Journal: Frontiers in Genetics

Article Title: Whole exome sequencing and bioinformatics reveal PMAIP1 and PDGFRL as immune-related gene markers in follicular thyroid carcinoma

doi: 10.3389/fgene.2025.1509245

Figure Lengend Snippet: Identification of hub genes. (A) The expression level of hub genes between FTC and FTA in microarray data; (B) The mRNA levels of the hub genes were compared between FTC cell lines and NTHY-ORI3-1; (C) PPI analysis of PDGFRL; (D) PPI analysis of PMAIP1. (* P < 0.05, ** P < 0.01, *** P < 0.001)

Article Snippet: The rabbit polyclonal antibody against PMAIP1 (A9801) was from ABclonal Technology Co, Ltd. (Wuhan, China).

Techniques: Expressing, Microarray

Journal: Frontiers in Genetics

Article Title: Whole exome sequencing and bioinformatics reveal PMAIP1 and PDGFRL as immune-related gene markers in follicular thyroid carcinoma

doi: 10.3389/fgene.2025.1509245

Figure Lengend Snippet: IHC was used to detect the difference expression of PMAIP1 and PDGFRL between FTC and FTA. (A) The protein level of PDGFRL in FTA and FTC; (B) The protein level of PMAIP1 in FTA and FTC. (** P < 0.01, *** P < 0.001)

Article Snippet: The rabbit polyclonal antibody against PMAIP1 (A9801) was from ABclonal Technology Co, Ltd. (Wuhan, China).

Techniques: Expressing

Journal: Cell Death and Differentiation

Article Title: MARCH5-dependent degradation of MCL1/NOXA complexes defines susceptibility to antimitotic drug treatment

doi: 10.1038/s41418-020-0503-6

Figure Lengend Snippet: a Parental HCT116 (WT) and HCT116-allBCL2KO (allKO) cells transduced with empty vector controls (EV), NOXA, MCL1 or both NOXA and MCL1 were transfected with the indicated siRNAs. The cells were left asynchronous (Asy) or synchronized by double thymidine block, released into paclitaxel and harvested at the indicated time points once cells entered mitotic arrest (M). For NOXA a short, an intermediate and a long exposure are shown. b HCT116-allBCL2KO (HCT116-allKO) cells transduced with expression vectors carrying MCL1, NOXA, NOXA-L29E or no cDNA were transfected with the indicated siRNAs. After 48 h cells were prepared for immunoblot analysis. Numbers below the blots show the quantification of the respective bands. Quantification was normalized to the GAPDH signal and to the single-transduced MCL1 or NOXA sample transfected with the control siRNA (GL2). The asteriks indicates unspecific bands in the MARCH5 blot.

Article Snippet: Antibodies used were: rabbit anti MARCH5 (Millipore, Burlington, MA, USA, 06–1036, 1:500), mouse anti NOXA (clone 114C307, Rockland Immunochemicals, Limerick, PA, USA, 200-301-H98, 1:500), rabbit anti MCL1 (Santa Cruz Biotechnology, Dallas, TX, USA, sc-819, 1:1000, discontinued), rabbit anti PARP1 (Cell Signaling, Danvers, MA, USA, #9542, 1:1000), rabbit anti CASP3 (Cell Signaling #9662, 1:1000), rabbit anti BIM (Enzo Life Sciences, Farmingdale, NY, USA, ADI-AAP-330-E, 1:500), mouse anti Ubiquitin (clone P4D1, Cell Signaling #3936, 1:1000), rabbit anti GAPDH (clone 14C10, Cell Signaling #2118, 1:5000), mouse anti HSP 90 (clone F8, Santa Cruz Biotechnology, sc-13119, 1:1000), rabbit anti BCLX (clone 54H6, Cell Signaling #2764, 1:1000), mouse anti BCL2 (clone S100, gift from Andreas Strasser, 1 µg/ml), goat anti rabbit IgG-HRP (Dako, Glostrup, Denmark, P0448, 1:5000), and rabbit anti mouse-IgG-HRP (Dako P0161, 1:5000).

Techniques: Transduction, Plasmid Preparation, Transfection, Blocking Assay, Expressing, Western Blot

Journal: Cell Death and Differentiation

Article Title: MARCH5-dependent degradation of MCL1/NOXA complexes defines susceptibility to antimitotic drug treatment

doi: 10.1038/s41418-020-0503-6

Figure Lengend Snippet: a Immunoblots of U2OS cells transfected with either a control siRNA targeting luciferase (GL2), GL2 and MARCH5 siRNA (siMARCH5 #1), GL2 and a different MARCH5 siRNA (siMARCH5 #2), GL2 and NOXA siRNA (siNOXA) or NOXA and MARCH5 #1 siRNA. Cells were left asynchronous or synchronized by double thymidine block, released into paclitaxel and harvested at the indicated time points for immunoblot analysis. The full length (fl) form of caspase-3 is shown. b Cell fate profiles of U2OS cells arrested in mitosis. Each horizontal bar indicates the duration of mitotic arrest of a single cell. Numbers to the left of the fate profiles indicated how many cells died or underwent slippage at the end of mitotic arrest. U2OS cells were transfected with the indicated siRNAs, synchronized by a single thymidine block followed by release into paclitaxel and the start of live cell imaging. c Box blots with 5–95% whiskers of the mitotic duration of U2OS cells that died during mitotic arrest (black bars in fate profiles) shown in b). One-way ANOVA was used with Holm–Sidak’s multiple comparisons test to compare the mitotic durations of the siMARCH5 sample against all other knockdowns. The p -values for selected populations are shown with p < 0.05 considered statistically significant.

Article Snippet: Antibodies used were: rabbit anti MARCH5 (Millipore, Burlington, MA, USA, 06–1036, 1:500), mouse anti NOXA (clone 114C307, Rockland Immunochemicals, Limerick, PA, USA, 200-301-H98, 1:500), rabbit anti MCL1 (Santa Cruz Biotechnology, Dallas, TX, USA, sc-819, 1:1000, discontinued), rabbit anti PARP1 (Cell Signaling, Danvers, MA, USA, #9542, 1:1000), rabbit anti CASP3 (Cell Signaling #9662, 1:1000), rabbit anti BIM (Enzo Life Sciences, Farmingdale, NY, USA, ADI-AAP-330-E, 1:500), mouse anti Ubiquitin (clone P4D1, Cell Signaling #3936, 1:1000), rabbit anti GAPDH (clone 14C10, Cell Signaling #2118, 1:5000), mouse anti HSP 90 (clone F8, Santa Cruz Biotechnology, sc-13119, 1:1000), rabbit anti BCLX (clone 54H6, Cell Signaling #2764, 1:1000), mouse anti BCL2 (clone S100, gift from Andreas Strasser, 1 µg/ml), goat anti rabbit IgG-HRP (Dako, Glostrup, Denmark, P0448, 1:5000), and rabbit anti mouse-IgG-HRP (Dako P0161, 1:5000).

Techniques: Western Blot, Transfection, Luciferase, Blocking Assay, Live Cell Imaging

Journal: Cell Death and Differentiation

Article Title: MARCH5-dependent degradation of MCL1/NOXA complexes defines susceptibility to antimitotic drug treatment

doi: 10.1038/s41418-020-0503-6

Figure Lengend Snippet: a Parental HeLaS3 (WT) and two clonal HeLaS3 MARCH5-KO lines created with two different small guide RNAs targeting MARCH5 (MARCH5-KO#1 and -KO#2) were treated with ABT737, etoposide (Eto), or staurosporine (STS) for 24 h. For the UV-R treatment cells were subjected to 2 mJ of UV-R and then incubated for 24 h. Cells were then harvested and propidium iodide uptake was used to measure cell death by flow cytometry. All data displayed are mean ± s.d. from five independent experiments, indicated as dots. Two-way ANOVA was used with Holm–Sidak’s multiple comparisons test to compare the WT as the control group against the two MARCH-KO lines for each treatment. All comparisons not indicated are statistically not significant ( p > 0.05). b Parental HeLaS3 and HeLaS3 MARCH5-KO cells were transfected with either control siRNA targeting luciferase (GL2), NOXA and GL2 siRNA (siNOXA), MCL1 and GL2 siRNA (siMCL1) or NOXA and MCL1 siRNA (DKD) for 48 h. Cells were then harvested and prepared for immunoblot analysis. The full length (fl) and the cleaved (cl) form of caspase-3 are shown. c Parental HeLaS3 and two independent HeLaS3 MARCH5-KO clones were treated with Cycloheximide (CHX), harvested after the indicated time points and prepared for immunoblot analysis. Numbers below the blots show the quantification of the respective bands. Quantification was normalized to the GAPDH signal and to the untreated sample (0) of the respective genotype.

Article Snippet: Antibodies used were: rabbit anti MARCH5 (Millipore, Burlington, MA, USA, 06–1036, 1:500), mouse anti NOXA (clone 114C307, Rockland Immunochemicals, Limerick, PA, USA, 200-301-H98, 1:500), rabbit anti MCL1 (Santa Cruz Biotechnology, Dallas, TX, USA, sc-819, 1:1000, discontinued), rabbit anti PARP1 (Cell Signaling, Danvers, MA, USA, #9542, 1:1000), rabbit anti CASP3 (Cell Signaling #9662, 1:1000), rabbit anti BIM (Enzo Life Sciences, Farmingdale, NY, USA, ADI-AAP-330-E, 1:500), mouse anti Ubiquitin (clone P4D1, Cell Signaling #3936, 1:1000), rabbit anti GAPDH (clone 14C10, Cell Signaling #2118, 1:5000), mouse anti HSP 90 (clone F8, Santa Cruz Biotechnology, sc-13119, 1:1000), rabbit anti BCLX (clone 54H6, Cell Signaling #2764, 1:1000), mouse anti BCL2 (clone S100, gift from Andreas Strasser, 1 µg/ml), goat anti rabbit IgG-HRP (Dako, Glostrup, Denmark, P0448, 1:5000), and rabbit anti mouse-IgG-HRP (Dako P0161, 1:5000).

Techniques: Incubation, Flow Cytometry, Transfection, Luciferase, Western Blot, Clone Assay

Journal: Cell Death and Differentiation

Article Title: MARCH5-dependent degradation of MCL1/NOXA complexes defines susceptibility to antimitotic drug treatment

doi: 10.1038/s41418-020-0503-6

Figure Lengend Snippet: a Input, elution and unbound fraction of a denaturing immunoprecipitation of MCL1 in U2OS Flag-MARCH5 overexpressing cells were analyzed by immunoblot. Doxycycline treatment for 24 h was used to induce overexpression of Flag-MARCH5 and MG132 treatment for 2 h to enrich for ubiquitinated proteins. For MCL1 a short and a long exposure are shown. Numbers below the blots show the quantification of the respective bands. In the long MCL1 exposure the bands with a higher molecular weight (red boxes) than the unmodified MCL1 signal (arrow) were quantified by normalizing the signal in the red boxes to the respective input signal shown in the short exposure of MCL1. The short exposure itself was normalized to GAPDH and the input with MG132 treatment. b Input, elution and unbound fraction of a TUBE assay in U2OS Flag-MARCH5 cells were analyzed by immunoblot. Doxycycline treatment for 24 h was used to induce overexpression of Flag-MARCH5 and MG132 treatment for 2 h to enrich for ubiquitinated proteins. For NOXA and MCL1 a short and a long exposure are shown. In the long exposure, the bands with a higher molecular weight (red boxes) than the unmodified MCL1 or NOXA signal (arrow) were quantified by normalizing the signal to the respective input signal shown in the short exposures. The short exposures themselves were normalized to GAPDH and the input with MG132 treatment.

Article Snippet: Antibodies used were: rabbit anti MARCH5 (Millipore, Burlington, MA, USA, 06–1036, 1:500), mouse anti NOXA (clone 114C307, Rockland Immunochemicals, Limerick, PA, USA, 200-301-H98, 1:500), rabbit anti MCL1 (Santa Cruz Biotechnology, Dallas, TX, USA, sc-819, 1:1000, discontinued), rabbit anti PARP1 (Cell Signaling, Danvers, MA, USA, #9542, 1:1000), rabbit anti CASP3 (Cell Signaling #9662, 1:1000), rabbit anti BIM (Enzo Life Sciences, Farmingdale, NY, USA, ADI-AAP-330-E, 1:500), mouse anti Ubiquitin (clone P4D1, Cell Signaling #3936, 1:1000), rabbit anti GAPDH (clone 14C10, Cell Signaling #2118, 1:5000), mouse anti HSP 90 (clone F8, Santa Cruz Biotechnology, sc-13119, 1:1000), rabbit anti BCLX (clone 54H6, Cell Signaling #2764, 1:1000), mouse anti BCL2 (clone S100, gift from Andreas Strasser, 1 µg/ml), goat anti rabbit IgG-HRP (Dako, Glostrup, Denmark, P0448, 1:5000), and rabbit anti mouse-IgG-HRP (Dako P0161, 1:5000).

Techniques: Immunoprecipitation, Western Blot, Over Expression, Molecular Weight

Journal: Cell Death and Differentiation

Article Title: MARCH5-dependent degradation of MCL1/NOXA complexes defines susceptibility to antimitotic drug treatment

doi: 10.1038/s41418-020-0503-6

Figure Lengend Snippet: a Parental HeLaS3 and two independent HeLaS3 MARCH5-KO clones were either left asynchronous (Asy) or synchronized by double thymidine block and released into paclitaxel. Once cells were mitotic (M), they were treated with Cycloheximide (CHX) or solvent control (DMSO) for the indicated times in minutes. Numbers below the blots show the quantification of the respective bands. Quantification was normalized to the GAPDH signal and to the early mitotic arrest sample without CHX (M) of the respective genotype. b Same as in a) with U2OS cells and two U2OS MARCH5-KO bulks generated with two different guide RNAs. For NOXA a short and a long exposure are shown. c Same as in a) with A549 cells and two A549 MARCH5-KO bulks generaed with two different guide RNAs.

Article Snippet: Antibodies used were: rabbit anti MARCH5 (Millipore, Burlington, MA, USA, 06–1036, 1:500), mouse anti NOXA (clone 114C307, Rockland Immunochemicals, Limerick, PA, USA, 200-301-H98, 1:500), rabbit anti MCL1 (Santa Cruz Biotechnology, Dallas, TX, USA, sc-819, 1:1000, discontinued), rabbit anti PARP1 (Cell Signaling, Danvers, MA, USA, #9542, 1:1000), rabbit anti CASP3 (Cell Signaling #9662, 1:1000), rabbit anti BIM (Enzo Life Sciences, Farmingdale, NY, USA, ADI-AAP-330-E, 1:500), mouse anti Ubiquitin (clone P4D1, Cell Signaling #3936, 1:1000), rabbit anti GAPDH (clone 14C10, Cell Signaling #2118, 1:5000), mouse anti HSP 90 (clone F8, Santa Cruz Biotechnology, sc-13119, 1:1000), rabbit anti BCLX (clone 54H6, Cell Signaling #2764, 1:1000), mouse anti BCL2 (clone S100, gift from Andreas Strasser, 1 µg/ml), goat anti rabbit IgG-HRP (Dako, Glostrup, Denmark, P0448, 1:5000), and rabbit anti mouse-IgG-HRP (Dako P0161, 1:5000).

Techniques: Clone Assay, Blocking Assay, Generated

Journal: Oncogene

Article Title: SOX30, a novel epigenetic silenced tumor suppressor, promotes tumor cell apoptosis by transcriptional activating p53 in lung cancer

doi: 10.1038/onc.2014.370

Figure Lengend Snippet: SOX30 accelerates expression of p53 signaling pathway-related genes. ( a ) Ectopic expression of SOX30 significantly ( P <0.01) enhances endogenous expression of P53, P21, BAX and PMAIP1 in A549 and H460 cells. The mRNA expression was examined by qRT–PCR, and ACTIN was used as an internal control. * P <0.05; ** P <0.01. ( b ) The protein levels of P53, P21, BAX and PMAIP1 were detected by WB after restoration of SOX30 in A549 and H460 cells. ACTIN was used as an internal control. ( c , d ) The mRNA and protein expression levels of SOX30 and P53 in xenograft tumors were examined by qRT–PCR and WB. ACTIN was used as an internal control. ( e ) SOX30 increases the number of terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL)-positive H460 cells in xenograft tumors. Two (1 and 2) representative images were randomly selected from each sample (H460-vector and H460-SOX30). The arrows indicate apoptotic cells.

Article Snippet: Primary antibodies were SOX30 rabbit polyclonal antibody (1:1000; Santa Cruz Biotechnology), p53 mouse polyclonal antibody (1:1000; Santa Cruz Biotechnology), BAX rabbit polyclonal antibody (1:1000; Santa Cruz Biotechnology), P21 rabbit polyclonal antibody (1:1000; Cell Signaling Technology, Boston, MA, USA) and PMAIP1 (NOXA) rabbit polyclonal antibody (1:1000; Santa Cruz Biotechnology).

Techniques: Expressing, Quantitative RT-PCR, End Labeling, TUNEL Assay, Plasmid Preparation

Journal: Cell Death Discovery

Article Title: TFF3-dependent resistance of human colorectal adenocarcinoma cells HT-29/B6 to apoptosis is mediated by miR-491-5p regulation of lncRNA PRINS

doi: 10.1038/cddiscovery.2016.106

Figure Lengend Snippet: PRINS interacts with PMAIP1 and determines its availability. ( a – c ) Volcano plots show significantly dysregulated apoptosis-relevant genes in nonsense, miR-491-5p or siPRINS-transfected HT-29/B6/TFF3 cells compared with nonsense-transfected mock controls (green: significantly downregulated; red: significantly upregulated). ( d ) PRINS localisation was determined by Stellaris FISH, arrows show focal PRINS signals (red: PRINS, blue: DAPI, scale bars indicate 25 nm). ( e ) PMAIP1 localisation was determined by IF (green: PMAIP1, yellow: f-actin, blue: DAPI, scale bars indicate 25 nm). ( f ) Colocalisation of PRINS with PMAIP1 was determined by combination of FISH and IF (red: PRINS, green: PMAIP1, blue: DAPI, scale bars indicate 25 nm). ( g and h ) Co-IP using the PMAIP1-specific antibody (CST, #D8L7U), experiments show specific interaction of PRINS with PMAIP1 proved by PCR (pulldown sample). Lysate supernatant (sn) and diverse washes were taken as controls. GAPDH serves as control for assay specificity. ( i ) PRINS knockdown in HT-29/B6/TFF3 cells causes accumulation of PMAIP1 with no differences in FOXK1 and 2. Black borders indicate cropped area of the western blot. MWs were determined with the Fusion Capt Advance Software (Vilber Lourmat) according to the MW marker Page Ruler Plus Prestained Protein Ladder (Thermo Fisher Scientific GmbH). ( j ) Densitometric analysis of section i was performed using the Fusion Capt Advance Software (Vilber Lourmat). Columns show normalised means (protein of interest/GAPDH) of three independent replicates and bars represent the S.D. Values were log2 transformed.

Article Snippet: The precleared lysate was incubated overnight at 4 °C with anti-PMAIP1 antibody (CST, #D8L7U) using a 1 : 100 dilution.

Techniques: Transfection, Co-Immunoprecipitation Assay, Control, Knockdown, Western Blot, Software, Marker, Transformation Assay