Journal: Oncology Reports

Article Title: KLF4, DAPK1 and SPG20 promoter methylation is not affected by DNMT1 silencing and hypomethylating drugs in lymphoma cells

doi: 10.3892/or.2021.8221

Figure Lengend Snippet: Quantitative promoter methylation in two diffuse large B cell lymphoma cell lines and effects of hypomethylating agents. (A) Toledo and NU-DUL-1 cell lines were analyzed via EpiTect Methyl II qPCR. Data are reported as % promoter methylation and error bars represent SD (n=3). (B) Immunoblotting showing DNMT1, PARP and β-actin after 5AZA treatment for 48 h. (C) Immunoblotting showing DNMT1 and PARP and β-actin after decitabine treatment for 48 h. (D) Immunoblotting showing DNMT1, PARP and β-actin after 5AZA treatments for 7 days. (E) Immunoblotting showing DNMT1 and PARP and β-actin after decitabine treatments for 7 days. n=2. DNMT1, DNA methyltransferase 1; PARP, poly (ADP-ribose) polymerase; NT, not treated; KLF4, Krüppel-like factor 4; DAPK1, death associated protein 1; SPG20, spastic paraplegia 20; 5AZA, 5′-aza-cytidine; decitabine, 5′-aza-2-deoxycitidine.

Article Snippet: Rabbit primary antibodies against DNMT1 (cat. no. 5032), DNMT3a (clone D23G1; cat. no. 3598), SPG20 (cat. no. 13520S), KLF4 (cat. no. 12173) and poly (ADP-ribose) polymerase (PARP; cat. no. 9532) were purchased from Cell Signaling Technology, Inc. Anti-DAPK1 (cat. no. MA1-24696) was purchased from Thermo Fisher Scientific, Inc. All antibodies were used at a final dilution of 1:2,000.

Techniques: Methylation, Western Blot

Journal: Oncology Reports

Article Title: KLF4, DAPK1 and SPG20 promoter methylation is not affected by DNMT1 silencing and hypomethylating drugs in lymphoma cells

doi: 10.3892/or.2021.8221

Figure Lengend Snippet: Global chromatin methylation, gene-specific quantitative methylation and gene expression after 5AZA and decitabine treatment. (A) Chromatin methylation after 48 h or 7 days from a single administration of the indicated drug. ***P<0.0001 (n=3; one-way ANOVA). (B) Quantitative promoter methylation of KLF4, DAPK1 and SPG20 after the administration of either 1 µM 5AZA or decitabine 0.5 µM for 48 h. (C) Gene expression of the selected targets upon treatment with hypomethylating drugs (cutoff, 37 cycles). n=2/3. (D) KLF4, DAPK1 and SPG20 protein expression levels after treatment with hypomethylating drugs for 48 h. n=3. The blots of GNE-587170 protein extracts are presented as positive controls. NT, not treated; KLF4, Krüppel-like factor 4; DAPK1, death associated protein 1; SPG20, spastic paraplegia 20; 5AZA, 5′-aza-cytidine; decitabine, 5′-aza-2-deoxycitidine.

Article Snippet: Rabbit primary antibodies against DNMT1 (cat. no. 5032), DNMT3a (clone D23G1; cat. no. 3598), SPG20 (cat. no. 13520S), KLF4 (cat. no. 12173) and poly (ADP-ribose) polymerase (PARP; cat. no. 9532) were purchased from Cell Signaling Technology, Inc. Anti-DAPK1 (cat. no. MA1-24696) was purchased from Thermo Fisher Scientific, Inc. All antibodies were used at a final dilution of 1:2,000.

Techniques: Methylation, Expressing

Journal: Oncology Reports

Article Title: KLF4, DAPK1 and SPG20 promoter methylation is not affected by DNMT1 silencing and hypomethylating drugs in lymphoma cells

doi: 10.3892/or.2021.8221

Figure Lengend Snippet: Effects of DNMT1- and DNMT3a-selective silencing on gene-specific quantitative methylation and expression. (A) Silencing efficiency after 48 h from transfection in Toledo and NU-DUL-1 cell lines as determined by RT-qPCR. *P<0.05, **P<0.01, ***P<0.0001 (n=3; one-way ANOVA). (B) Silencing efficiency 48 h after transfection in Toledo and NU-DUL-1 cell lines as determined by immunoblotting (n=2). (C) Gene expression levels of KLF4, DAPK1 and SPG20 as determined by RT-qPCR (n=2). (D) Quantitative promoter methylation in the panel of target loci 48 h after DNMT1 silencing. (E) Quantitative promoter methylation in the panel of target loci 48 h after DNMT3a silencing. (F) Quantitative promoter methylation in the panel of target loci 48 h after DNMT1 + DNMT3a silencing (n=2/3). (G) Ki67 staining of the transfected cells after 48 h incubation as determined via immunocytochemistry (magnification, ×40). (H) Cell Counting Kit-8 viability assay of the transfected cells after 48 h incubation. *P<0.05 (one-way ANOVA). DNMT, DNA methyltransferase; RT-qPCR, reverse transcription-quantitative PCR; KLF4, Krüppel-like factor 4; DAPK1, death associated protein 1; SPG20, spastic paraplegia 20; si, small interfering RNA.

Article Snippet: Rabbit primary antibodies against DNMT1 (cat. no. 5032), DNMT3a (clone D23G1; cat. no. 3598), SPG20 (cat. no. 13520S), KLF4 (cat. no. 12173) and poly (ADP-ribose) polymerase (PARP; cat. no. 9532) were purchased from Cell Signaling Technology, Inc. Anti-DAPK1 (cat. no. MA1-24696) was purchased from Thermo Fisher Scientific, Inc. All antibodies were used at a final dilution of 1:2,000.

Techniques: Methylation, Expressing, Transfection, Quantitative RT-PCR, Western Blot, Staining, Incubation, Immunocytochemistry, Cell Counting, Viability Assay, Real-time Polymerase Chain Reaction, Small Interfering RNA

Journal: Oncology Reports

Article Title: KLF4, DAPK1 and SPG20 promoter methylation is not affected by DNMT1 silencing and hypomethylating drugs in lymphoma cells

doi: 10.3892/or.2021.8221

Figure Lengend Snippet: SPG20 is epigenetically regulated in human PBMCs. (A) Methylation profile of the selected targets in four representative PBMCs from healthy donors. (B) Overall methylation profiles of 20 PBMCs. UM corresponds to a % of demethylation ≥91%; lower percentages are specified in each box. (C) SPG20 and DNMT1 protein levels in 20 representative PBMCs from healthy donors (data are representative of a total of 27 independent PBMCs samples). SPG20, spastic paraplegia 20; PBMCs, peripheral blood mononuclear cells; UM, unmethylated; DNMT1, DNA methyltransferase 1; KLF4, Krüppel-like factor 4; DAPK1, death associated protein 1.

Article Snippet: Rabbit primary antibodies against DNMT1 (cat. no. 5032), DNMT3a (clone D23G1; cat. no. 3598), SPG20 (cat. no. 13520S), KLF4 (cat. no. 12173) and poly (ADP-ribose) polymerase (PARP; cat. no. 9532) were purchased from Cell Signaling Technology, Inc. Anti-DAPK1 (cat. no. MA1-24696) was purchased from Thermo Fisher Scientific, Inc. All antibodies were used at a final dilution of 1:2,000.

Techniques: Methylation