Journal: International Journal of Molecular Sciences

Article Title: Insight into the Interactome of Intramitochondrial PKA Using Biotinylation-Proximity Labeling

doi: 10.3390/ijms21218283

Figure Lengend Snippet: Protein kinase A (PKA) catalytic subunit α is localized within mitochondria of HeLa cells. ( A ) Representative immunoblots ( n = 3) of PKA catalytic (cat) subunit, PKA regulatory (reg) subunit, the mitochondrial protein SDHa and of the cytosolic protein α-Tubulin with corresponding total protein load (TPL) in total cell lysate (TCL), cytosolic (Cyto) and mitochondrial (Mito) fractions isolated from HeLa cells. ( B ) Representative immunoblots ( n = 3) of PKA cat, PKA reg, SDHa and TOM20 with corresponding total protein load (TPL) in mitochondrial fractions isolated from HeLa cells and treated as indicated. ( C ) Representative micrographs ( n = 6) of HeLa cells expressing empty vector (pcDNA), PKA-Myc or mt-PKA-Myc and labelled with anti-Myc (green) and anti-TOM20 (red) as a mitochondrial marker. Scale bars = 5 μm. ( D ) Representative immunoblots ( n = 3) of Myc, SDHa and α-Tubulin with corresponding total protein load (TPL) in TCL, cyto and mito fractions isolated from HeLa cells expressing empty vector (pcDNA), PKA-Myc or mt-PKA-Myc. ( E ) Representative immunoblots ( n = 3) of Myc, TOM20, SDHa and SOD2 with corresponding total protein load (TPL) in mitochondrial enriched fractions isolated from HeLa cells expressing empty vector (pcDNA), PKA-Myc or mt-PKA-Myc and treated as indicated. ( F ) Representative immunoblots ( n = 3) of Myc, PKA Cat, ATP5a, MFN2 and ERp57 with corresponding TPL in pellet and supernatant (SN) obtained after treatment of mitochondria isolated from HeLa cells expressing PKA-Myc or mt-PKA-Myc and treated as indicated. ( G ) Representative immunoblots ( n = 5) of phospho-PKA substrates and Myc with corresponding total protein load (TPL) in mitochondrial enriched fractions isolated from HeLa cells expressing empty vector (pcDNA), PKA-Myc or mt-PKA-Myc.

Article Snippet: Protein immunodetection was performed using primary antibodies directed against PKA C-α (Cell Signaling; 4782S), PKA RI-α/β (Cell Signaling; 3927), Phospho-PKA Substrate (Cell Signaling; 9624S), α-Tubulin (Cell Signaling; 2144) SDHa (Abcam; ab14715), SOD2 (Santa Cruz; sc-30080), NDUF9a (Abcam; ab14713), Cytochrome c (Abcam; ab110325), TOM20 (Santa Cruz; (F-10) sc-17764), UQCRC2 (Abcam; ab14745), PAM16 (Abcam; Ab184157), Hsp60 (Santa Cruz; sc-13115), Hsp70 (Santa Cruz; sc-24), V5 (Cell Signaling; 13202S), Myc (Cell Signaling; 2276S), ATP5a (Abcam; ab14730), MFN2 (Cell signaling; 9482S), ERp57 (R&Dsystems; AF8219).

Techniques: Western Blot, Isolation, Expressing, Plasmid Preparation, Marker

Journal: Journal of Inflammation Research

Article Title: Significance of Immunogenic Cell Death-Related Prognostic Gene Signature in Cervical Cancer Prognosis and Anti-Tumor Immunity

doi: 10.2147/JIR.S410140

Figure Lengend Snippet: Expression of the PDIA3 gene in cervical cancer (CC) and its prognostic value. ( A ) The cell types of IPGs distribution in the single-cell analysis of the GSE168652 database were described. ( B ) UMAP visualization of PDIA3 in major cell subpopulations of cervical cancer. ( C ) The expression levels of HPV16- and HPV18-positive groups were compared with normal groups in the GSE6791 database (* p < 0.05, ** p < 0.01, and *** p < 0.001). ( D ) Cox survival analysis showing hazard ratios for different IPGs. ( E ) Differences in PDIA3 expression between normal and cancer tissue samples (* p < 0.05). ( F and G ) Kaplan–Meier curves showing differences in overall survival ( F ) and progression-free survival ( G ) between PDIA3 high and low expression groups; ( H – J ) PDIA3 protein expression in normal cervical tissue samples and CC tissue samples. Normal cervical tissue ( H ), cervical squamous cancer ( I ), and cervical adenocarcinoma tissue ( J ).

Article Snippet: PDIA3 antibody (Proteintech, Wuhan, China), GAPDH antibody (CST, Shanghai, China), goat anti-rabbit IgG antibody secondary antibody (Zsbio, Beijing, China), and goat anti-mouse IgG H&L/Cy3 (Beyotime, Shanghai, China).

Techniques: Expressing, Single-cell Analysis

Journal: Journal of Inflammation Research

Article Title: Significance of Immunogenic Cell Death-Related Prognostic Gene Signature in Cervical Cancer Prognosis and Anti-Tumor Immunity

doi: 10.2147/JIR.S410140

Figure Lengend Snippet: PDIA3 is highly expressed in cervical cancer (CC) tissues and cell lines. ( A and B ) Western blot analysis reveals a high expression of PDIA3 in CC cell lines. ( C ) Distribution of PDIA3 in CC cell lines. ( D ) Immunohistochemistry reveals PDIA3 expression in CC tissues, with normal cervical tissue serving as a positive control (200×). ( E ) IPGs mRNA expression relative to GAPDH in cervical cancer samples comparing (* p < 0.05, ** p < 0.01, and *** p < 0.001).

Article Snippet: PDIA3 antibody (Proteintech, Wuhan, China), GAPDH antibody (CST, Shanghai, China), goat anti-rabbit IgG antibody secondary antibody (Zsbio, Beijing, China), and goat anti-mouse IgG H&L/Cy3 (Beyotime, Shanghai, China).

Techniques: Western Blot, Expressing, Immunohistochemistry, Positive Control

Journal: Brain Pathology

Article Title: Endoplasmic reticulum‐stress and unfolded protein response‐activation in immune‐mediated necrotizing myopathy

doi: 10.1111/bpa.13084

Figure Lengend Snippet: Proteins found by unbiased proteomic profiling of IMNM patient‐derived muscle biopsies.

Article Snippet: The TaqMan® Gene Exp Assay from Life Technologies/ThermoFisher are listed as follows: ATF4 Hs00909569_g1, ATF6 Hs00232586_m1, HSPA5 (BiP) Hs00607129_gH, CALR Hs00376764_m1, CANX Hs01558409_m1, EDEM1 Hs00976004_m1, ERN2 (EIF2) Hs01086607_m1, HSP90B1 (GRP94) Hs00427665_g1, HYOU1 (GRP170) Hs00197328_m1, ERN2/IRE1 Hs01086607_m1, PDIA2 Hs00429010_m1, PDIA3 Hs00607126_m1, EIF2AK3 (PERK) Hs00984003_m1, PGK1 Hs99999906_m1, SIL1 Hs00223835_m1, XBP1 Hs00231936_m1.

Techniques:

Journal: Brain Pathology

Article Title: Endoplasmic reticulum‐stress and unfolded protein response‐activation in immune‐mediated necrotizing myopathy

doi: 10.1111/bpa.13084

Figure Lengend Snippet: Unfolded protein response‐chaperones are activated in immune‐mediated necrotizing myopathies (IMNM) patient's muscles. (A) Immunohistochemical staining reveals strong accumulation of BiP and Co‐chaperones SIL1 and GRP170/HYOU1 in IMNM patient's muscles independent of the underlying auto‐antibody subtype. (B) Gene expression analysis of BIP , SIL1 , and GRP170 shows no significant elevation as compared to non‐diseased control (NDC); however, BIP seems to be elevated in single patients. (C) Protein levels of GRP94 were increased on immune cells in the endomysium and on some myofibers, while there was a marked immunochistochemical reaction on myofibers of both PDIs, with subsarcolemmal accentuation for PDIA1. Original magnification 400×. (D) Gene expression analysis of GRP94 showed no significant elevation as compared to NDC, while EDEM1 , PDIA2 , and PDIA3 were significantly elevated in IMNM when compared to NDC. (E) Calreticulin is strongly stained on immune cells. Original magnification 400×. (F) Transcriptional analysis showed no elevated expression of CALR nor CANX . The level of signigficance was set to * p < 0.05, ** p < 0.01, *** p < 0.001

Article Snippet: The TaqMan® Gene Exp Assay from Life Technologies/ThermoFisher are listed as follows: ATF4 Hs00909569_g1, ATF6 Hs00232586_m1, HSPA5 (BiP) Hs00607129_gH, CALR Hs00376764_m1, CANX Hs01558409_m1, EDEM1 Hs00976004_m1, ERN2 (EIF2) Hs01086607_m1, HSP90B1 (GRP94) Hs00427665_g1, HYOU1 (GRP170) Hs00197328_m1, ERN2/IRE1 Hs01086607_m1, PDIA2 Hs00429010_m1, PDIA3 Hs00607126_m1, EIF2AK3 (PERK) Hs00984003_m1, PGK1 Hs99999906_m1, SIL1 Hs00223835_m1, XBP1 Hs00231936_m1.

Techniques: Muscles, Immunohistochemical staining, Staining, Expressing, Control

Journal: OncoTargets and therapy

Article Title: Expression of protein disulfide isomerase A3 precursor in colorectal cancer

doi: 10.2147/OTT.S154452

Figure Lengend Snippet: Mass spectrometry results of protein disulfide isomerase A3 precursor (PDIA3). Mass spectrogram of segments of TFSHELSDFGLESTAGEIPVVAIR ( A ), FVMQEEFSRDGK ( B ), MDATANDVPSPYEVR ( C ) and ELSDFISYLQR ( D ) verified the presence of PDIA3 in colorectal cancer tissue.

Article Snippet: PDIA3 expression was quantified by real-time PCR method using the real-time PCR master mix kit (Genecopoeia). β-Actin was used as an internal reference.

Techniques: Mass Spectrometry

Journal: OncoTargets and therapy

Article Title: Expression of protein disulfide isomerase A3 precursor in colorectal cancer

doi: 10.2147/OTT.S154452

Figure Lengend Snippet: Expression of protein disulfide isomerase A3 precursor (PDIA3) protein in colorectal cancer tissues and cell lines. ( A ) Western blotting assays showed that PDIA3 was expressed in carcinoma tissues (1) and adjacent tissues (2) (upper panel) and quantitated plot (lower panel). * P <0.05 compared with adjacent tissues. ( B ) Immunofluorescence microscopy showed PDIA3 expression in SW480, HCT116, CACO2, NCM460 and HT-29 cells at 100× magnification. ( C ) Western blotting assays showed PDIA3 expression in carcinoma cell lines. Abbreviation: DAPI, 4′,6-diamidino-2-phenylindole.

Article Snippet: PDIA3 expression was quantified by real-time PCR method using the real-time PCR master mix kit (Genecopoeia). β-Actin was used as an internal reference.

Techniques: Expressing, Western Blot, Immunofluorescence, Microscopy

Journal: OncoTargets and therapy

Article Title: Expression of protein disulfide isomerase A3 precursor in colorectal cancer

doi: 10.2147/OTT.S154452

Figure Lengend Snippet: Protein disulfide isomerase A3 precursor-small interfering RNA (PDIA3-siRNA) reduced PDIA3 expression in SW480 and HCT116 cells. ( A ) SW480 cells were transfected with two siRNA-PDIA3 vectors. ( B ) HCT116 cells were transfected with two siRNA-PDIA3 vectors. ( C ) Western blotting assays showed that PDIA3-siRNA reduced PDIA3 expression. ( D ) Real-time polymerase chain reaction showed that PDIA3-siRNA reduced PDIA3 expression. Note: * P <0.05 compared with the controls. Magnification was set at 100×. siRNA-1: 5′-GGACAAGACUGUGGCAUAUTTAUAUGCCACAGUCUUGUCC TT-3′; siRNA-2: 5′-CAGCCAACAAGAAGCUAAATTUUUAGCUUCUUGUUG GCUGTT-3′. Abbreviation: NC, scrambled control.

Article Snippet: PDIA3 expression was quantified by real-time PCR method using the real-time PCR master mix kit (Genecopoeia). β-Actin was used as an internal reference.

Techniques: Small Interfering RNA, Expressing, Transfection, Western Blot, Real-time Polymerase Chain Reaction, Control

Journal: OncoTargets and therapy

Article Title: Expression of protein disulfide isomerase A3 precursor in colorectal cancer

doi: 10.2147/OTT.S154452

Figure Lengend Snippet: Effect of protein disulfide isomerase A3 precursor (PDIA3) knockdown on cell growth. Cell death was measured by Cell Counting Kit-8 assay. SW480 ( A ) and HCT116 ( B ) cells were incubated with two different small interfering RNA (siRNA)-PDIA3 for 12, 24 and 48 hours, respectively. SW480 cells treated with or without siRNA-PDIA3 were observed under a transmission electron microscope set at 400× magnification ( C ). Notes: siRNA-1: 5′-GGACAAGACUGUGGCAUAUTTAUAUGCCACAGUCUUGUCC TT-3′; siRNA-2: 5′-CAGCCAACAAGAAGCUAAATTUUUAGCUUCUUGUUG GCUGTT-3′.

Article Snippet: PDIA3 expression was quantified by real-time PCR method using the real-time PCR master mix kit (Genecopoeia). β-Actin was used as an internal reference.

Techniques: Knockdown, Cell Counting, Incubation, Small Interfering RNA, Transmission Assay, Microscopy

Journal: OncoTargets and therapy

Article Title: Expression of protein disulfide isomerase A3 precursor in colorectal cancer

doi: 10.2147/OTT.S154452

Figure Lengend Snippet: Effect of protein disulfide isomerase A3 precursor (PDIA3) knockdown on apoptosis. ( A ) SW480 cells were treated with small interfering RNA (siRNA)-PDIA3, and then the cells were stained with TUNEL and DAPI, magnification set at 100×. ( B ) The averaged apoptosis index indicated significant apoptotic cell death in SW480 cells with or without siRNA-PDIA3. ( C ) Bax and Bcl-2 expression after siRNA-PDIA3 treatments. ( D ) Quantification data of Bcl-2 expression. ( E ) Quantification data of Bax expression. Notes: Data were expressed as mean ± SEM, * P <0.01 compared with the controls. siRNA-1: 5′-GGACAAGACUGUGGCAUAUTTAUAUGCCACAGUCUUGUCC TT-3′; siRNA-2: 5′-CAGCCAACAAGAAGCUAAATTUUUAGCUUCUUGUUG GCUGTT-3′. Abbreviations: DAPI, 4′,6-diamidino-2-phenylindole; NC, scrambled control; SEM, standard error of the mean; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling.

Article Snippet: PDIA3 expression was quantified by real-time PCR method using the real-time PCR master mix kit (Genecopoeia). β-Actin was used as an internal reference.

Techniques: Knockdown, Small Interfering RNA, Staining, TUNEL Assay, Expressing, Control

Journal: eLife

Article Title: Co-movement of astral microtubules, organelles and F-actin by dynein and actomyosin forces in frog egg cytoplasm

doi: 10.7554/eLife.60047

Figure Lengend Snippet:

Article Snippet: The ER was also probed with labeled anti-Protein disulfide-isomerase A3 (PDIA3) (Boster Bio #PB9772).

Techniques: Labeling, Recombinant, Expressing, Software

Journal: International journal of oncology

Article Title: Downregulation of protein disulfide‑isomerase A3 expression inhibits cell proliferation and induces apoptosis through STAT3 signaling in hepatocellular carcinoma.

doi: 10.3892/ijo.2019.4710

Figure Lengend Snippet: Figure 1. Association between PDIA3 expression and cell proliferation and apoptosis in HCC tissues. (A) Representative histological and immunohistochemistry staining of two HCC tissues (x600 magnification). The tissues were examined by (a and b) H&E staining, immunohistochemistry staining with antibodies against (c and d) PDIA3 and (e and f) Ki‑67, and (g and h) the TUNEL assay. The HCC tissue of case 17 exhibits high expression of PDIA3 and that of case 19 shows low expression. Scale bar, 50 µm. Scatter plots of (B) Ki‑67 index values and (C) the percentage of TUNEL positive cells in HCC tissues with high (score, 4≤) and low (score, 4>) PDIA3 expression. ***P<0.001 (Mann‑Whitney U‑test). PDIA3, protein disulfide‑isomerase A3; Ki‑67, proliferation marker protein Ki‑67; TUNEL, terminal deoxynucleotidyl‑transferase‑mediated dUTP nick end labeling; H&E, hematoxylin and eosin; HCC, hepatocellular carcinoma.

Article Snippet: The qPCR was performed for PDIA3 and 18S rRNA (as an internal standard) using the StepOnePlus Real-Time PCR system with TaqMan probes and primers (18S, cat. no. Hs 03928990_g1; PDIA3, cat. no. Hs 04194196_g1) (all Thermo Fisher Scientific, Inc.).

Techniques: Expressing, Immunohistochemistry, Staining, TUNEL Assay, Mann-Whitney U-Test, Marker, End Labeling

Journal: International journal of oncology

Article Title: Downregulation of protein disulfide‑isomerase A3 expression inhibits cell proliferation and induces apoptosis through STAT3 signaling in hepatocellular carcinoma.

doi: 10.3892/ijo.2019.4710

Figure Lengend Snippet: Figure 2. Effects of PDIA3 knockdown on cell proliferation, apoptosis and cell cycle in HCC cell lines. (A) Representative western blots of PDIA3 protein expression in normal hepatocyte THLE‑2 and HCC Huh‑7 and HuH‑1 cells. (B) Relative quantification of the PDIA3 protein expression. The data are expressed as the mean ± SEM. ***P<0.001 vs. THLE‑2 (one‑way ANOVA). (C) Representative western blots of the expression of PDIA3 in Huh‑7 and HuH‑1 cells lines transfected Ctrl si, PDIA3 si‑1 and PDIA3 si‑2. (D) Cell proliferation of the HCC cells transfected with Ctrl si, PDIA3 si‑1 and PDIA3 si‑2 was evalu ated using the WST‑8 cell counting reagent. At the indicated time points, the number of cells per well was estimated by measuring the absorbance (450 nm). The data are expressed as the mean ± SEM. ***P<0.001 vs. Ctrl si at each time point (two‑way ANOVA). (E) Representative plots of the flow cytometry analysis of apoptosis. The apoptotic cells were counted by flow cytometry following double staining with annexin V and ethidium homodimer III. Q1 represents the necrotic cells; Q2 the late apoptotic cells; Q3 the live cells; and Q4 the early apoptotic cells. (F) The percentage of live, apoptotic and necrotic cells in the groups transfected with siRNAs. The data are expressed as the mean ± SEM. ***P<0.001 vs. Ctrl si (two‑way ANOVA). (G) Representative histograms of flow cytometry analysis of the cell cycle, following propidium iodide staining. P4 represents the SubG1 (fragmented DNA); P5 the G0/G1 phase; P6 the S phase; and P7 the G2/M phase. (H) The percentage of cells in each cell cycle phase following siRNA treatment. ***P<0.001 vs. Ctrl si (two‑way ANOVA). These results were from ≥3 independent experiments. PDIA3, protein disulfide‑isomerase A3; HCC, hepatocellular carcinoma; PDIA si‑1/PDIA si‑2, PDIA3 siRNA; Ctrl si, control siRNA; ANOVA, analysis of variance; SEM, standard error of the mean.

Article Snippet: The qPCR was performed for PDIA3 and 18S rRNA (as an internal standard) using the StepOnePlus Real-Time PCR system with TaqMan probes and primers (18S, cat. no. Hs 03928990_g1; PDIA3, cat. no. Hs 04194196_g1) (all Thermo Fisher Scientific, Inc.).

Techniques: Knockdown, Western Blot, Expressing, Quantitative Proteomics, Transfection, Cell Counting, Flow Cytometry, Double Staining, Staining, Control

Journal: International journal of oncology

Article Title: Downregulation of protein disulfide‑isomerase A3 expression inhibits cell proliferation and induces apoptosis through STAT3 signaling in hepatocellular carcinoma.

doi: 10.3892/ijo.2019.4710

Figure Lengend Snippet: Figure 3. Endoplasmic reticulum stress levels in HCC cells with knockdown of PDIA3 expression. Huh‑7 and HuH‑1 cells were transfected with Ctrl si, PDIA3 si‑1 and PDIA3 si‑2 for 96 h. Total protein was extracted for immunoblotting with antibodies against PDIA3, GRP78 and β‑actin. The images are representative western blots demonstrating GRP78 protein expression following downregulation of PDIA3 expression in HCC cells. PDIA3, protein disulfide‑isomerase A3; HCC, hepatocellular carcinoma; Untr, untreated control; PDIA si‑1/PDIA si‑2, PDIA3 siRNA; Ctrl si, control siRNA; GRP78, 78 kDa glucose‑regulated protein.

Article Snippet: The qPCR was performed for PDIA3 and 18S rRNA (as an internal standard) using the StepOnePlus Real-Time PCR system with TaqMan probes and primers (18S, cat. no. Hs 03928990_g1; PDIA3, cat. no. Hs 04194196_g1) (all Thermo Fisher Scientific, Inc.).

Techniques: Knockdown, Expressing, Transfection, Western Blot, Control

Journal: International journal of oncology

Article Title: Downregulation of protein disulfide‑isomerase A3 expression inhibits cell proliferation and induces apoptosis through STAT3 signaling in hepatocellular carcinoma.

doi: 10.3892/ijo.2019.4710

Figure Lengend Snippet: Figure 4. Association of PDIA3 and STAT3 in hepatocellular carcinoma cells. (A) Immunofluorescence staining for PDIA3 (red), STAT3 (green) and the nucleus (DAPI; blue) in Huh‑7 cells (x1,000 magnification). The yellow color in the merged image indicates the colocalization of PDIA3 and STAT3. (B) A co‑immunoprecipitation assay was performed using anti‑PDIA3 or anti‑STAT3 antibodies, and immunoblotting with anti‑STAT3 or anti‑PDIA3 antibodies, respectively. Normal mouse IgG was used for the Ctrl. (C) Huh‑7 and HuH‑1 cells were transfected with Ctrl si, PDIA3 si‑1 and PDIA3 si‑2 for 96 h. Total proteins were extracted and subjected to immunoblotting with antibodies against PDIA3, STAT3, P‑STAT3 (Tyr705) and β‑actin. Input represents the total cell lysate. PDIA3, protein disulfide‑isomerase A3; STAT3, signal transducer and activator of transcription 3; P‑STAT3, phosphorylated STAT3; Untr, untreated control; PDIA si‑1/PDIA si‑2, PDIA3 siRNA; Ctrl si, control siRNA; Ctrl, control.

Article Snippet: The qPCR was performed for PDIA3 and 18S rRNA (as an internal standard) using the StepOnePlus Real-Time PCR system with TaqMan probes and primers (18S, cat. no. Hs 03928990_g1; PDIA3, cat. no. Hs 04194196_g1) (all Thermo Fisher Scientific, Inc.).

Techniques: Immunofluorescence, Staining, Co-Immunoprecipitation Assay, Western Blot, Transfection, Control

Journal: International journal of oncology

Article Title: Downregulation of protein disulfide‑isomerase A3 expression inhibits cell proliferation and induces apoptosis through STAT3 signaling in hepatocellular carcinoma.

doi: 10.3892/ijo.2019.4710

Figure Lengend Snippet: Figure 5. Downstream targets of signal transducer and activator of transcrip tion 3 in hepatocellular carcinoma cells with silenced PDIA3. Huh‑7 and HuH‑1 cells were transfected with Ctrl si, PDIA3 si‑1 and PDIA3 si‑2 for 96 h. (A) Total protein was extracted for immunoblotting with antibodies against PDIA3, survivin, XIAP, Mcl‑1, Bcl‑2, Bcl‑XL, cyclin D1, p53 and β‑actin. (B) Relative quantification of the protein expression. The data are expressed as the mean ± standard error of the mean. **P<0.01, ***P<0.001, vs. Ctrl si (two‑way analysis of variance). PDIA3, protein disulfide‑isomerase A3; XIAP, X‑linked inhibitor of apoptosis protein; Mcl‑1, induced myeloid leu kemia cell differentiation protein Mcl‑1; Bcl‑2, B‑cell lymphoma 2; Bcl‑XL, Bcl‑2‑like protein 1; p53, cellular tumor antigen p53; Untr, untreated con trol; PDIA si‑1/PDIA si‑2, PDIA3 siRNA; Ctrl si, control siRNA, N.D., not detected.

Article Snippet: The qPCR was performed for PDIA3 and 18S rRNA (as an internal standard) using the StepOnePlus Real-Time PCR system with TaqMan probes and primers (18S, cat. no. Hs 03928990_g1; PDIA3, cat. no. Hs 04194196_g1) (all Thermo Fisher Scientific, Inc.).

Techniques: Transfection, Western Blot, Quantitative Proteomics, Expressing, Cell Differentiation, Control

Journal: International journal of oncology

Article Title: Downregulation of protein disulfide‑isomerase A3 expression inhibits cell proliferation and induces apoptosis through STAT3 signaling in hepatocellular carcinoma.

doi: 10.3892/ijo.2019.4710

Figure Lengend Snippet: Figure 6. Effects of PDIA3 knockdown on HCC cell proliferation in the presence of AG490. (A) Representative western blots of P‑STAT3 (Tyr705) levels following treatment with 100 µM AG490 (a tyrosine‑protein kinase JAK/STAT3 signaling inhibitor) or control (0.1% DMSO) for 3 h in HCC Huh‑7 and HuH‑1 cells. Total protein was extracted and subjected to immu noblotting with antibodies against P‑STAT3, STAT3 and β‑actin. (B) Cell proliferation was evaluated using the WST‑8 cell counting reagent. Huh‑7 and HuH‑1 cells were transfected with Ctrl si, PDIA3 si‑1 and PDIA3 si‑2, and treated with 25 µM AG490. At the indicated time points, the number of cells per well was estimated by measuring the absorbance at 450 nm. The data are expressed as the mean ± standard error of the mean. ###P<0.001 vs. Ctrl si; *P<0.05, PDIA3 si‑1 + AG490 vs. Ctrl si + AG490; ***P<0.001, PDIA3 si‑1 + AG490 vs. Ctrl si + AG490 (two‑way analysis of variance). The results are from ≥3 independent experiments. PDIA3, protein disul fide‑isomerase A3; STAT3, signal transducer and activator of transcription 3; P‑STAT3, phosphorylated STAT3; HCC, hepatocellular carcinoma; PDIA si‑1/PDIA si‑2, PDIA3 siRNA; Ctrl si, control siRNA.

Article Snippet: The qPCR was performed for PDIA3 and 18S rRNA (as an internal standard) using the StepOnePlus Real-Time PCR system with TaqMan probes and primers (18S, cat. no. Hs 03928990_g1; PDIA3, cat. no. Hs 04194196_g1) (all Thermo Fisher Scientific, Inc.).

Techniques: Knockdown, Western Blot, Control, Cell Counting, Transfection

Journal: International journal of oncology

Article Title: Downregulation of protein disulfide‑isomerase A3 expression inhibits cell proliferation and induces apoptosis through STAT3 signaling in hepatocellular carcinoma.

doi: 10.3892/ijo.2019.4710

Figure Lengend Snippet: Figure 7. Association between P‑STAT3 and PDIA3 expression, Ki‑67 index and apoptotic cells in HCC tissues. (A) Representative histological and immu nohistochemical staining of two HCC tissues (x600 magnification). The HCC tissues were examined by (a and b) H&E staining, and immunohistochemistry staining with (c and d) anti‑P‑STAT3 (Tyr705) antibody and (e and f) anti‑PDIA3 antibody. The HCC tissue of case 20 is P‑STAT3 positive (a, c and e) and that of case 31 is P‑STAT3 negative (b, d and f). Scale bars, 50 µm. (B) The association between P‑STAT3 status and PDIA3 expression in tissues from 35 HCC cases. Values are expressed as the mean ± standard deviation. ***P<0.001 (Mann‑Whitney U‑test). Scatter plots of percentages of (C) the Ki‑67 index and (D) TUNEL‑positive cells in HCC tissues positive (≥10% positive tumor cells) or negative (<10% positive tumor cells) for P‑STAT3. ***P<0.001 (Mann‑Whitney U‑test). PDIA3, protein disulfide‑isomerase A3; STAT3, signal transducer and activator of transcription 3; P‑STAT3, phosphorylated STAT3; Ki‑67, proliferation marker protein Ki‑67; TUNEL, terminal deoxynucleotidyl‑transferase‑mediated dUTP nick end labeling; H&E, hematoxylin and eosin; HCC, hepatocellular carcinoma.

Article Snippet: The qPCR was performed for PDIA3 and 18S rRNA (as an internal standard) using the StepOnePlus Real-Time PCR system with TaqMan probes and primers (18S, cat. no. Hs 03928990_g1; PDIA3, cat. no. Hs 04194196_g1) (all Thermo Fisher Scientific, Inc.).

Techniques: Expressing, Staining, Immunohistochemistry, Standard Deviation, Mann-Whitney U-Test, Marker, TUNEL Assay, End Labeling

Journal: International journal of oncology

Article Title: Downregulation of protein disulfide‑isomerase A3 expression inhibits cell proliferation and induces apoptosis through STAT3 signaling in hepatocellular carcinoma.

doi: 10.3892/ijo.2019.4710

Figure Lengend Snippet: Figure 8. Flow diagram of the association of PDIA3 and the STAT3 signaling pathway in HCC. The STAT3 signaling pathway modulates cell survival in HCC. The progress of STAT3 signaling requires the expression of PDIA3. HCC, hepatocellular carcinoma; PDIA3, protein disulfide‑isomerase A3; STAT3, signal transducer and activator of transcription 3; XIAP, X‑linked inhibitor of apoptosis protein; Mcl‑1, induced myeloid leukemia cell differ entiation protein Mcl‑1; Bcl‑XL, Bcl‑2‑like protein 1.

Article Snippet: The qPCR was performed for PDIA3 and 18S rRNA (as an internal standard) using the StepOnePlus Real-Time PCR system with TaqMan probes and primers (18S, cat. no. Hs 03928990_g1; PDIA3, cat. no. Hs 04194196_g1) (all Thermo Fisher Scientific, Inc.).

Techniques: Expressing