pml iv  (Addgene inc)

Journal: Nature Immunology

Article Title: Chemosensor receptors are lipid-detecting regulators of macrophage function in cancer

doi: 10.1038/s41590-025-02191-x

Figure Lengend Snippet: a , Experimental scheme of genome-wide CRISPR–Cas9 Knockout GecKO v2 Library B screening in primary murine macrophages. b , Representative plot of backbone (LGP) or library-B-infected macrophages exposed or not to conditioned media from Pten −/− Trp53 −/− tumor cells. c , Two independent experiments were performed. Graphs show the correlations between the distribution of the guides found in the CD206 − MHCII + population (MHCII) and in the CD206 bright MHCII − population (CD206) from the two experiments. Lib1, library 1; Lib2, library 2. d , Volcano plot showing genes related to the differentially enriched sgRNA guides from CD206 − MHCII + versus CD206 bright MHCII − cells. Negative regulators of the CD206 bright MHCII − population are shown in light blue, and positive regulators are shown in red. log 2 FC ± 0.56, P < 0.005. Statistical analyses and comparisons from NGS output were performed with MAGeCK. e , Western blot analysis showing the percentage of expression of total STAT6. Two independent sgRNA guides (g1 and g2) were utilized to silence Stat6 in macrophages ( n = 2). f , g , FACS analysis of control (LGP) and Stat6 -silenced (g1 and g2) macrophages following exposure to Pten −/− Trp53 −/− conditioned media, with events gated on F4/80 + CD11b + cells: LGP n = 5, g1 n = 5, g2 n = 6 ( f ); LGP n = 4, g1 n = 4, g2 n = 4 ( g ). h , Proliferation of CD8 + T cells exposed to supernatant from Untr. and CM-tr. macrophages: bar graph shows the number of divisions. i , Scratch assay: graph and curves showing the distance (μm) covered by tumor cells over time after exposure to supernatant from Untr. or CM-tr. macrophages ( n = 8). Statistical analyses were performed using two-tailed unpaired Student’s t -test. Values are presented as the mean ± s.e.m. All replicates represent biological replicates. Schematic in a created using BioRender.com .

Article Snippet: The mouse GeCKO v2 Library B (Addgene) was used: this library consists of 62,804 sgRNAs constructs, with three sgRNAs targeting each of the 20,661 genes of the mouse genome.

Techniques: Genome Wide, CRISPR, Knock-Out, Infection, Western Blot, Expressing, Control, Wound Healing Assay, Two Tailed Test

Journal: Cell Death Discovery

Article Title: USP22 regulates APL differentiation via PML-RARα stabilization and IFN repression

doi: 10.1038/s41420-024-01894-8

Figure Lengend Snippet: A Western blot analysis of basal PML and USP22 expression in control (non-human target; n.h.t) and USP22 knockout (KO) HT-29 cells. The major (120 kDa) PML isoform is indicated with a red arrow. Vinculin served as loading control. Representative blots of at least two different independent experiments are shown. B Densitometric quantification of gray level intensities of the major (120 kDa) PML isoform (indicated with red arrow) detected by Western blot analysis of PML in n.h.t and USP22 KO HT-29 cells from ( A ), normalized against loading control intensities. Mean and SEM of three independent experiments are shown. C Basal mRNA expression levels of all PML isoforms in n.h.t and USP22 KO HT-29 cells using qRT-PCR. Gene expression was normalized against 28S mRNA and is presented as x-fold mRNA expression compared to n.h.t. Mean and SEM of three independent experiments are shown. D Western blot analysis of n.h.t and USP22 KO HEK293T cells transiently transfected with plasmids expressing PML-HA isoform IV and mCherry, prior to treatment with 20 μg/ml cycloheximide (CHX) for the indicated timepoints. β-Actin served as loading control. Representative blots of at least three different independent experiments are shown. E Densitometric quantification of gray level intensities of PML-HA isoform IV detected by Western blot analysis of HA in n.h.t and USP22 KO HEK293T cells in the presence of CHX for the indicated timepoints, normalized against loading control intensities. Mean and SEM of three independent biological replicates are shown.

Article Snippet: For transient expression, full-length human PML isoform IV (mRNA transcript variant 6, NM_002675.4) was subcloned into the pSBbi-blasticidin plasmid [ ] (Addgene plasmid #60526) using the TOPOTM TA CloningTM Kit (Thermo Scientific), according to manufacturer recommendations.

Techniques: Western Blot, Expressing, Control, Knock-Out, Quantitative RT-PCR, Gene Expression, Transfection

Journal: Cell Death Discovery

Article Title: USP22 regulates APL differentiation via PML-RARα stabilization and IFN repression

doi: 10.1038/s41420-024-01894-8

Figure Lengend Snippet: A Schematic overview of PML mRNA (blue) with approximate exon spans indicated, as well as the PML (black) and PML-RARα protein (yellow) with relevant amino acids indicated (K, lysine; A, alanine; I, isoleucine; S, serine). RBCC/Trim motif, RING-B-box1-B-box2-Coiled-Coil domain; NLS, nuclear localization signal; SIM, SUMO-interacting motif. B Western blot analysis of HEK293T cells transiently transfected with plasmids expressing wild-type (WT) and K394R HA-tagged PML isoform IV. Co-transfection with GFP plasmid served as transfection control. GAPDH served as loading control. Representative blots of at least two different independent experiments are shown. C Western blot analysis of HEK293T cells transiently transfected with plasmids expressing WT and K394R HA-PML isoform IV, prior to treatment with 20 μg/ml CHX for the indicated timepoints. Co-transfection with GFP plasmid served as transfection control. GAPDH served as loading control. Representative blots of at least two different independent experiments are shown. D Densitometric quantification of gray level intensities of WT and K394R HA-PML isoform IV detected by Western blot analysis of HA in HEK293T cells in the presence of CHX for the indicated timepoints, normalized against loading control intensities. Mean and SEM of three independent biological replicates are shown. E Western blot analysis of anti-ubiquitin (Ub), -HA and -PML on anti-HA-immunoprecipitated fractions of denatured lysates of HEK293T cells transiently transfected with empty vector (EV) or plasmids expressing WT and K394R HA-PML isoform IV. ß-Actin served as loading control. Representative blots of at least two different independent experiments are shown.

Article Snippet: For transient expression, full-length human PML isoform IV (mRNA transcript variant 6, NM_002675.4) was subcloned into the pSBbi-blasticidin plasmid [ ] (Addgene plasmid #60526) using the TOPOTM TA CloningTM Kit (Thermo Scientific), according to manufacturer recommendations.

Techniques: Western Blot, Transfection, Expressing, Cotransfection, Plasmid Preparation, Control, Ubiquitin Proteomics, Immunoprecipitation

Journal: Cell Death Discovery

Article Title: USP22 regulates APL differentiation via PML-RARα stabilization and IFN repression

doi: 10.1038/s41420-024-01894-8

Figure Lengend Snippet: A Western blot analysis of basal PML-RARα and USP22 expression in wild-type (wt), control (non-human target; n.h.t) and USP22 knockout (KO) NB4 acute promyelocytic leukemia (APL) cells. β-Actin served as loading control. Representative blots of at least two different independent experiments are shown. B Densitometric quantification of gray level intensities of the major (130 kDa) PML-RARα isoform detected by Western blot analysis in wt, n.h.t and USP22 KO NB4 APL cells, normalized against loading control intensities. Mean and SEM of three independent experiments are shown. C Basal mRNA expression levels of the PML-RARα long isoform in wt, n.h.t. and USP22 KO NB4 APL cells using qRT-PCR. Gene expression was normalized against 28S mRNA and is presented as x-fold mRNA expression compared to n.h.t. Mean and SEM of three independent experiments are shown. D Western blot analysis of wt, n.h.t and USP22 KO NB4 APL cells treated with 20 μg/ml CHX for the indicated timepoints. GAPDH served as loading control. Representative blots of at least two different independent experiments are shown. Red dashed line indicates protein stability at 50% compared to untreated controls. E Densitometric quantification of gray level intensities of the major (130 kDa) PML-RARα isoform detected by Western blot analysis in wt, n.h.t and USP22 KO NB4 APL cells treated with 20 μg/ml CHX for the indicated timepoints, normalized against loading control intensities. Mean and SEM of three independent experiments are shown. F Western blot analysis of n.h.t and USP22 KO HEK293T cells, transiently transfected with plasmids encoding the long isoform of PML-RARα and treated with 20 μg/ml CHX for the indicated timepoints. Co-transfection with GFP plasmid served as transfection control. GAPDH served as loading control. Representative blots of at least two different independent experiments are shown. Red dashed line indicates protein stability at 50% compared to untreated controls. G Densitometric quantification of gray level intensities of the long isoform of PML-RARα transiently transfected in n.h.t and USP22 KO HEK293T cells treated with 20 μg/ml CHX for the indicated timepoints, normalized against loading control intensities. Mean and SEM of three independent experiments are shown. H Western blot analysis of HEK293T cells, transiently transfected with plasmids encoding the long isoform of wt (PML-RARα) and K394R (KR) PML-RARα (PML-K394R-RARα) and treated with 20 μg/ml CHX for the indicated timepoints. Co-transfection with GFP plasmid served as transfection control. GAPDH served as loading control. Representative blots of at least two different independent experiments are shown. Red dashed line indicates protein stability at 50% compared to untreated controls. I Densitometric quantification of gray level intensities of the long isoform of wt (PML-RARα) and K394R (KR) PML-RARα (PML-K394R-RARα) transiently transfected in HEK293T cells treated with 20 μg/ml CHX for the indicated timepoints, normalized against loading control intensities. Mean and SEM of three independent experiments are shown.

Article Snippet: For transient expression, full-length human PML isoform IV (mRNA transcript variant 6, NM_002675.4) was subcloned into the pSBbi-blasticidin plasmid [ ] (Addgene plasmid #60526) using the TOPOTM TA CloningTM Kit (Thermo Scientific), according to manufacturer recommendations.

Techniques: Western Blot, Expressing, Control, Knock-Out, Quantitative RT-PCR, Gene Expression, Transfection, Cotransfection, Plasmid Preparation