Journal: Cancer Cell International

Article Title: KHDRBS3 accelerates glycolysis and promotes malignancy of hepatocellular carcinoma via upregulating 14-3-3ζ

doi: 10.1186/s12935-023-03085-4

Figure Lengend Snippet: Sequence of primers for qRT-PCR

Article Snippet: Slides were blocked in 1% BSA (Sangon, Shanghai, China) for 15 min and then incubated with KHDRBS3 antibody (Santa Cruz Biotechnology, sc-374,461, Shanghai, China) diluted (1:50) in PBS at 4 °C overnight.

Techniques: Sequencing

Journal: Cancer Cell International

Article Title: KHDRBS3 accelerates glycolysis and promotes malignancy of hepatocellular carcinoma via upregulating 14-3-3ζ

doi: 10.1186/s12935-023-03085-4

Figure Lengend Snippet: KHDRBS3 is elevated in human HCC tissues and is associated with poor clinal outcomes. ( A ) The expression profiles of KHDRBS3 gene in HCC on GEPIA database. ( B ) The expression of KHDRBS3 gene in three GEO datasets. ( C ) The overall survival (OS), recurrence-free survival (RFS), progression-free survival (PFS) and disease-specific survival (DSS) analysis in HCC patients with different KHDRBS3 expression. ( D-E ) KHDRBS3 expression in human HCC and adjacent tissues were detected by qPCR and Western blotting. Error bars represent standard deviation. * P < 0.05 vs. Para-tumor group; ** P < 0.01 vs. Normal group

Article Snippet: Slides were blocked in 1% BSA (Sangon, Shanghai, China) for 15 min and then incubated with KHDRBS3 antibody (Santa Cruz Biotechnology, sc-374,461, Shanghai, China) diluted (1:50) in PBS at 4 °C overnight.

Techniques: Expressing, Western Blot, Standard Deviation

Journal: Cancer Cell International

Article Title: KHDRBS3 accelerates glycolysis and promotes malignancy of hepatocellular carcinoma via upregulating 14-3-3ζ

doi: 10.1186/s12935-023-03085-4

Figure Lengend Snippet: Representative images of KHDRBS3 immunohistochemical staining in HCC and adjacent tissues

Article Snippet: Slides were blocked in 1% BSA (Sangon, Shanghai, China) for 15 min and then incubated with KHDRBS3 antibody (Santa Cruz Biotechnology, sc-374,461, Shanghai, China) diluted (1:50) in PBS at 4 °C overnight.

Techniques: Immunohistochemical staining, Staining

Journal: Cancer Cell International

Article Title: KHDRBS3 accelerates glycolysis and promotes malignancy of hepatocellular carcinoma via upregulating 14-3-3ζ

doi: 10.1186/s12935-023-03085-4

Figure Lengend Snippet: KHDRBS3 promotes cell proliferation in vitro and tumor growth in vivo. ( A ) The efficiency of knockdown and overexpression of KHDRBS3 was examined by Western blotting analysis. ( B-C ) MTT cell proliferation assay and colony formation assay in indicated cells. ( D-F ) Image, volume and weights of xenograft tumors derived from the Huh7 cells. ( G ) The protein levels of KHDRBS3 expression in xenograft tumors. Error bars represent standard deviation. ** P < 0.01 vs. Vector group or NC − sh group

Article Snippet: Slides were blocked in 1% BSA (Sangon, Shanghai, China) for 15 min and then incubated with KHDRBS3 antibody (Santa Cruz Biotechnology, sc-374,461, Shanghai, China) diluted (1:50) in PBS at 4 °C overnight.

Techniques: In Vitro, In Vivo, Knockdown, Over Expression, Western Blot, MTT Cell Proliferation, Colony Assay, Derivative Assay, Expressing, Standard Deviation, Plasmid Preparation

Journal: Cancer Cell International

Article Title: KHDRBS3 accelerates glycolysis and promotes malignancy of hepatocellular carcinoma via upregulating 14-3-3ζ

doi: 10.1186/s12935-023-03085-4

Figure Lengend Snippet: The knockdown of KHDRBS3 induces apoptosis and inhibits glycolysis of HCC cells. ( A ) Apoptosis was analyzed using flow cytometry with Annexin V-PI staining double staining. ( B-C ) Measurement of glucose consumption and lactate production in cell culture medium. Error bars represent standard deviation. * P < 0.05, ** P < 0.01 vs. NC − sh group

Article Snippet: Slides were blocked in 1% BSA (Sangon, Shanghai, China) for 15 min and then incubated with KHDRBS3 antibody (Santa Cruz Biotechnology, sc-374,461, Shanghai, China) diluted (1:50) in PBS at 4 °C overnight.

Techniques: Knockdown, Flow Cytometry, Staining, Double Staining, Cell Culture, Standard Deviation

Journal: Cancer Cell International

Article Title: KHDRBS3 accelerates glycolysis and promotes malignancy of hepatocellular carcinoma via upregulating 14-3-3ζ

doi: 10.1186/s12935-023-03085-4

Figure Lengend Snippet: KHDRBS3 enhances chemoresistance of HCC cells. ( A ) Cell viability of HCC cells upon treatment with different concentrations of doxorubicin was assessed by MTT. ( B ) Flow cytometry analysis of indicated cells with doxorubicin (1 µmol) treatment. Error bars represent standard deviation. ** P < 0.01 vs. Vector group or NC − sh group

Article Snippet: Slides were blocked in 1% BSA (Sangon, Shanghai, China) for 15 min and then incubated with KHDRBS3 antibody (Santa Cruz Biotechnology, sc-374,461, Shanghai, China) diluted (1:50) in PBS at 4 °C overnight.

Techniques: Flow Cytometry, Standard Deviation, Plasmid Preparation

Journal: Cancer Cell International

Article Title: KHDRBS3 accelerates glycolysis and promotes malignancy of hepatocellular carcinoma via upregulating 14-3-3ζ

doi: 10.1186/s12935-023-03085-4

Figure Lengend Snippet: KHDRBS3 binds to YWHAZ and upregulates its expression. ( A ) Relative mRNA levels of c-Myc, GLUT1, LDHA, YWHAZ, HOXB9, CRABP1 and DLL4 in stable KHDRBS3 knockdown or overexpressing Huh7 cells. ( B ) Protein levels of c-Myc, GLUT1, LDHA and 14-3-3ζ in infected Huh7 cells were evaluated by western blotting. ( C ) RIP assay was performed to detect the enrichment of c-Myc, GLUT1, LDHA and YWHAZ in the anti-KHDRBS3 IP group. ( D ) RNA pull-down assay determined the binding between KHDRBS3 and YWHAZ. Error bars represent standard deviation. ** P < 0.01 vs. Vector group or NC − sh group

Article Snippet: Slides were blocked in 1% BSA (Sangon, Shanghai, China) for 15 min and then incubated with KHDRBS3 antibody (Santa Cruz Biotechnology, sc-374,461, Shanghai, China) diluted (1:50) in PBS at 4 °C overnight.

Techniques: Expressing, Knockdown, Infection, Western Blot, Pull Down Assay, Binding Assay, Standard Deviation, Plasmid Preparation

Journal: Cancer Cell International

Article Title: KHDRBS3 accelerates glycolysis and promotes malignancy of hepatocellular carcinoma via upregulating 14-3-3ζ

doi: 10.1186/s12935-023-03085-4

Figure Lengend Snippet: KHDRBS3 promotes malignant progression of HCC cells via regulating 14-3-3ζ expression. ( A ) 14-3-3ζ protein levels in Huh7 cells were measured by western blotting. ( B ) Cell proliferation was assessed using MTT assay. ( C ) Cell apoptosis was analyzed by flow cytometry. ( D-E ) Glucose consumption and lactate production were measured in Huh7 cells. Error bars represent standard deviation. * P < 0.05, ** P < 0.01 vs. KHDRBS3 + NC − si group; # P < 0.05, ## P < 0.01 vs. NC − si group

Article Snippet: Slides were blocked in 1% BSA (Sangon, Shanghai, China) for 15 min and then incubated with KHDRBS3 antibody (Santa Cruz Biotechnology, sc-374,461, Shanghai, China) diluted (1:50) in PBS at 4 °C overnight.

Techniques: Expressing, Western Blot, MTT Assay, Flow Cytometry, Standard Deviation

Journal: Cancer Cell International

Article Title: KHDRBS3 accelerates glycolysis and promotes malignancy of hepatocellular carcinoma via upregulating 14-3-3ζ

doi: 10.1186/s12935-023-03085-4

Figure Lengend Snippet: Diagram of the mechanism of KHDRBS3 promoting HCC progression. KHDRBS3 upregulates 14-3-3ζ expression by binding to YWHAZ, resulting in promotion of glycolysis, cell proliferation, chemoresistance and inhibition of apoptosis

Article Snippet: Slides were blocked in 1% BSA (Sangon, Shanghai, China) for 15 min and then incubated with KHDRBS3 antibody (Santa Cruz Biotechnology, sc-374,461, Shanghai, China) diluted (1:50) in PBS at 4 °C overnight.

Techniques: Expressing, Binding Assay, Inhibition

Journal: Life sciences

Article Title: KHDRBS3 facilitates self-renewal and temozolomide resistance of glioblastoma cell lines.

doi: 10.1016/j.lfs.2024.123132

Figure Lengend Snippet: Fig. 4. Expression of the gene encoding KHDRBS3 is up-regulated in glioblastoma-derived neurospheres. (A) The differential expression levels for proteins signif icantly up-regulated in spheroids over monolayer culture and for proteins significantly down-regulated in butyrate treatment over monolayer culture (p < 0.05). (B and C) KHDRBS3 and BAG3 mRNA expression in neurospheres of four glioblastoma cell lines including U251, LN229, U87 and M059J. The numbers indicate mean of fold-change. ACTB was used as internal control. The relative value was expressed as the mean ± SD (n = 4). The statistical analyses were tested by One-way ANOVA. ** p < 0.01; *** p < 0.005.

Article Snippet: Then, non-specific binding was blocked by incubating the membrane with 5 % bovine serum albumin (BSA) in TBS-T (20 mM Tris, 137 mM NaCl, 0.1 % Tween 20) for 1 h. The membrane was rinse twice with TBS-T and probed with KHDRBS3 rabbit polyclonal antibody (Proteintech, 13,563–1-AP, 1:1000 dilution) or β-actin mouse polyclonal antibody (Sigma-Aldrich, A1978,1:1000 dilution) at 4 ◦C overnight.

Techniques: Expressing, Derivative Assay, Quantitative Proteomics, Control

Journal: Life sciences

Article Title: KHDRBS3 facilitates self-renewal and temozolomide resistance of glioblastoma cell lines.

doi: 10.1016/j.lfs.2024.123132

Figure Lengend Snippet: Fig. 5. Depletion of KHDRBS3 expression decreased proliferation of U251 and LN229 cells. (A) Realtime PCR analysis of KHDRBS3 in U251 and LN229 cells which were transfected with 50 nM of negative control siRNA (si-CTL) or KHDRBS3-specific siRNA (si-KHDRBS3) for 48 h. ACTB was used as internal control. The relative expression value was expressed as the mean ± SD from three independent experiments. (B) Western blot analysis of KHDRBS3. Band intensities were analyzed and compared using imageJ software. (C) si-CTL and si-KHDRBS3 treated U251 and LN229 were grown under monolayer culture up to 6 days. Cell number was counted at the indicated time points. (D) Cell proliferation was determined by MTT assay at the indicated time points. Data is represented as the mean ± SD from three independent experiments. The statistical analyses were tested by One-way ANOVA. ** p < 0.01; *** p < 0.005.

Article Snippet: Then, non-specific binding was blocked by incubating the membrane with 5 % bovine serum albumin (BSA) in TBS-T (20 mM Tris, 137 mM NaCl, 0.1 % Tween 20) for 1 h. The membrane was rinse twice with TBS-T and probed with KHDRBS3 rabbit polyclonal antibody (Proteintech, 13,563–1-AP, 1:1000 dilution) or β-actin mouse polyclonal antibody (Sigma-Aldrich, A1978,1:1000 dilution) at 4 ◦C overnight.

Techniques: Expressing, Transfection, Negative Control, Control, Western Blot, Software, MTT Assay

Journal: Life sciences

Article Title: KHDRBS3 facilitates self-renewal and temozolomide resistance of glioblastoma cell lines.

doi: 10.1016/j.lfs.2024.123132

Figure Lengend Snippet: Fig. 6. KHDRBS3 facilitates neurosphere formation of U251 and LN229 cells. Sphere formation assay was performed in U251 (top) and LN229 (bottom) cells transfected with control (si-CTL) or KHDRBS3 siRNA (si-KHDRBS3). Spheres were photographed by light microscopy at days 3, 5 and 7 (magnification: 10×, scale bar: 200 μm). The number of spheres was counted in and represented as the mean ± SD from three independent experiments. The statistical analyses were tested by One-way ANOVA. * p < 0.05; ** p < 0.01; *** p < 0.005.

Article Snippet: Then, non-specific binding was blocked by incubating the membrane with 5 % bovine serum albumin (BSA) in TBS-T (20 mM Tris, 137 mM NaCl, 0.1 % Tween 20) for 1 h. The membrane was rinse twice with TBS-T and probed with KHDRBS3 rabbit polyclonal antibody (Proteintech, 13,563–1-AP, 1:1000 dilution) or β-actin mouse polyclonal antibody (Sigma-Aldrich, A1978,1:1000 dilution) at 4 ◦C overnight.

Techniques: Tube Formation Assay, Transfection, Control, Light Microscopy

Journal: Life sciences

Article Title: KHDRBS3 facilitates self-renewal and temozolomide resistance of glioblastoma cell lines.

doi: 10.1016/j.lfs.2024.123132

Figure Lengend Snippet: Fig. 7. KHDRBS3 promotes cell migration of U251 and LN229. Wound healing assay of U251 (left) and LN229 (right) transfected with si-CTL or si-KHDRBS3 was performed. Cells were seeded at density of 2.5 × 105 cells/well and incubated in normal culture media for 24 h prior wound scratching. The scratch-wounds were photographed at 0 and 24 h of incubation. The percentage of cell migration was calculated according to the reduction of wound area at 24 h compared with the area at 0 h. Error bars represent SD from three independent experiments. The statistical analyses were tested by One-way ANOVA. * p < 0.05; *** p < 0.005.

Article Snippet: Then, non-specific binding was blocked by incubating the membrane with 5 % bovine serum albumin (BSA) in TBS-T (20 mM Tris, 137 mM NaCl, 0.1 % Tween 20) for 1 h. The membrane was rinse twice with TBS-T and probed with KHDRBS3 rabbit polyclonal antibody (Proteintech, 13,563–1-AP, 1:1000 dilution) or β-actin mouse polyclonal antibody (Sigma-Aldrich, A1978,1:1000 dilution) at 4 ◦C overnight.

Techniques: Migration, Wound Healing Assay, Transfection, Incubation

Journal: Life sciences

Article Title: KHDRBS3 facilitates self-renewal and temozolomide resistance of glioblastoma cell lines.

doi: 10.1016/j.lfs.2024.123132

Figure Lengend Snippet: Fig. 8. KHDRBS3 facilitates expression of genes involved in cancer stem cells of the glioblastoma cell lines U251 and LN229. Expression of the genes encoding SOX2, PAX6, CD133 and CD44 was determined by realtime PCR in neurospheres derived from U251 and LN229 cells treated with si-CTL or si-KHDRBS3. ACTB was used as internal control. The relative value was expressed as the mean ± SD from three independent experiments. The statistical analyses were tested by One-way ANOVA. *** p < 0.005.

Article Snippet: Then, non-specific binding was blocked by incubating the membrane with 5 % bovine serum albumin (BSA) in TBS-T (20 mM Tris, 137 mM NaCl, 0.1 % Tween 20) for 1 h. The membrane was rinse twice with TBS-T and probed with KHDRBS3 rabbit polyclonal antibody (Proteintech, 13,563–1-AP, 1:1000 dilution) or β-actin mouse polyclonal antibody (Sigma-Aldrich, A1978,1:1000 dilution) at 4 ◦C overnight.

Techniques: Expressing, Derivative Assay, Control

Journal: Life sciences

Article Title: KHDRBS3 facilitates self-renewal and temozolomide resistance of glioblastoma cell lines.

doi: 10.1016/j.lfs.2024.123132

Figure Lengend Snippet: Fig. 9. Silencing of KHDRBS3 promotes TMZ sensitivity in glioblastoma. (A) U251 and LN229 cells transfected with si-CTL or si-KHDRBS3 were cultured in 96-well plates and treated with the indicated concentrations of TMZ (0–400 μM) for 48 h. The viability of cells treated with TMZ was determined by MTT assay. (B) Left panel: representative FACS analyses of apoptotic cell death of si-CTL and si-KHDRBS3 treated U251 cells in response to 100 and 200 μM TMZ. FITC-conjugated Annexin V and PI were used as the early-stage and late-stage apoptotic markers. Right panel: the histogram displayed percentages of apoptotic cells, including early and late apoptotic cells. Error bars represent SD from three independent experiments. The statistical analyses were tested by One-way ANOVA. ** p < 0.01; *** p < 0.005.

Article Snippet: Then, non-specific binding was blocked by incubating the membrane with 5 % bovine serum albumin (BSA) in TBS-T (20 mM Tris, 137 mM NaCl, 0.1 % Tween 20) for 1 h. The membrane was rinse twice with TBS-T and probed with KHDRBS3 rabbit polyclonal antibody (Proteintech, 13,563–1-AP, 1:1000 dilution) or β-actin mouse polyclonal antibody (Sigma-Aldrich, A1978,1:1000 dilution) at 4 ◦C overnight.

Techniques: Transfection, Cell Culture, MTT Assay