Journal: Molecular and cellular endocrinology

Article Title: SDHB and SDHD silenced pheochromocytoma spheroids respond differently to tumour microenvironment and their aggressiveness is inhibited by impairing stroma metabolism.

doi: 10.1016/j.mce.2022.111594

Figure Lengend Snippet: Fig. 1. Characterization of stable SDHB and SDHD silenced cell lines. (A) Representative blots of the expression of mitochondrial SDHA, B and D subunits. (B) Densitometric analysis of western blot bands, performed by Bio-Rad imaging and analysis software (Quantity One), showed significant differences in the SDHB and SDHD subunit expression levels in SDHB (light grey) and in SDHD silenced cells (dark grey) respectively, compared to Wt (black). Bars are the means of three independent preparations ± SD, ***p < 0.001. (C) Representative traces of SDH enzymatic activity measured in cell homogenates. The silenced SDHB and SDHD cells (dotted and continuous lines, respectively) showed a similar decrease of the SDH activity, significantly different compared with Wt (dashed line). (D) Histogram represents the SDH activity expressed as the percentages. SDHB and SDHD silenced cells (light and dark grey, respectively) showed a significantly decreased of SDH activity compared to Wt (black). Bars are the means of three independent experiments (each of them conducted in duplicate samples) ± SD, ***p < 0.01. (E) The bar graph represents the means of intracellular succinate/fumarate ratio ± SD, measured by GC/MS, in three independent experiments with two replicates. SDHB silenced cells (light grey) showed a significant increase of the metabolites ratio compared with both Wt (black) and to SDHD silenced cells (dark grey), *p < 0.05, **p < 0.01. (F) Representative immunoblot of HIF1α expression in Wt, SDHB and SDHD silenced cells. (G) Optical density analysis of western blot bands. Actin im munoblots was used as loading control. For all the analyses One-way ANOVA post-test Bonferroni was used.

Article Snippet: Bound antibodies were detected using ECL reagents (Immobilon) and analysed with a Bio-Rad ChemiDoc Imaging System (Quantity One) for dedicated chemiluminescent image acquisition.

Techniques: Expressing, Western Blot, Imaging, Software, Activity Assay, Gas Chromatography-Mass Spectrometry, Control

Journal: Nature Communications

Article Title: CDKN1A is a target for phagocytosis-mediated cellular immunotherapy in acute leukemia

doi: 10.1038/s41467-022-34548-3

Figure Lengend Snippet: a p21 expression in control (siCo.) or p21-silenced (sip21) MDMs after 24 h silencing. b Confocal micrograph of siCo. or sip21 CMFDA + MDMs cocultured with CMTMR + Jurkat cells. c – e Percentages of Jurkat ( c ), MOLT4 ( d ) or patient CD34 + AML ( e ) phagocytosis by siCo. or sip21 MDMs (** p = 0.0078, * p = 0.0312, **** p = 6.1e-5). f Phagocytosis (Fluorescence Units (FU)) of pHrodo Green E. coli bioparticle by siCo. or sip21 MDMs. g Percentages of CrCD47MOLT4 or CrCo.MOLT4 phagocytosis by MDMs transduced with control (Co.TD) or p21-expressing (p21TD) lentiviral vectors (LVs) after 72 h of transduction (** p = 0.0061, ** p = 0.0053, ** p = 0.0048). h – m SIRPα and p21 proteins ( h , k ) or mRNAs ( i , j , l , m ) in siCo. or sip21 MDMs (** p = 0.0024, ** p = 0.0020) ( h – j ) or in Co.TD-MDMs or p21TD-MDMs (** p = 0.0063, * p = 0.0291) ( k – m ). n ChIP-qPCR assays performed with control MDMs, Co.TD-MDMs (** p = 0.0016) or p21TD-MDMs (** p = 0.0017, * p = 0.0194) immunoprecipitated with control IgG or anti-p21 antibodies and analyzed by qPCR at SIRPα promoter. o , p Luciferase activities in siCo. or sip21 MDMs ( o ) or in Co.TD-MDMs or p21TD-MDMs ( p ) transduced with a lentiviral vector expressing a luciferase reporter gene under the control of the SIRPα promoter (* p = 0.0431, * p = 0.0458). q – s MDMs derived from monocytes transduced with Co.TD and/or p21TD LVs and/or SIRPα-expressing LVs (SIRPαTD) assessed for the indicated proteins ( q ) and for phagocytosis of mCherry + MOLT4 cells (** p = 0.0079, * p = 0.0472, *** p = 0.0002, *** p = 0.0004) ( r , s ). In a , b , h , k , q and r , the data are representative of n = 3 donors. In c , d , e and o , p , the data are donor matched from n = 8, n = 6, n = 9, n = 4 and n = 3 donors. In e , the CD34 + cells were from n = 6 AML patients. In i , j and g , l , m , n , s , the data are presented as the mean±SEM from n = 5 and n = 3 donors. Exact p-values are indicated and determined with two-tailed paired Wilcoxon ( c , d , e ), two- ( i , j , l , n ) or one-tailed ( m ) ratio-paired t, and one-tailed paired t ( o , p ) tests and two-way ANOVA with Sidak’s ( g ) or one-way ANOVA Tukey’s ( s ) multiple comparison tests. Source data are provided as a Source Data file.

Article Snippet: The RT-qPCR data were analyzed with qPCR CFX Maestro Version 4.0.2325.0418. (Biorad).

Techniques: Expressing, Control, Fluorescence, Transduction, ChIP-qPCR, Immunoprecipitation, Luciferase, Plasmid Preparation, Derivative Assay, Two Tailed Test, One-tailed Test, Comparison