Journal: Bioengineered

Article Title: Deleted in lymphocytic leukemia 2 induces retinoic acid receptor beta promoter methylation and mitogen activated kinase-like protein activation to enhance viability and mobility of colorectal cancer cells

doi: 10.1080/21655979.2022.2076482

Figure Lengend Snippet: DLEU2 activates the MAPK-signaling pathway via enhancing RARB promoter methylation. (a-b) target genes of RARB showing an over 0.5 correlation coefficient with RARB and the KEGG pathway analysis based on these genes; (c) RARB expression in cells after oe-DLEU2 or sh-DLEU2 transfections detected by RT-qPCR (two-way ANOVA) (DLEU2: SW480: oe-DLEU2 vs. NC: p < 0.0001; sh-DLEU2 vs. NC: p = 0.0025; HT29: oe-DLEU2 vs. NC: p < 0.0001; sh-DLEU2 vs. NC. p = 0.0087; RARB: SW480: oe-DLEU2 vs. NC: p = 0.0024, sh-DLEU2 vs. NC: p < 0.0001; HT29: oe-DLEU2 vs. NC: p = 0.001, sh-DLEU2 vs. NC: p < 0.0001); (d) methylation level in the RARB promoter examined by qMSP (two-way ANOVA) (SW480: oe-DLEU2 vs. NC: p < 0.0001, sh-DLEU2 vs. NC p = 0.0013; HT29: oe-DLEU2 vs. NC p < 0.0001, sh-DLEU2 vs. NC: p = 0.0033); (e) phosphorylation and protein levels of Raf, p38, and ERK in CRC cells determined by immunoblot analysis (two-way ANOVA) (see p values in ). Repetition = 3.

Article Snippet: The primary antibodies were anti-RARB (1:1,000, GTX66626, GeneTex, CA, USA), anti-pRaf (Ser299) (1:1,000, #4431S, Cell Signaling Technology (CST), Beverly, MA, USA), anti-Raf (1:1,000, #4432S, CST), anti-p-p38 (1:2,000, GTX133460, GeneTex), anti-p38 (1:1,000, GTX110720, CST), anti-p-ERK1/2 (1:1,000, ab201015, Abcam), anti-ERK1/2 (1:10,000, ab184699, Abcam), and anti-GAPDH (1:10,000, ab181603, Abcam) [ ].

Techniques: Methylation, Expressing, Transfection, Quantitative RT-PCR, Phospho-proteomics, Western Blot

Journal: Bioengineered

Article Title: Deleted in lymphocytic leukemia 2 induces retinoic acid receptor beta promoter methylation and mitogen activated kinase-like protein activation to enhance viability and mobility of colorectal cancer cells

doi: 10.1080/21655979.2022.2076482

Figure Lengend Snippet: P values of the phosphorylation extent of the Raf, p38, and ERK between the oe-DLEU2 and NC groups in SW480 and HT29 cells

Article Snippet: The primary antibodies were anti-RARB (1:1,000, GTX66626, GeneTex, CA, USA), anti-pRaf (Ser299) (1:1,000, #4431S, Cell Signaling Technology (CST), Beverly, MA, USA), anti-Raf (1:1,000, #4432S, CST), anti-p-p38 (1:2,000, GTX133460, GeneTex), anti-p38 (1:1,000, GTX110720, CST), anti-p-ERK1/2 (1:1,000, ab201015, Abcam), anti-ERK1/2 (1:10,000, ab184699, Abcam), and anti-GAPDH (1:10,000, ab181603, Abcam) [ ].

Techniques: Phospho-proteomics

Journal: Bioengineered

Article Title: Deleted in lymphocytic leukemia 2 induces retinoic acid receptor beta promoter methylation and mitogen activated kinase-like protein activation to enhance viability and mobility of colorectal cancer cells

doi: 10.1080/21655979.2022.2076482

Figure Lengend Snippet: RARB silencing blocks the inhibiting role of sh-DLEU2 in CRC cells. (a) RARB expression in SW480 and HT29 cells after sh-RARB transfection detected by RT-qPCR (DLEU2: SW480: sh-DLEU2 + sh-NC vs. sh-DLEU2 + sh-RARB: p = 0.9657; HT29: sh-DLEU2 + sh-NC vs. sh-DLEU2 + sh-RARB: p = 0.8714; RARB: SW480: sh-DLEU2 + sh-NC vs. sh-DLEU2 + sh-RARB: p = 0.0006; HT29: sh-DLEU2 + sh-NC vs. sh-DLEU2 + sh-RARB: p = 0.0001); (b) viability of SW480 and HT29 cells examined by the CCK-8 method (two-way ANOVA) (SW480: 0 h: p = 0.9931; 24 h: p = 0.4837; 48 h: p = 0.0069; 72 h: p = 0.0001; HT29: 0 h: p = 0.9947; 24 h: p = 0.2835; 48 h: p = 0.0013; 72 h: p < 0.0001); (c-d) migration and invasiveness of SW480 and HT29 cells measured by Transwell assays, respectively (two-way ANOVA) (C: SW480: sh-DLEU2 + sh-NC vs. sh-DLEU2 + sh-RARB: p = 0.0005; HT29: sh-DLEU2 + sh-NC vs. sh-DLEU2 + sh-RARB p = 0.0002; D: SW480: sh-DLEU2 + sh-NC vs. sh-DLEU2 + sh-RARB: p = 0.001; HT29: sh-DLEU2 + sh-NC vs. sh-DLEU2 + sh-RARB: p = 0.0006); (e) apoptosis of SW480 and HT29 cells detected by flow cytometry (two-way ANOVA) (SW480: sh-DLEU2 + sh-NC vs. sh-DLEU2 + sh-RARB: p = 0.0001; HT29: sh-DLEU2 + sh-NC vs. sh-DLEU2 + sh-RARB: p = 0.0003); (f) phosphorylation and protein levels of Raf, p38, and ERK in CRC cells determined by immunoblot analysis (two-way ANOVA) (see p values in ); (g) p38 phosphorylation in CRC cells after Dehydrocorydaline treatment examined by immunoblot analysis (sh-DLEU2 + DMSO vs. sh-DLEU2 + Dehydrocorydaline: SW480: p = 0.0006; HT29: p = 0.0052); (h) proliferation of cells examined by the CCK-8 assay (sh-DLEU2 + DMSO vs. sh-DEU2 + Dehydrocorydaline: SW480: 0 h: p = 0.9990, 24 h: p = 0.6695, 48 h: p < 0.0001, 72 h: p < 0.0001; HT29: 0 h, p = 0.9997, 24 h: p = 0.0603, 48 h: p < 0.0001, 72 h, p < 0.0001); (i-j) migration (i, sh-DLEU2 + DMSO vs. sh-DEU2 + Dehydrocorydaline: SW480: p < 0.0001; HT29: p < 0.0001) and invasiveness (j, sh-DLEU2 + DMSO vs. sh-DEU2 + Dehydrocorydaline: SW480: p = 0.001; HT29: p = 0.0017) of cells analyzed by Transwell assays; (k) apoptosis of cells examined by flow cytometry (sh-DLEU2 + DMSO vs. sh-DEU2 + Dehydrocorydaline: SW480: p = 0.0005; HT29: p = 0.0004). Repetition = 3.

Article Snippet: The primary antibodies were anti-RARB (1:1,000, GTX66626, GeneTex, CA, USA), anti-pRaf (Ser299) (1:1,000, #4431S, Cell Signaling Technology (CST), Beverly, MA, USA), anti-Raf (1:1,000, #4432S, CST), anti-p-p38 (1:2,000, GTX133460, GeneTex), anti-p38 (1:1,000, GTX110720, CST), anti-p-ERK1/2 (1:1,000, ab201015, Abcam), anti-ERK1/2 (1:10,000, ab184699, Abcam), and anti-GAPDH (1:10,000, ab181603, Abcam) [ ].

Techniques: Expressing, Transfection, Quantitative RT-PCR, CCK-8 Assay, Migration, Flow Cytometry, Phospho-proteomics, Western Blot

Journal: Bioengineered

Article Title: Deleted in lymphocytic leukemia 2 induces retinoic acid receptor beta promoter methylation and mitogen activated kinase-like protein activation to enhance viability and mobility of colorectal cancer cells

doi: 10.1080/21655979.2022.2076482

Figure Lengend Snippet: P values of the phosphorylation extent of the Raf, p38, and ERK between the sh-DLEU2 + sh-NC and sh-DLEU2 + sh-RARB groups in SW480 and HT29 cells

Article Snippet: The primary antibodies were anti-RARB (1:1,000, GTX66626, GeneTex, CA, USA), anti-pRaf (Ser299) (1:1,000, #4431S, Cell Signaling Technology (CST), Beverly, MA, USA), anti-Raf (1:1,000, #4432S, CST), anti-p-p38 (1:2,000, GTX133460, GeneTex), anti-p38 (1:1,000, GTX110720, CST), anti-p-ERK1/2 (1:1,000, ab201015, Abcam), anti-ERK1/2 (1:10,000, ab184699, Abcam), and anti-GAPDH (1:10,000, ab181603, Abcam) [ ].

Techniques: Phospho-proteomics

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: Raf/ERK drives the proliferative and invasive phenotype of BMPR2-silenced pulmonary artery endothelial cells

doi: 10.1152/ajplung.00303.2015

Figure Lengend Snippet: Aberrant activation of the Raf/ERK signaling pathway in BMPR2-silenced HPAECs. Lysates from control HPAECs and BMPR2-deficient HPAECs were resolved by SDS-PAGE and immunoblotted. pRAF1 (serine 338) and total RAF1 (3 independent experiments in 3 different donors) (A): pARAF (Ser-445) and total ARAF (n = 4) (B), pBRAF (Ser-445) and total BRAF (n = 4) (C), pERK, and total ERK (n = 6) (D), pp38 and total p38 (n = 4) (E), pJNK and total JNK (n = 4) (F), and BIM (BCL2L11; n = 4) (G). Densitometric analyses are normalized to the loading controls and the corresponding siControl condition. Representative blots are shown, and data are presented as means ± SE; *P < 0.05; **P < 0.01. H: schematic diagram of dysregulated Ras/Raf/ERK signaling resulting from BMPR2 loss-of-function. Red (up-regulated) and green (down-regulated) shading indicates the directional changes of significantly regulated transcripts [false discovery rates (FDR) < 0.01; no FC cutoff] by microarray. Black arrows (up or down) indicate experimentally determined directional changes in protein expression and/or activation state.

Article Snippet: Antibodies against p-ERK (no. 9101, 1:1,000), total ERK (no. 9102, 1:1,000), p-p38 (no. 9211, 1:400), total p38 (no. 9212, 1:800), p-JNK (no. 4671, 1:400), total JNK (no. 9258, 1:1,000), p-RAF-1 (no. 9427, 1:1,000), p-ARAF (no. 4431, 1:500), p-BRAF (no. 2696, 1:500), were purchased from Cell Signaling (Danvers, MA).

Techniques: Activation Assay, Control, SDS Page, Microarray, Expressing