enolase2 or Bnip3 and relative ODD-luciferase activities.

Article Snippet: Protein extracts were prepared using 1% Triton buffer containing protease inhibitor, separated by SDS-PAGE, transferred onto nitrocellulose membranes and probed with antibodies against GFP (Cell Signaling Technology; 2555), MnSOD (Sigma-Aldrich; HPA001814), catalase (Sigma-Aldrich; C0979), GPX1 (Cell Signaling Technology; 3286S and Novus Biologicals; NBP1-33620), GPX4 (LSBio; LS B1596), PRDX3 (Novus Biologicals; NBP2-19777), Luciferase (Santa Cruz Biotechnology; sc-74548), HIF1α (Novus Biologicals; NB100-479) and citrate synthase (Cell Signaling Technology; 14309S).

Techniques: Expressing, Control, Luciferase

Journal: Journal of cancer research and therapeutics

Article Title: PRDX3 promotes resistance to cisplatin in gastric cancer cells.

doi: 10.4103/jcrt.jcrt_970_22

Figure Lengend Snippet: Figure 2: PRDX3 is downregulated by siRNA in AGS and MKN‑74 cells. (a) Relative mRNA expression of PRDX3 in AGS after transfection with siNC and four siRNAs. (b) Relative mRNA expression of PRDX3 in MKN‑74 after transfection with siNC and four siRNAs. (c) Relative PRDX3 protein expression in AGS after transfection with siNC and siPRDX3. (d) Relative PRDX3 protein expression in MKN‑74 after transfection with siNC and siPRDX3. (e) Western blot bands of PRDX3 protein in AGS and MKN‑74 after transfection with siNC and siPRDX3

Article Snippet: PRDX3 interfering RNA (siPRDX3 group) and negative control (siNC group) were transfected according to the instructions of the siTran1.0 transfection kit (Origene), and the blank control group was not transfected.

Techniques: Expressing, Transfection, Western Blot

Journal: Journal of cancer research and therapeutics

Article Title: PRDX3 promotes resistance to cisplatin in gastric cancer cells.

doi: 10.4103/jcrt.jcrt_970_22

Figure Lengend Snippet: Figure 1: PRDX3 expression level in adjacent noncancer and gastric cancer tissue (original magnification, ×400). (a) Immunohistochemistry images of normal gastric tissue. (b) Immunohistochemistry images of moderately differentiated adenocarcinoma. (c) Immunohistochemistry images of poorly differentiated adenocarcinoma. (d) PRDX3 expression in gastric cancer tissue was significantly higher than in adjacent noncancer tissue. **P < 0.01

Article Snippet: PRDX3 interfering RNA (siPRDX3 group) and negative control (siNC group) were transfected according to the instructions of the siTran1.0 transfection kit (Origene), and the blank control group was not transfected.

Techniques: Expressing, Immunohistochemistry

Journal: Journal of cancer research and therapeutics

Article Title: PRDX3 promotes resistance to cisplatin in gastric cancer cells.

doi: 10.4103/jcrt.jcrt_970_22

Figure Lengend Snippet: Figure 3: IC50 values of each group after PRDX3 knockdown in AGS and MKN‑74 cells. (a) Control (CON) group of AGS. (b) siNC group of AGS. (c) siPRDX3 group of AGS. (d) CON group of MKN‑74. (e) siNC group of MKN‑74. (f) siPRDX3 group for MKN‑74. (g) Comparative histogram of IC50 in each group in AGS cells. (h) Comparative histogram of IC50 in each group in MKN‑74 cells. **P < 0.01

Article Snippet: PRDX3 interfering RNA (siPRDX3 group) and negative control (siNC group) were transfected according to the instructions of the siTran1.0 transfection kit (Origene), and the blank control group was not transfected.

Techniques: Knockdown, Control

Journal: Journal of cancer research and therapeutics

Article Title: PRDX3 promotes resistance to cisplatin in gastric cancer cells.

doi: 10.4103/jcrt.jcrt_970_22

Figure Lengend Snippet: Figure 5: Expression level of the related proteins in the apoptosis pathway in AGS and MKN‑74 cells transfected with siPRDX3 after cisplatin treatment. ***P < 0.001. (a) Western blot bands. (b) Relative expression of cleaved caspase‑3 protein in each group of AGS cells. (c) Relative expression of Bax protein in each group of AGS cells. (d) Relative expression of PRDX3 protein in each group of AGS cells. (e) Relative expression of cleaved caspase‑3 protein in each group of MKN‑74 cells. (f) Relative expression of Bax protein in each group of MKN‑74 cells. (g) Relative expression of PRDX3 protein in each group of MKN‑74 cells

Article Snippet: PRDX3 interfering RNA (siPRDX3 group) and negative control (siNC group) were transfected according to the instructions of the siTran1.0 transfection kit (Origene), and the blank control group was not transfected.

Techniques: Expressing, Transfection, Western Blot