Journal: Frontiers in Immunology

Article Title: PP2Cδ Controls the Differentiation and Function of Dendritic Cells Through Regulating the NSD2/mTORC2/ACLY Pathway

doi: 10.3389/fimmu.2021.751409

Figure Lengend Snippet: PP2Cδ represses Rictor expression through restraining the level of methyltransferase NSD2. (A, B) Immunoblotting analysis for the total or phosphorylated proteins as indicated in WT or PP2Cδ -/- DCs stimulated with LPS (100 ng/mL) for the indicated time periods. (C, D) Immunoblotting of Rictor and Raptor (C) , or H3K36me2 and H3K27me3 (D) in WT or PP2Cδ -/- DCs stimulated with LPS (100 ng/mL) for the indicated time periods. (E) ChIP test of NSD2 enrichment at the Rictor locus in WT or PP2Cδ -/- DCs with or without LPS stimulation. IgG as a negative control. (F) Immunoblotting of the indicated molecules in WT or PP2Cδ -/- DCs transfected with NSD2-targeted siRNA or non-specific control nucleotides (NC). (G-K) WT or PP2Cδ -/- DCs were transfected with NSD2 siRNA or NC, followed by stimulation with LPS. DCs were then subjected to the analysis of apoptotic rate (G) ; activation markers expression (H) , cytokines production (I) , T cell-secreted cytokines (J) and induction of T cells differentiation (K) . The data below the lanes represent the bands densities relative to that of the loading control Actin. Shown are representative images and the data from three independent experiments are expressed as means ± SD, with two or three technical replicates. *P < 0.05, **P < 0.01, ***P < 0.001 by student’s t test.

Article Snippet: Anti-AKT antibody (4691S), antibody to AKT phosphorylated at Ser473 (4060S), antibody to AKT phosphorylated at Thr308 (13038), anti-mTOR antibody (2983S), antibody to mTOR phosphorylated at Ser2448 (5536S), anti-PP2Cδ antibody (11901S), anti-FoxO1 antibody (2880), anti-Rictor antibody (9476S), anti-Raptor antibody (2280S), anti-H3K36me2 antibody (2901S), anti-H3K27me3 antibody (9733S), antibody to ACLY phosphorylated at Ser455 (4331S), anti-Phospho-p70S6Kinase (97596S), anti-p70S6 Kinase (9202s), anti-Phospho-4E-BP1 (2855), anti-4E-BP1 (9644S), anti-H3K9/14Ac antibody (9677S), anti-H3K27Ac antibody (8173S) were from Cell Signaling technology; anti-NSD2 antibody (ab75359), anti-Phospho-serine (ab9332), anti-ACLY (ab40793) were from Abcam; anti-CUL4B antibody (20882-1-AP) was from Proteintech; anti-NDRG1 antibody (abs136385) and antibody to NDRG1 phosphorylated at Thr346 (abs140323) were from Absin.

Techniques: Expressing, Western Blot, Negative Control, Transfection, Control, Activation Assay

Journal: Neuron

Article Title: The Dynamic Epigenetic Landscape of the Retina During Development, Reprogramming, and Tumorigenesis

doi: 10.1016/j.neuron.2017.04.022

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Rabbit polyclonal Anti-H3K36me3 , Active Motif , Cat# 61101, RRID:AB_2615073.

Techniques: Recombinant, Sequencing, Derivative Assay, Software, Expressing

Journal: Cell Death & Disease

Article Title: SETD2 mutation in renal clear cell carcinoma suppress autophagy via regulation of ATG12

doi: 10.1038/s41419-020-2266-x

Figure Lengend Snippet: Immunoblot analysis of the histone modifying enzyme SETD2 ( a ) and its histone target H3K36me3 ( b ) expression levels in ACHN and CAKI-1 renal clear carcinoma cells confirmed the SETD2 deficiency in the last named RCC cell line. c Immunoblot analysis of LC3 shows decreased expression level in SETD2 -deficient CAKI-1 cells as compared with SETD2- wild-type ACHN cells. Treatment with Bafilomycin A1 (BafA1, 40 nM) for 4 h shows a significant decrease in LC3-II lipidation in CAKI-1 cells as compared with ACHN cells. d Quantification of LC3-II expression compared with the expression of the housekeeping gene, actin, with and without BafA1 treatment in both cell lines. e Quantification of LC3-II/LC3-I ratio to monitor autophagic flux in ACHN and CAKI-1 cells with and without BafA1 treatment demonstrates that cells that lacks SETD2 expression exhibit a decreased autophagic flux. f Analysis of MAP1LC3B mRNA expression by qPCR in SETD2 -deficient CAKI-1 cells and SETD2-competent ACHN cells. Bars display the mean of four ( d , e ) or three ( f ) experiments, error bars represent SEM; *** p ≤ 0.001; ** p ≤ 0.01; * p ≤ 0.05.

Article Snippet: H3K36me3 (rabbit pAb) , RRID:AB_1950412 , Western blot , Cell Signaling (4909).

Techniques: Western Blot, Expressing

Journal: Cell Death & Disease

Article Title: SETD2 mutation in renal clear cell carcinoma suppress autophagy via regulation of ATG12

doi: 10.1038/s41419-020-2266-x

Figure Lengend Snippet: a Human kidney tissue microarrays including 30 tissues classified as ccRCC tumors (highlighted in light gray) were proceed for immunohistochemical staining for H3K36me3 ( b ) and ATG12 protein ( c ) and revealed low H3K36me3 ( d ) but high ATG12 ( e ) protein expressions. HeLa were used as a positive control for H3K36me3 protein expression in b . f , g SETD2 and ATG12 high versus low gene expression levels (expressed in FPKM and with a cut off of 5.37 for SETD2 and 4.66 for ATG12 ) in tumor tissue derived from ccRCC patients at the time of diagnosis and their correlation to patient survival, i.e., follow up after diagnosis (expressed in years) was extracted from the pathology atlas of the human cancer transcriptome available at the Human Protein Atlas ( www.proteinatlas.org ). Survival analysis, prognosis of each group of patients examined using Kaplan–Meier survival estimators, and survival outcomes of the groups compared by log-rank tests revealed that SETD2 should be considered as a favorable prognostic gene and ATG12 as an unfavorable prognostic gene for ccRCC patients.

Article Snippet: H3K36me3 (rabbit pAb) , RRID:AB_1950412 , Western blot , Cell Signaling (4909).

Techniques: Immunohistochemical staining, Staining, Positive Control, Expressing, Gene Expression, Derivative Assay, Biomarker Discovery

Journal: Cell Death & Disease

Article Title: SETD2 mutation in renal clear cell carcinoma suppress autophagy via regulation of ATG12

doi: 10.1038/s41419-020-2266-x

Figure Lengend Snippet: Antibody used in this study.

Article Snippet: H3K36me3 (rabbit pAb) , RRID:AB_1950412 , Western blot , Cell Signaling (4909).

Techniques: Western Blot