Journal: Heliyon

Article Title: Dexmedetomidine suppressed the biological behavior of RAW264.7 cells treated with LPS by down-regulating HOTAIR

doi: 10.1016/j.heliyon.2024.e27690

Figure Lengend Snippet: The primers of qRT-PCR used in this study.

Article Snippet: Antibodies were used, including rabbit anti-YTHDF1 monoclonal antibody (1:1000，Cell signaling technology), mouse anti-GAPDH monoclonal antibody (1:1000,Abcam).

Techniques:

Journal: Heliyon

Article Title: Dexmedetomidine suppressed the biological behavior of RAW264.7 cells treated with LPS by down-regulating HOTAIR

doi: 10.1016/j.heliyon.2024.e27690

Figure Lengend Snippet: Dexmedetomidine up-regulated the m6A level of HOTAIR in LPS-treated RAW264.7 cells. (A) The expression of HOTAIR m6A level was detect by MeRIP-qPCR. (B) HOTAIR was significantly enriched by YTHDF1 compared with IgG. (C) The expression of YTHDF1 was detect by qRT-PCR and WB in LPS-treated RAW264.7 cells. RAW264.7 cells were transfected with siYTHDF1 (300 nM) or pYTHDF1 (10 μg) in a 100-mm dish, and the cell lysate was divided into three parts: one for WB to determine the expression of YTHDF1 (D), and the other two for qRT-PCR to assess HOTAIR expression (E) and MeRIP-qPCR to determine the m6A level on HOTAIR (F).

Article Snippet: Antibodies were used, including rabbit anti-YTHDF1 monoclonal antibody (1:1000，Cell signaling technology), mouse anti-GAPDH monoclonal antibody (1:1000,Abcam).

Techniques: Expressing, Quantitative RT-PCR, Transfection

Journal: Heliyon

Article Title: Dexmedetomidine suppressed the biological behavior of RAW264.7 cells treated with LPS by down-regulating HOTAIR

doi: 10.1016/j.heliyon.2024.e27690

Figure Lengend Snippet: The primers of qRT-PCR used in this study.

Article Snippet: The beads (Thermo Scientific, USA) were combined with rabbit anti-YTHDF1 antibody (Cell signaling technology) and anti-rabbit IgG antibody (Cell Signaling Technology), and washed twice with RIP wash buffer.

Techniques:

Journal: Heliyon

Article Title: Dexmedetomidine suppressed the biological behavior of RAW264.7 cells treated with LPS by down-regulating HOTAIR

doi: 10.1016/j.heliyon.2024.e27690

Figure Lengend Snippet: Dexmedetomidine up-regulated the m6A level of HOTAIR in LPS-treated RAW264.7 cells. (A) The expression of HOTAIR m6A level was detect by MeRIP-qPCR. (B) HOTAIR was significantly enriched by YTHDF1 compared with IgG. (C) The expression of YTHDF1 was detect by qRT-PCR and WB in LPS-treated RAW264.7 cells. RAW264.7 cells were transfected with siYTHDF1 (300 nM) or pYTHDF1 (10 μg) in a 100-mm dish, and the cell lysate was divided into three parts: one for WB to determine the expression of YTHDF1 (D), and the other two for qRT-PCR to assess HOTAIR expression (E) and MeRIP-qPCR to determine the m6A level on HOTAIR (F).

Article Snippet: The beads (Thermo Scientific, USA) were combined with rabbit anti-YTHDF1 antibody (Cell signaling technology) and anti-rabbit IgG antibody (Cell Signaling Technology), and washed twice with RIP wash buffer.

Techniques: Expressing, Quantitative RT-PCR, Transfection

Journal: Molecular Therapy Oncolytics

Article Title: Targeting YTHDF1 effectively re-sensitizes cisplatin-resistant colon cancer cells by modulating GLS-mediated glutamine metabolism

doi: 10.1016/j.omto.2021.01.001

Figure Lengend Snippet: Increased YTHDF1 expression is associated with CRC (A) Expression levels of YTHDF1 from 50 colon tumor tissues and their matched normal colon tissues were detected by qRT-PCR. (B) YTHDF1 protein expression levels were detected from eight CRC tumors and their matched normal colon tissues by western blot. β-Actin was a loading control. (C) Representative immunohistochemical staining of YTHDF1 protein expression levels from colon tumor tissues and their matched normal colon tissues. (D) mRNA expression levels of YTHDF1 in normal colon epithelial cells and five CRC cell lines were detected by qRT-PCR. (E and F) DLD-1 (E) and LoVo (F) cells were transfected with control or YTHDF1 overexpression plasmid for 48 h, followed by cisplatin treatments at the indicated concentrations. Cell viability was determined by MTT and clonogenic assays. Columns include mean of three independent experiments; data are presented as mean ± SD. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.

Article Snippet: Vector containing the wild-type open reading frame (ORF) clone of Homo sapiens protein YTHDF1 was purchased from Origene. sh-YTHDF1 and control shRNA were constructed by GenePharma (Shanghai, China); si-GLS1 and control small interfering RNA (siRNA) were constructed by Hanbio (Shanghai, China); and CHX, cisplatin, MG-132, and BPTES were purchased from Sigma-Aldrich (Shanghai, China).

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Control, Immunohistochemical staining, Staining, Transfection, Over Expression, Plasmid Preparation

Journal: Molecular Therapy Oncolytics

Article Title: Targeting YTHDF1 effectively re-sensitizes cisplatin-resistant colon cancer cells by modulating GLS-mediated glutamine metabolism

doi: 10.1016/j.omto.2021.01.001

Figure Lengend Snippet: Positive correlation between YTHDF1 and cisplatin resistance in colon cancer cells (A) LoVo CDDP R cells were established according to the description in . (B and C) LoVo parental and CDDP R cells were treated with cisplatin at the indicated concentrations for 48 h, and cell viability was measured by an MTT assay, clonogenic assay (B), and annexin V assay (C). (D and E) The protein (D) and mRNA (E) expression levels of EGFR were measured in DLD-1 parental and 5-fluorouracil (5-Fu)-resistant cells. (F) YTHDF1 was stably knocked down in LoVo CDDP R cells. Cell were treated with cisplatin at the indicated concentrations for 48 h, and cell viability was measured by an MTT assay. Columns include mean of three independent experiments; data are presented as mean ± SD. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.

Article Snippet: Vector containing the wild-type open reading frame (ORF) clone of Homo sapiens protein YTHDF1 was purchased from Origene. sh-YTHDF1 and control shRNA were constructed by GenePharma (Shanghai, China); si-GLS1 and control small interfering RNA (siRNA) were constructed by Hanbio (Shanghai, China); and CHX, cisplatin, MG-132, and BPTES were purchased from Sigma-Aldrich (Shanghai, China).

Techniques: MTT Assay, Clonogenic Assay, Annexin V Assay, Expressing, Stable Transfection

Journal: Molecular Therapy Oncolytics

Article Title: Targeting YTHDF1 effectively re-sensitizes cisplatin-resistant colon cancer cells by modulating GLS-mediated glutamine metabolism

doi: 10.1016/j.omto.2021.01.001

Figure Lengend Snippet: YTHDF1 directly binds with GLS1 to promote its protein translation (A and B) LoVo and DLD-1 cells were transfected with control, wild-type (WT) YTHDF1, or YTH domain mutant YTHDF1 for 48 h (A), and glutamine uptake and GLS activity (B) were detected. (C) Predicted YTHDF1 binding motif on the 3′ UTR of GLS1 mRNA. (D) RNA immunoprecipitation (RIP) was performed in LoVo and DLD-1 cells using anti-IgG control or anti-YTHDF1 antibody. (E and F) GLS1 abundance in the immunoprecipitated fraction was measured by agarose gel electrophoresis and (E) qRT-PCR. (F) An RNA pull-down assay was performed in LoVo and DLD-1 cells. The biotin-labeled 3′ UTR of WT or binding motif mutant GLS1 was incubated with proteins extracted from cells. The YTHDF1 protein, which was pulled down by the GLS1 binding motif, was detected by western blot. β-Actin was used as a negative control. (G) LoVo and DLD-1 cells were transfected with control shRNA or YTHDF1 shRNA for 48 h, and RIP experiments were performed using an anti-IgG control or anti-YTHDF1 antibody. GLS1 and β-actin mRNA abundance levels in the immunoprecipitated fraction were measured by agarose gel electrophoresis and (H) qRT-PCR. (I and J) Reporter constructs containing the WT or the binding motif mutant (Mut) GLS1 3′ UTR were co-transfected with control shRNA or GLS1 shRNA into LoVo and DLD-1 cells. Luciferase activities were measured using a Dual-Luciferase reporter assay kit. (K) LoVo and DLD-1 cells were treated with 10 μg/mL CHX for 0 and 6 h, and the relative GLS1 protein expression was measured by western blot. (L) LoVo and DLD-1 cells were treated with 50 nM MG-132 for 0 and 6 h, and the relative GLS1 protein expression was measured by western blot. β-Actin was used as an internal control. Data are presented as mean ± SD. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.

Article Snippet: Vector containing the wild-type open reading frame (ORF) clone of Homo sapiens protein YTHDF1 was purchased from Origene. sh-YTHDF1 and control shRNA were constructed by GenePharma (Shanghai, China); si-GLS1 and control small interfering RNA (siRNA) were constructed by Hanbio (Shanghai, China); and CHX, cisplatin, MG-132, and BPTES were purchased from Sigma-Aldrich (Shanghai, China).

Techniques: Transfection, Control, Mutagenesis, Activity Assay, Binding Assay, RNA Immunoprecipitation, Immunoprecipitation, Agarose Gel Electrophoresis, Quantitative RT-PCR, Pull Down Assay, Labeling, Incubation, Western Blot, Negative Control, shRNA, Construct, Luciferase, Reporter Assay, Expressing

Journal: Molecular Therapy Oncolytics

Article Title: Targeting YTHDF1 effectively re-sensitizes cisplatin-resistant colon cancer cells by modulating GLS-mediated glutamine metabolism

doi: 10.1016/j.omto.2021.01.001

Figure Lengend Snippet: YTHDF1-mediated cisplatin resistance is through promoting glutamine metabolism (A) DLD-1 cells were transfected with control or YTHDF1 overexpression plasmid for 48 h. Cells were treated without or with BPTES for 24 h. The GLS activity was measured. (B) The above cells were treated with cisplatin at 0, 2.5, 5, 10, 20, or 40 μM for 48 h. Cell viability and death were assessed by an MTT assay and (C) annexin V assay, respectively. (D) LoVo cells were transfected with control or YTHDF1 overexpression plasmid for 48 h. Cells were treated without or with BPTES for 24 h. The GLS activity was measured. (E) The above cells were treated with cisplatin at 0, 2, 4, 8, 16, or 32 μM for 48 h. Cell viability and death were assessed by an MTT assay and (F) annexin V assay, respectively. Data are presented as mean ± SD. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.

Article Snippet: Vector containing the wild-type open reading frame (ORF) clone of Homo sapiens protein YTHDF1 was purchased from Origene. sh-YTHDF1 and control shRNA were constructed by GenePharma (Shanghai, China); si-GLS1 and control small interfering RNA (siRNA) were constructed by Hanbio (Shanghai, China); and CHX, cisplatin, MG-132, and BPTES were purchased from Sigma-Aldrich (Shanghai, China).

Techniques: Transfection, Control, Over Expression, Plasmid Preparation, Activity Assay, MTT Assay, Annexin V Assay

Journal: Molecular Therapy Oncolytics

Article Title: Targeting YTHDF1 effectively re-sensitizes cisplatin-resistant colon cancer cells by modulating GLS-mediated glutamine metabolism

doi: 10.1016/j.omto.2021.01.001

Figure Lengend Snippet: In vivo blocking YTHDF1-mediated glutamine metabolism sensitizes colon cancer cells to cisplatin (A) LoVo CDDP R cells were transfected with control shRNA or YTHDF1 shRNA. Cells were subcutaneously injected into nude mice for developing xenograft tumors. Mice without or with YTHDF1 silencing xenograft tumors were grouped and treated with control saline or cisplatin via intraperitoneal injection twice a week. Mice survival rates and (B) tumor growth were examined. (C) A total of 60 xenograft tumors from the above-treated mice of each experiment group (15 for each group). (D) Xenograft tumors from mice were dissected, and the protein expression levels of GLS1 were examined by western blot and quantified. Data are presented as mean ± SD. ∗p < 0.05, ∗∗∗p < 0.001.

Article Snippet: Vector containing the wild-type open reading frame (ORF) clone of Homo sapiens protein YTHDF1 was purchased from Origene. sh-YTHDF1 and control shRNA were constructed by GenePharma (Shanghai, China); si-GLS1 and control small interfering RNA (siRNA) were constructed by Hanbio (Shanghai, China); and CHX, cisplatin, MG-132, and BPTES were purchased from Sigma-Aldrich (Shanghai, China).

Techniques: In Vivo, Blocking Assay, Transfection, Control, shRNA, Injection, Saline, Expressing, Western Blot