Journal: Current protocols

Article Title: O-GlcNAc engineering on a target protein in cells with nanobody-OGT and -splitOGA

doi: 10.1002/cpz1.117

Figure Lengend Snippet: Number of wells and DNA amounts can be scaled up accordingly for additional replicates or material.

Article Snippet: . pcDNA3.1-GFP-Flag-Nup62-EPEA (Addgene, cat. No. 168101). pcDNA3.1-empty vector (Invitrogen, cat. No. V79020). pcDNA3.1-myc-OGA(1–400) (Addgene, cat. No. 168095). pcDNA3.1-myc-OGA(1–400, D174N) (Addgene, cat. No. 168097). pcDNA3.1-HA-nLag6-(EAAAK)4-OGA(544–706) (Addgene, cat. No. 168190).

Techniques: Plasmid Preparation

Journal: ACS Omega

Article Title: Plk3 Enhances Cisplatin Sensitivity of Nonsmall-Cell Lung Cancer Cells through Inhibition of the PI3K/AKT Pathway via Stabilizing PTEN

doi: 10.1021/acsomega.3c07271

Figure Lengend Snippet: Plk3 expression is downregulated in NSCLC clinical samples and cell lines. (A–C) Differential gene expression levels of Plk3, GSK3B, and CSNK2A2 between normal, LUAD, and LUSC subtypes were analyzed using GEPIA2 based on the TCGA and GTEx databases. (D) RT-qPCR assay was performed to measure the Plk3 mRNA levels in different cell lines. (E,F) Western blot analysis was conducted for the detection of Plk3 protein levels in different cell lines. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: The recombinant plasmids pcDNA3.1/Plk3 (Plk3-OE), pcDNA3.1/PTEN (PTEN-OE), small interfering RNA targeting Plk3 (si-Plk3), si-PTEN, and negative control (si-NC) were obtained from Guangzhou RiboBio Co., Ltd. (RiboBio, Guangzhou, China).

Techniques: Expressing, Gene Expression, Quantitative RT-PCR, Western Blot

Journal: ACS Omega

Article Title: Plk3 Enhances Cisplatin Sensitivity of Nonsmall-Cell Lung Cancer Cells through Inhibition of the PI3K/AKT Pathway via Stabilizing PTEN

doi: 10.1021/acsomega.3c07271

Figure Lengend Snippet: Overexpression of Plk3 enhances the DDP sensitivity of DDP-resistant NSCLC cells. (A–C) Plk3-overexpressing A549/DDP cells and H1299/DDP cells were, respectively, constructed. Results of RT-qPCR and Western blot showed that Plk3 expression was dramatically upregulated in A549/DDP cells and H1299/DDP cells after transfection with the Plk3-OE plasmid. (D,E) Plk3 suppressed the DDP resistance of A549/DDP cells and H1299/DDP cells, as shown by reduced IC 50 values of DDP. (F,G) Flow cytometry demonstrated that Plk3 overexpression promoted A549/DDP and H1299/DDP cell apoptosis. (H,I) The mRNA levels of ABCB1 and the protein levels of p-gp (encoded by the ABCB1 gene) were downregulated by Plk3, as shown by RT-qPCR and Western blot. ** p < 0.01, *** p < 0.001.

Article Snippet: The recombinant plasmids pcDNA3.1/Plk3 (Plk3-OE), pcDNA3.1/PTEN (PTEN-OE), small interfering RNA targeting Plk3 (si-Plk3), si-PTEN, and negative control (si-NC) were obtained from Guangzhou RiboBio Co., Ltd. (RiboBio, Guangzhou, China).

Techniques: Over Expression, Construct, Quantitative RT-PCR, Western Blot, Expressing, Transfection, Plasmid Preparation, Flow Cytometry

Journal: ACS Omega

Article Title: Plk3 Enhances Cisplatin Sensitivity of Nonsmall-Cell Lung Cancer Cells through Inhibition of the PI3K/AKT Pathway via Stabilizing PTEN

doi: 10.1021/acsomega.3c07271

Figure Lengend Snippet: Knockdown of Plk3 decreases the DDP sensitivity of NSCLC cells via regulating PI3K/AKT signaling. (A,B) Plk3-silencing A549/DDP and H1299/DDP cell lines were respectively constructed through transfection with si-Plk3. RT-qPCR and Western blot showed that Plk3 expression was dramatically downregulated after transfection with si-Plk3. (C,D) A549 and H1299 cells transfected with si-NC or si-Plk3 were incubated with MK-2206 (10 μM) for 24 h in the presence of 2 μg/mL DDP. Plk3-silencing A549 and H1299 cells showed higher levels of the p-Akt/Akt ratio, which could be partially reversed by MK2206. (E) Flow cytometry showed that the si-Plk3-caused decrease in cell apoptosis was mitigated by MK2206. (F,G) Knockdown of Plk3 caused an obvious increase in the IC 50 values of DDP in A549 and H1299 cells. While interfering with MK2206 blocked the effects of si-Plk3 on the IC 50 values of DDP. (H–J) A549 and H1299 cells transfected with si-Plk3 showed increased mRNA levels of ABCB1 and protein levels of p-gp, which could be attenuated by MK2206. ** p < 0.01, *** p < 0.001.

Article Snippet: The recombinant plasmids pcDNA3.1/Plk3 (Plk3-OE), pcDNA3.1/PTEN (PTEN-OE), small interfering RNA targeting Plk3 (si-Plk3), si-PTEN, and negative control (si-NC) were obtained from Guangzhou RiboBio Co., Ltd. (RiboBio, Guangzhou, China).

Techniques: Knockdown, Construct, Transfection, Quantitative RT-PCR, Western Blot, Expressing, Incubation, Flow Cytometry

Journal: ACS Omega

Article Title: Plk3 Enhances Cisplatin Sensitivity of Nonsmall-Cell Lung Cancer Cells through Inhibition of the PI3K/AKT Pathway via Stabilizing PTEN

doi: 10.1021/acsomega.3c07271

Figure Lengend Snippet: Plk3 phosphorylates and stabilizes PTEN in both DDP-sensitive NSCLC cells and DDP-resistant NSCLC cells. (A,B) For CHX treatment, the cells were respectively incubated with CHX (0.1 mg/mL) for 0, 1, 4, or 8 h. A western blot was then performed to measure PTEN protein degradation. (C–E) Western blot also showed that Plk3 overexpression induced expression levels of PTEN and p-PTEN in A549/DDP cells, and Plk3 knockdown suppressed PTEN and p-PTEN expression in H1299 cells. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: The recombinant plasmids pcDNA3.1/Plk3 (Plk3-OE), pcDNA3.1/PTEN (PTEN-OE), small interfering RNA targeting Plk3 (si-Plk3), si-PTEN, and negative control (si-NC) were obtained from Guangzhou RiboBio Co., Ltd. (RiboBio, Guangzhou, China).

Techniques: Incubation, Western Blot, Over Expression, Expressing, Knockdown

Journal: ACS Omega

Article Title: Plk3 Enhances Cisplatin Sensitivity of Nonsmall-Cell Lung Cancer Cells through Inhibition of the PI3K/AKT Pathway via Stabilizing PTEN

doi: 10.1021/acsomega.3c07271

Figure Lengend Snippet: Plk3 inhibits PI3K/AKT signaling by regulating PTEN. (A,B) Decreased p-Akt/Akt ratio caused by Plk3 overexpression was elevated after transfection with si-PTEN. (C,D) Knockdown of Plk3 significantly increased the p-Akt/Akt ratio, which could be reversed by PTEN overexpression. ** p < 0.01, *** p < 0.001.

Article Snippet: The recombinant plasmids pcDNA3.1/Plk3 (Plk3-OE), pcDNA3.1/PTEN (PTEN-OE), small interfering RNA targeting Plk3 (si-Plk3), si-PTEN, and negative control (si-NC) were obtained from Guangzhou RiboBio Co., Ltd. (RiboBio, Guangzhou, China).

Techniques: Over Expression, Transfection, Knockdown