Journal: Oncology Letters

Article Title: Knockdown of high mobility group box 3 impairs cell viability and colony formation but increases apoptosis in A549 human non-small cell lung cancer cells

doi: 10.3892/ol.2019.9927

Figure Lengend Snippet: HMGB3 knockdown in A549 cells by shRNA transfection. (A) Western blotting result demonstrating that transfection with shHMGB3-1 or shHMGB3-2 significantly decreased the protein expression level of HMGB3 in A549 cells. (B) Reverse transcription-quantitative polymerase chain reaction result showing that transfection with shHMGB3-1 or shHMGB3-2 significantly decreased HMGB3 mRNA expression level in A549 cells. The mRNA expression level in each transfection group was normalized to that in NT cells. (C) Fluorescent intensity analysis of the western blotting result. The fluorescent intensity of the HMGB3 band in each shHMGB3 transfection group was normalized to that in control cells transfected with non-targeting shRNA (shNC). Data in panel (B) and (C) represents three independent repeats. *P<0.05; ****P<0.0001. shRNA, short hairpin RNA; HMGB3, high mobility group box 3; NT, non-transfected control; shHMGB3, shRNA targeting HMGB3.

Article Snippet: Human HMGB3 and GAPDH mRNA expression in cells of each experimental group was evaluated by RT-qPCR using a custom-made RT-qPCR kit (cat. no. QP001) purchased from Genecopoeia, Inc., following the manufacturer's instructions.

Techniques: Knockdown, shRNA, Transfection, Western Blot, Expressing, Reverse Transcription, Real-time Polymerase Chain Reaction, Control

Journal: Oncology Letters

Article Title: Knockdown of high mobility group box 3 impairs cell viability and colony formation but increases apoptosis in A549 human non-small cell lung cancer cells

doi: 10.3892/ol.2019.9927

Figure Lengend Snippet: Increasing apoptosis in A549 or H1299 NSCLC cells following HMGB3 knockdown. (A) Representative flow cytometry data showing an increased population of apoptotic cells in A549 or H1299 cells following HMGB3 knockdown. Annexin-V positively stained cells were considered apoptotic cells. (B) Percentage of apoptotic cells was increased in A549 OR H1299 cells following HMGB3 knockdown. (C) Western blotting detecting the expression level of apoptosis-associated proteins in A549 or H1299 cells with different treatment. (D) Bax over Bcl-2 ratio was increased by HMGB3 knockdown in A549 or H1299 cells. (E and F), increased (E) caspase-9 activation and (F) unchanged caspase-8 activation in A549 or H1299 cells following HMGB3 knockdown. Western blotting data analysis was performed by comparing the gray scale of protein bands revealed by western blotting following normalization to β-actin. Data in panel (B) and (D-F) each represents at least 3 independent repeats. *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001. shRNA, short hairpin RNA; HMGB3, high mobility group box 3; NT, non-transfected control; shHMGB3, shRNA targeting HMGB3; shNC, negative control shRNA.

Article Snippet: Human HMGB3 and GAPDH mRNA expression in cells of each experimental group was evaluated by RT-qPCR using a custom-made RT-qPCR kit (cat. no. QP001) purchased from Genecopoeia, Inc., following the manufacturer's instructions.

Techniques: Knockdown, Flow Cytometry, Staining, Western Blot, Expressing, Activation Assay, shRNA, Transfection, Control, Negative Control

Journal: Oncology Letters

Article Title: Knockdown of high mobility group box 3 impairs cell viability and colony formation but increases apoptosis in A549 human non-small cell lung cancer cells

doi: 10.3892/ol.2019.9927

Figure Lengend Snippet: Colony forming ability and cell viability of non-small cell lung cancer cells were decreased following HMGB3 knockdown. (A) Typical result of colony forming assay showing a decreased colony forming ability of A549 or H1299 cells following HMGB3 knockdown. (B) The CFE of A549 cells was decreased following HMGB3 knockdown. (C) Cell viability of A549 or H1299 was decreased following HMGB3 knockdown. Data in (B and C) are from three independent repeats. Statistical significance test was performed on data obtained on the last time point. ****P<0.0001. shRNA, short hairpin RNA; HMGB3, high mobility group box 3; NT, non-transfected control; shHMGB3, shRNA targeting HMGB3; shNC, negative control shRNA; OD, optical density; CFE, colony formation efficiency.

Article Snippet: Human HMGB3 and GAPDH mRNA expression in cells of each experimental group was evaluated by RT-qPCR using a custom-made RT-qPCR kit (cat. no. QP001) purchased from Genecopoeia, Inc., following the manufacturer's instructions.

Techniques: Knockdown, shRNA, Transfection, Control, Negative Control

Journal: Oncology Letters

Article Title: Knockdown of high mobility group box 3 impairs cell viability and colony formation but increases apoptosis in A549 human non-small cell lung cancer cells

doi: 10.3892/ol.2019.9927

Figure Lengend Snippet: Investigation on proteins potentially interacting with HMGB3. (A) Proteins that may interact with HMGB3 were obtained using STRING online database with the confidence score set at 0.4; each node represent a protein, and each edge between two nodes represents the protein-protein interaction. The width of each edge represents the confidence score of that protein-protein interaction. (B) GO annotation of predicted HMGB3 interacting proteins; number of genes enriched to each GO term was plotted to the upper x-axis in box-plot fashion, and the -log2 (p) of each annotated term was plotted to the lower x-axis in dotted-linear graph fashion. HMGB3, high mobility group box 3; GO, Gene Ontology.

Article Snippet: Human HMGB3 and GAPDH mRNA expression in cells of each experimental group was evaluated by RT-qPCR using a custom-made RT-qPCR kit (cat. no. QP001) purchased from Genecopoeia, Inc., following the manufacturer's instructions.

Techniques:

Journal: Oncology Letters

Article Title: Knockdown of high mobility group box 3 impairs cell viability and colony formation but increases apoptosis in A549 human non-small cell lung cancer cells

doi: 10.3892/ol.2019.9927

Figure Lengend Snippet: Gene Ontology enrichment analysis of predicted HMGB3 interacting proteins.

Article Snippet: Human HMGB3 and GAPDH mRNA expression in cells of each experimental group was evaluated by RT-qPCR using a custom-made RT-qPCR kit (cat. no. QP001) purchased from Genecopoeia, Inc., following the manufacturer's instructions.

Techniques: Membrane, Binding Assay, RNA Binding Assay, Activity Assay, Phospho-proteomics, Virus

Journal: Cancer Medicine

Article Title: Construction and validation of a prognostic model based on 11 lymph node metastasis‐related genes for overall survival in endometrial cancer

doi: 10.1002/cam4.4844

Figure Lengend Snippet: Eleven lymph node metastasis associated genes and corresponding coefficient value

Article Snippet: The knockdown efficiency was proved by western blot using primary antibodies against HMGB3 (1:500; Cat. No. AF5507; R&D Systems, Inc.), and GAPDH (1,1,000; Cat. No. 5174S; Cell Signaling Technology).

Techniques:

Journal: Cancer Medicine

Article Title: Construction and validation of a prognostic model based on 11 lymph node metastasis‐related genes for overall survival in endometrial cancer

doi: 10.1002/cam4.4844

Figure Lengend Snippet: In vitro functional validation of the HMGB3. (A). The relative expression of HMGB3 in GEPIA. (B). Knockdown efficiency of HMGB3 by two small interferon RNA transfection. (C). Proliferative effect of HMGB3 on Ishikawa evaluated by Cell Counting Kit‐8 tests. (D). Effects of HMGB3 on the invasion of Ishikawa cells evaluated by Transwell assays. (E). Statistical analysis of the Transwell invasion. (F) Effects of MMP12 on the migration of Ishikawa cells evaluated by gap closure assays. (G) Statistical analysis of the gap closure. * p < 0.05, ** p < 0.01.

Article Snippet: The knockdown efficiency was proved by western blot using primary antibodies against HMGB3 (1:500; Cat. No. AF5507; R&D Systems, Inc.), and GAPDH (1,1,000; Cat. No. 5174S; Cell Signaling Technology).

Techniques: In Vitro, Functional Assay, Biomarker Discovery, Expressing, Knockdown, Transfection, Cell Counting, Migration

Journal: Bioengineered

Article Title: LINC00857 promotes colorectal cancer progression by sponging miR-150-5p and upregulating HMGB3 (high mobility group box 3) expression

doi: 10.1080/21655979.2021.2003941

Figure Lengend Snippet: LINC00857 enhances HMGB3 expression via sponging miR-150-5p. (a) Transfection of miR-150-5p mimic increased miR-150-5p level, as determined by qRT-PCR assay. (b) miR-150-5p interacts with 3ʹ non-coding region of HMGB3. Bioinformatics analysis of binding site between LINC00857 and miR-150-5p using Starbase bioinformatic tool (left panel); WT or MUT luciferase reporter plasmids were transfected into SW480 and HCT116 cells for 48 h in the presence of miR-150-5p mimic or miR-NC. The luciferase activity was measured in each group (right panel). (c & d) NC inhibitor, miR-150-5p inhibitor, miR-NC, or miR-150-5p mimic was transfected into SW480 and HCT116 cells for 48 h. qRT-PCR and Western blot was used to determined HMGB3 expression level. (e & f) HMGB3 mRNA and protein expression was determined in cells of the following groups: sh-NC, sh-LINC00857, sh-LINC00857 + miR-150-5p inhibitor. qRT-PCR and Western blot was used to determined HMGB3 expression level. (g) HMGB3 expression in CRC tumor and non-carcinoma tissues was detected by PCR analysis (n = 50 pairs). (h) The expression levels of LINC0085 7 and HMGB3 in CRC tumors showed a significant positive correlation. (i) HMGB3 expression level was negatively correlated with miR-150-5p expression level in CRC tumors. *P < 0.05, **P < 0.01, ***P < 0.001

Article Snippet: Afterward, the membranes were blocked by 5% milk for 1 hour, followed by incubation with anti-HMGB3 antibody (R&D Systems, Minneapolis, MN, USA, Cat# MAB5507, 1:1000 dilution) and anti-GAPDH antibody (Santa Cruz Biotechnology, TX, USA, Cat# sc-365062, 1:1000 dilution) overnight at 4ʹ.

Techniques: Expressing, Transfection, Quantitative RT-PCR, Binding Assay, Luciferase, Activity Assay, Western Blot