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