Journal: Nature Communications

Article Title: Adipocyte lysoplasmalogenase TMEM86A regulates plasmalogen homeostasis and protein kinase A-dependent energy metabolism

doi: 10.1038/s41467-022-31805-3

Figure Lengend Snippet: a Heatmap of RNA-seq data showing the effects of 8 weeks of high-fat diet (HFD) feeding on genes involved in ether lipid metabolism (GO: 0046485) in gWAT. (NCD: normal chow diet, *| fold change (FC) | > 2; p < 0.05) b – d qPCR analysis of Tmem86a and Tmem86b in BAT, iWAT and gWAT of mice fed a NCD or HFD for 8 weeks. n = 6. e – g Immunoblot analysis of TMEM86A expression. n = 6. h Analysis of TMEM86A expression in human adipose tissue (non-obesity (NO): n = 9; insulin-sensitive obesity (OIS): n = 21; insulin-resistant obesity (OIR): n = 18), determined by publicly available transcriptomic data (Gene Expression Omnibus (GEO) repository, accession number GSE94753). qPCR analysis of Tmem86a expression in adipocytes and stromal vascular fraction (SVF; F4/80+, PDGFRα+, or F4/80-PDGFRα-) isolated from iWAT ( i ) and gWAT ( j ) of mice. n = 3. Each point represents a biological replicate. Data are presented as the mean ± SEM. Statistical significance was determined using the unpaired, two-tailed t -test in a – j . Source data are provided as a Source Data file.

Article Snippet: For overexpression of TMEM86A in C3H10T1/2 adipocytes, mouse Tmem86a (NM_026436) ORF Clone was purchased from Origene.

Techniques: RNA Sequencing Assay, Western Blot, Expressing, Isolation, Two Tailed Test

Journal: Nature Communications

Article Title: Adipocyte lysoplasmalogenase TMEM86A regulates plasmalogen homeostasis and protein kinase A-dependent energy metabolism

doi: 10.1038/s41467-022-31805-3

Figure Lengend Snippet: a Immunoblot analysis of TMEM86A expression in C3H10T1/2 adipocytes overexpressing TMEM86A (OE) or controls (mock) n = 3. Data are presented as the mean ± SEM. b – d PCA, volcano plot, and heatmap of phospholipid profiling of TMEM86A-overexpressing C3H10T1/2 adipocytes (OE) or controls (mock). e , f Normalized intensity of lysoplasmalogens and plasmalogens from phospholipid profiling of TMEM86A-overexpressing C3H10T1/2 adipocytes or controls. n = 3. (Intensity was normalized by median, *FDR < 0.05). Data are presented as the mean ± SEM. g LPE P-18:0 consumption levels of TMEM86A-overexpressing C3H10T1/2 adipocytes or controls. Cells were treated with LPE P-18:0 (1 μM) and then LPE P-18:0 in conditioned media were measured by LC-MS at indicated time. LPE P-18:0 consumption was calculated by subtracting LPE P-18:0 peak area at each time point from the initial value followed by normalization with a total protein quantity of cells. n = 3. Data are presented as the mean ± SD. h AlphaFold 3D model of human TMEM86A protein (AF-Q5BLD1-F1-model_v2.pdb). i Representative images of C3H10T1/2 adipocytes overexpressing GFP-tagged TMEM86A, stained with ER-Tracker. Nuclei and lipid were counterstained with DAPI and LipidTox, respectively. Scale bars = 10 μm. n = 3. j Immunoblot analysis of TMEM86A expression in HEK293T cells overexpressing WT, D82A mutant, or D190A mutant TMEM86A. n = 3. k LPE P-18:0 consumption levels of HEK293T cells overexpressing WT and mutant TMEM86A. n = 3. (WT OE + LPE P-18:0 vs. D82A OE + LPE P-18:0: p = 0.036525, p = 0.002498, p = 0.001757, p = 0.000735, or p = 0.001335, respectively for 2, 4, 6, 8, or 12 h; WT OE + LPE P-18:0 vs. D190A OE + LPE P-18:0: p = 0.019168, p = 0.003337, p = 0.00967, p = 0.02689, or p = 0.251998, respectively for 2, 4, 6, 8, or 12 h). p value was calculated by comparing each mutant group with WT OE + LPE P-18:0 as a control group. Data are presented as the mean ± SD. Each point represents a biological replicate. Statistical significance was determined using the unpaired, two-tailed t -test in a , e , f , g and using ANOVA (one-way, Bonferroni’s test) in k . Source data are provided as a Source Data file.

Article Snippet: For overexpression of TMEM86A in C3H10T1/2 adipocytes, mouse Tmem86a (NM_026436) ORF Clone was purchased from Origene.

Techniques: Western Blot, Expressing, Liquid Chromatography with Mass Spectroscopy, Staining, Mutagenesis, Two Tailed Test

Journal: Nature Communications

Article Title: Adipocyte lysoplasmalogenase TMEM86A regulates plasmalogen homeostasis and protein kinase A-dependent energy metabolism

doi: 10.1038/s41467-022-31805-3

Figure Lengend Snippet: a Schematic diagram showing generation of adipocyte-specific TMEM86A knockout mice (TMEM86A AKO). b – e Immunoblot analysis of TMEM86A expression in adipose tissue of WT and TMEM86A AKO mice. n = 6. f PCA plots showing a separation of clusters of WT and TMEM86A AKO samples. Heatmap analysis ( g ) and volcano plots ( h – j ) of untargeted phospholipid profiling of BAT, iWAT and gWAT from WT and TMEM86A AKO mice. Lysoplasmalogens are highlighted in yellow in h – j . BAT WT: n = 11, KO: n = 12; iWAT WT: n = 13, KO: n = 13; gWAT WT: n = 12, KO: n = 14. k – p Normalized intensities of lysoplasmalogens in BAT, iWAT and gWAT of WT and TMEM86A AKO mice (Intensity was normalized by median, *FDR < 0.05). BAT WT: n = 11, KO: n = 12; iWAT WT: n = 13, KO: n = 13; gWAT WT: n = 12, KO: n = 14. (WT BAT vs. AKO BAT: p < 0.000001 for LPC P-18:0, LPC P-18:1, LPC P-20:0, LPE P-16:0, LPE P-18:0, LPE P-18:1, LPE P-18:2, LPE P-20:0, and LPE P-20:1; WT iWAT vs. AKO iWAT: p = 0.000003 for LPC P-16:0, p = 0.001422 for LPE P-16:0, p = 0.000835 for LPE P-18:1, p < 0.000001 for LPC P-18:0, LPC P-20:0, LPE P-18:0, LPE P-20:0, and LPE P-20:1; WT gWAT vs. AKO gWAT: p = 0.001322 for LPC P-16:0, p = 0.000694 for LPC P-20:0, p = 0.012532 for LPE P-16:0, p = 0.000865 for LPE P-18:1, p < 0.000001 for LPC P-18:0, LPE P-18:0, LPE P-20:0, and LPE P-20:1) Each point represents a biological replicate. Data are presented as the mean ± SEM. Statistical significance was determined using the unpaired, two-tailed t -test in a and k – p . Source data are provided as a Source Data file.

Article Snippet: For overexpression of TMEM86A in C3H10T1/2 adipocytes, mouse Tmem86a (NM_026436) ORF Clone was purchased from Origene.

Techniques: Knock-Out, Western Blot, Expressing, Two Tailed Test

Journal: Nature Communications

Article Title: Adipocyte lysoplasmalogenase TMEM86A regulates plasmalogen homeostasis and protein kinase A-dependent energy metabolism

doi: 10.1038/s41467-022-31805-3

Figure Lengend Snippet: a – d Immunoblot analysis of BAT, iWAT and gWAT of WT and TMEM86A AKO mice. n = 6. e – h Indirect calorimetry analysis. Arrows indicates CL316,243 (CL) injection. EE: energy expenditure; RER: respiratory exchange ratio; VO 2 : rate of oxygen consumption; VCO 2: rate of carbon dioxide production. n = 6. i , j Monitoring of food intake and activity. k Body fat and lean mass. n = 6. l Infrared thermal images showing skin temperature at room temperature or after 60 min of cold exposure (4 °C). n = 4. m , n Triphenyltetrazolium chloride (TTC) staining of BAT, iWAT, and gWAT and quantification. n = 6. o – q Electron microscopy of BAT, iWAT and gWAT. Boxed regions are magnified. n = 6. Each point represents a biological replicate. Data are presented as the mean ± SEM. Statistical significance was determined using the unpaired, two-tailed t -test in d – j and n . Source data are provided as a Source Data file.

Article Snippet: For overexpression of TMEM86A in C3H10T1/2 adipocytes, mouse Tmem86a (NM_026436) ORF Clone was purchased from Origene.

Techniques: Western Blot, Injection, Activity Assay, Staining, Electron Microscopy, Two Tailed Test

Journal: Nature Communications

Article Title: Adipocyte lysoplasmalogenase TMEM86A regulates plasmalogen homeostasis and protein kinase A-dependent energy metabolism

doi: 10.1038/s41467-022-31805-3

Figure Lengend Snippet: a – d Immunoblot analysis of adipocytes differentiated from precursors obtained from BAT, iWAT and gWAT of WT and TMEM86A AKO mice. n = 3. e , f Oxygen consumption rate measurement of adipocytes differentiated from precursors obtained from gWAT of WT and TMEM86A AKO mice. n = 3. g – i Immunoblot and qPCR analyses of TMEM86A overexpression effects in C3H10T1/2 adipocytes. n = 3. j , k Oxygen consumption rate of TMEM86A overexpressing C3H10T1/2 adipocytes or controls. n = 5. Each point represents a biological replicate. Data are presented as the mean ± SEM. Statistical significance was determined by the unpaired, two-tailed t -test in d – f , h – i , and k . Source data are provided as a Source Data file.

Article Snippet: For overexpression of TMEM86A in C3H10T1/2 adipocytes, mouse Tmem86a (NM_026436) ORF Clone was purchased from Origene.

Techniques: Western Blot, Over Expression, Two Tailed Test

Journal: Nature Communications

Article Title: Adipocyte lysoplasmalogenase TMEM86A regulates plasmalogen homeostasis and protein kinase A-dependent energy metabolism

doi: 10.1038/s41467-022-31805-3

Figure Lengend Snippet: a Body weight monitoring of WT and TMEM86A AKO mice during 8-weeks of HFD feeding. n = 6. (WT HFD vs. KO HFD: p = 0.015084, p = 0.006167, p = 0.042557, p = 0.026351, p = 0.019894, p = 0.040475, p = 0.004757, p = 0.010575, or p = 0.1215, respectively for week 0, 1, 2, 3, 4, 5, 6, 7, or 8; WT NCD vs. KO NCD: p = 0.039863, p = 0.010424, p = 0.003051, or p = 0.003318, respectively for week 5, 6, 7, or 8). b Weights of adipose tissue of WT and TMEM86A AKO mice fed a NCD or HFD. n = 6. c , d Intraperitoneal glucose tolerance test. n = 4. e – g Immunoblot analysis of BAT, iWAT and gWAT. n = 6. h H&E staining of paraffin sections of BAT, iWAT and gWAT. n = 6. Scale bar = 50 μm. i Immunoblot analysis of F4/80 in gWAT. n = 6. j Immunofluorescence staining of F4/80 in paraffin sections of gWAT. n = 6. Scale bar = 20 μm. k , l Flow cytometric analysis of CD45 + CD11b + CD64 + CD11C + cells (M1-like macrophages) and CD45 + CD11b + CD64 + CD206 + cells (M2-like macrophages) in gWAT. n = 3. Each point represents a biological replicate. Data are presented as mean ± SEM. Statistical significance was determined using the unpaired, two-tailed t -test in a , b , d , e – g , i , Source data are provided as a Source Data file.

Article Snippet: For overexpression of TMEM86A in C3H10T1/2 adipocytes, mouse Tmem86a (NM_026436) ORF Clone was purchased from Origene.

Techniques: Western Blot, Staining, Immunofluorescence, Two Tailed Test

Journal: Nature Communications

Article Title: Adipocyte lysoplasmalogenase TMEM86A regulates plasmalogen homeostasis and protein kinase A-dependent energy metabolism

doi: 10.1038/s41467-022-31805-3

Figure Lengend Snippet: a Concentration of LPE P-18:0 (LPE-P) in serum of TMEM86A AKO mice or WT mice treated with either VC or LPE-P for 2 weeks (i.p. 200 μg/kg/day). n = 4. b , c Intraperitoneal glucose tolerance test of WT mice fed a HFD treated intraperitoneally with VC or LPE-P for 2 weeks. n = 4. d – g Immunoblot analysis of BAT, iWAT and gWAT of WT mice fed a HFD treated with VC or LPE-P for 2 weeks (i.p. 200 μg/kg/day). n = 6. h Grouping of 48 individuals in quartile according to body mass index (BMI). i Normalized intensities of lysoplasmalogen LPE P-18:0 in human subcutaneous adipose tissue (Intensity was normalized by median, *FDR < 0.05). n = 48. j Correlation analysis of normalized intensity of LPE P-18:0 with BMI. n = 48; linear regression of correlation; R = −0.4717, p = 0.0007. Each point represents a biological replicate. Data are presented as mean ± SEM. Statistical significance was determined using the unpaired, two-tailed t -test in a , c , and g and using ANOVA (one-way, Tukey’s HSD post-hoc analysis) in h and i . Source data are provided as a Source Data file.

Article Snippet: For overexpression of TMEM86A in C3H10T1/2 adipocytes, mouse Tmem86a (NM_026436) ORF Clone was purchased from Origene.

Techniques: Concentration Assay, Western Blot, Two Tailed Test

Journal: Clinical and Experimental Immunology

Article Title: Gene silencing of non-obese diabetic receptor family (NLRP3) protects against the sepsis-induced hyper-bile acidaemia in a rat model

doi: 10.1111/cei.12457

Figure Lengend Snippet: The hepatic expression of non-obese diabetic-like receptor family (NLRP3). (a,b) The representative images of NLRP3 mRNA expression by real-time–polymerase chain reaction (RT–PCR) from the four groups at indicated time-points and the quantitative analysis of mRNA density. (c) NLRP3 mRNA levels in the four groups were also evaluated by quantitative (q)RT–PCR. (d,e) The representative images of NLRP3 protein expression by Western blot from the four groups and the quantitative analysis of protein density. NLRP3 shRNA represents the group administrated with NLRP3 shRNA and then subjected to caecal ligation and puncture (CLP); scrambled shRNA represents the group administered with scrambled NLRP3 shRNA and then subjected to CLP (the same throughout the paper). Data are expressed as percentages of the baseline value in the sham group. Data represent mean ± standard error of the mean, six per group; the experiments were repeated three times. *P < 0·05 versus baseline value in the sham group; **P < 0·001 versus baseline value in the sham group.

Article Snippet: Sprague–Dawley rats (Animal Model Institute of Xi'an, China); NLRP3 shRNA and scrambled shRNA plasmids (OriGene Technologies, Rockville, MD, USA); anti-NLRP3 antibody (Abcam, Cambridge, UK); horseradish peroxidase (HRP)-conjugated goat anti-rabbit immunoglobulin (Ig)G (Santa Cruz Biotechnology, Santa Cruz, CA, USA); anti-Bsep antibody (Sigma-Aldrich, St Louis, MO, USA); anti-Mrp2 antibody (Sigma-Aldrich); anti-Mrp3 antibody (Sigma-Aldrich); anti-Mrp4 antibody (Sigma-Aldrich); anti-CD68 antibody (Santa Cruz Biotechnology); anti-myeloperoxidase (MPO) antibody (Santa Cruz Biotechnology); IL-1β and IL-1β enzyme-linked immunosorbent assays (ELISA) (R&D Systems, Minneapolis, MN, USA); transmission electron microscopy (Phillips, Amsterdam, the Netherlands); high-performance liquid chromatography (LKB Bromma, Bromma, Sweden); bile acid standards (Sigma-Aldrich); RAW 264·7 cells (Cell Resource Center of Shanghai, Shanghai, China); lipopolysaccharide (LPS) (Sigma-Aldrich); propidium iodide (PI) (Invitrogen, Carlsbad, CA, USA); and 6-carboxyfluorescein-YVAD-fluoro-methylketone (Kamiya Biomedical Company, Seattle, WA, USA).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Western Blot, shRNA, Ligation

Journal: Clinical and Experimental Immunology

Article Title: Gene silencing of non-obese diabetic receptor family (NLRP3) protects against the sepsis-induced hyper-bile acidaemia in a rat model

doi: 10.1111/cei.12457

Figure Lengend Snippet: (a,b) Hepatic neutrophil infiltration was determined by myeloperoxidase (MPO) levels. The representative immunofluorescent stainings for neutrophils (green) at 12 h after surgery (original magnification ×100). (c,d) Serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels (IU/ml) were measured in rats from the four groups. (e,f) The representative images of interleukin (IL)-1β mRNA expression by real-time–polymerase chain reaction (RT–PCR) from the four groups at the indicated time-points and the quantitative analysis of mRNA density. (g,h) The representative images of IL-18 mRNA expression by RT–PCR from the four groups and the quantitative analysis of mRNA density. Data are expressed as percentages of the baseline value in the sham group. Data represent mean ± standard deviation, six per group; the experiments were repeated three times. *P < 0·05 versus baseline value in the sham group; **P < 0·001 versus baseline value in the sham group.

Article Snippet: Sprague–Dawley rats (Animal Model Institute of Xi'an, China); NLRP3 shRNA and scrambled shRNA plasmids (OriGene Technologies, Rockville, MD, USA); anti-NLRP3 antibody (Abcam, Cambridge, UK); horseradish peroxidase (HRP)-conjugated goat anti-rabbit immunoglobulin (Ig)G (Santa Cruz Biotechnology, Santa Cruz, CA, USA); anti-Bsep antibody (Sigma-Aldrich, St Louis, MO, USA); anti-Mrp2 antibody (Sigma-Aldrich); anti-Mrp3 antibody (Sigma-Aldrich); anti-Mrp4 antibody (Sigma-Aldrich); anti-CD68 antibody (Santa Cruz Biotechnology); anti-myeloperoxidase (MPO) antibody (Santa Cruz Biotechnology); IL-1β and IL-1β enzyme-linked immunosorbent assays (ELISA) (R&D Systems, Minneapolis, MN, USA); transmission electron microscopy (Phillips, Amsterdam, the Netherlands); high-performance liquid chromatography (LKB Bromma, Bromma, Sweden); bile acid standards (Sigma-Aldrich); RAW 264·7 cells (Cell Resource Center of Shanghai, Shanghai, China); lipopolysaccharide (LPS) (Sigma-Aldrich); propidium iodide (PI) (Invitrogen, Carlsbad, CA, USA); and 6-carboxyfluorescein-YVAD-fluoro-methylketone (Kamiya Biomedical Company, Seattle, WA, USA).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Standard Deviation

Journal: Clinical and Experimental Immunology

Article Title: Gene silencing of non-obese diabetic receptor family (NLRP3) protects against the sepsis-induced hyper-bile acidaemia in a rat model

doi: 10.1111/cei.12457

Figure Lengend Snippet: Gene silencing of non-obese diabetic-like receptor family (NLRP3) in-vitro cell experiments. RAW264·7 cells were transfected with NLRP3 shRNA or scrambled shRNA. The stably transfected cells were stimulated or not with lipopolysaccharide (LPS) (100 ng/ml) for different periods. (a,b) The cell lysates were collected and NLRP3 protein levels were measured by Western blot. The representative images of NLRP3 protein expression and the quantitative analysis of protein density. (c,d) The supernatants in cells culture media from the four groups were analysed for IL-1β and IL-18 by enzyme-linked immunosorbent assay (ELISA). (e) The representative fluorescence microscopy images of pyroptic cells (original magnification ×200). The caspase-1 (green) and propidium iodide (PI) (red) signals were captured, respectively. (f) The percentages of caspase-1 and PI double-positive cells from the four groups at 12 h and 48 h after stimulation. Data are expressed as percentages of the baseline value in the sham group. Data represent mean ± standard deviation; the experiments were repeated three times. *P < 0·05 versus baseline value in the sham group; **P < 0·001 versus baseline value in the sham group.

Article Snippet: Sprague–Dawley rats (Animal Model Institute of Xi'an, China); NLRP3 shRNA and scrambled shRNA plasmids (OriGene Technologies, Rockville, MD, USA); anti-NLRP3 antibody (Abcam, Cambridge, UK); horseradish peroxidase (HRP)-conjugated goat anti-rabbit immunoglobulin (Ig)G (Santa Cruz Biotechnology, Santa Cruz, CA, USA); anti-Bsep antibody (Sigma-Aldrich, St Louis, MO, USA); anti-Mrp2 antibody (Sigma-Aldrich); anti-Mrp3 antibody (Sigma-Aldrich); anti-Mrp4 antibody (Sigma-Aldrich); anti-CD68 antibody (Santa Cruz Biotechnology); anti-myeloperoxidase (MPO) antibody (Santa Cruz Biotechnology); IL-1β and IL-1β enzyme-linked immunosorbent assays (ELISA) (R&D Systems, Minneapolis, MN, USA); transmission electron microscopy (Phillips, Amsterdam, the Netherlands); high-performance liquid chromatography (LKB Bromma, Bromma, Sweden); bile acid standards (Sigma-Aldrich); RAW 264·7 cells (Cell Resource Center of Shanghai, Shanghai, China); lipopolysaccharide (LPS) (Sigma-Aldrich); propidium iodide (PI) (Invitrogen, Carlsbad, CA, USA); and 6-carboxyfluorescein-YVAD-fluoro-methylketone (Kamiya Biomedical Company, Seattle, WA, USA).

Techniques: In Vitro, Transfection, shRNA, Stable Transfection, Western Blot, Expressing, Enzyme-linked Immunosorbent Assay, Fluorescence, Microscopy, Standard Deviation

Journal: Nature protocols

Article Title: A 3D human neural cell culture system for modeling Alzheimer’s disease

doi: 10.1038/nprot.2015.065

Figure Lengend Snippet: Antibodies used for Western Blot analysis.

Article Snippet: Biochemical Extraction, Western Blot and Dot Blot list-behavior=unordered prefix-word= mark-type=disc max-label-size=0 HPLC-grade H 2 O (Fisher Scientific, cat. no. W5-1) 4x LDS sample loading buffer (Life Technologies, cat. no. NP0007) Invitrogen NuPage MES SDS Running Buffer (20X) (Life Technologies, cat. no. NP0002-02) Full range molecular weight Marker (Amersham, cat. no. GERPN800E) SuperBlock T20 (TBS) Blocking Buffer (Thermo Scientific cat. no. 37536) 50% glutaraldehyde solution (Electron Microscopy Sciences cat. no. 16316) Supersignal West Dura Chemiluminescence solution (Thermo Scientific cat. no. 34075AB) Supersignal West Femto Chemiluminescence solution (Thermo Scientific, cat. no. 34095AB) N-Lauroylsarcosine (Sigma-Aldrich, cat. no. SKU L5777) NaVO 3 (Sigma-Aldrich, cat. no. SKU 72060) NaF (Sigma-Aldrich, cat. no. 201154) Protease inhibitor mixture (Roche Life Science, cat. no. 04693159001) Phosphatase inhibitor cocktail (Thermo Scientific, cat no. 88667) 1,10-o-phenanthroline (PNT, EMD Millipore, cat. no. M1072250010) Phenylmethylsulfonyl fluoride (PMSF, Sigma-Aldrich, cat. no. P7626) Formic acid (Sigma-Aldrich, cat. no. 399388) Immun-Blot® PVDF Membrane (Bio-Rad, cat. no. 162-0177) Nitrocellulose Membranes (0.20 Micron, 7.9 cm x 10.5 cm) (Thermo Scientific, cat. no. PE1846871) Bio-Dot SF Filter Paper (Bio-rad, cat. no. 1620161) A summary of the used antibodies was shown in table ft1 table-wrap mode="anchored" t5 Table 2 caption a7 Useful markers for Antibody Host Supplier Cat. no. Dilution p-tau AT-8 Mouse Thermo Scientific MN1020 1:40 p-tau PHF-1 Mouse A gift from Dr. Davis N/A 1:1000 Total tau anti-Tau Rabbit DAKO A0024 1:2000 Abeta 6E10 Mouse Covance SIG-39300 1:400 Abeta MOAB-2 Mouse Biosensis M-1586-100 1:100 Neuron (dendritic) anti-MAP2 Rabbit Cell Signaling Technology 8717 1:200 Neuron (dendritic) anti-MAP2 Rabbit EMD Millipore AB5622 1:500 presenilin anti-Presenilin 1 Rabbit Cell Signaling Technology 5643 1:2000 BACE1 anti-BACE1 Rabbit Cell Signaling Technology 5606 1:1000 Amyloid Precursor Protein anti-C66 APP C-terminal Rabbit A gift from Dr. Kovacs N/A 1:2000 Neuron anti-NCAM Mouse Cell Signaling Technology 3576 1:1000 Neuron (synaptic) anti-synapsin I Rabbit Cell Signaling Technology 5297 1:500 ER anti-Calnexin Rabbit Cell Signaling Technology 2679 1:1000 Heat shock protein/control anti-HSP70 Rabbit Enzo Life Sciences ADI-SPA-812 1:1000 human Mitochondria anti-human mitochondrial antigen Mouse EMD Millipore MAB1273 1:500 Open in a separate window Antibodies used for Western Blot analysis.

Techniques: Western Blot

Journal: eLife

Article Title: TRIM28 promotes HIV-1 latency by SUMOylating CDK9 and inhibiting P-TEFb

doi: 10.7554/eLife.42426

Figure Lengend Snippet: ( A ) The knockdown efficiency of different SUMO4 siRNAs was confirmed by qPCR. Data represents mean ±SEM in triplicates. p-Vvalues were calculated by Student’s t -test. ***p<0.001. ( B ) The expression of SUMO4 in different cells was quantitated by qPCR and normalized to HEK293T group. β-actin mRNA was set as internal reference. ( C ) CD4 + T cells from three healthy donors were stimulated with PHA for 2 days or left untreated. One part of PHA-activated CD4 + T cells was washed for removing PHA and cultured in RPMI1640 which contained low IL-2 for 1 month. Then, resting CD4 + T cells were isolated from long-term cultured CD4 + T cells. Total RNAs from unstimulated (red), PHA-stimulated (green) and resting (blue) CD4 + T cells were extracted and proceeded to qPCR. SUMO4 from each group was quantitated and normalized to unstimulated group. β-actin mRNA was set as internal reference. ( D–H ) Data represented positive controls of siSUMO4-related ChIP. SUMO4 in TZM-bl cells was knocked down by siRNA targeting SUMO4 mRNA or treated with siNC. ChIP assays with antibodies against SUMO4, Histone H3, H3K9me3, H3K9Acetyl and H3K27me3 were performed for each group. For SUMO4 and Histone H3 ChIP, ChIP-qPCR DNA signals were normalized to Input of ‘B’ which represented the nucleosome-free region of HIV-1 LTR ( D–E ). ChIP-qPCR DNA signals were normalized to Input of the promoter of β-Globin for H3K9me3 ChIP ( F ). ChIP-qPCR DNA signals were normalized to Input of the promoter of GAPDH for H3K9Acetyl ChIP ( G ). ChIP-qPCR DNA signals were normalized to Input of the promoter of MYT1 for H3K27me3 ChIP ( H ). Data represents mean ±SEM in triplicates. p-Values were calculated by Student’s t -test. **p<0.01. ( I ) HA-tagged CDK9 was co-overexpressed with Flag-tagged SUMO1, Flag-tagged SUMO2 and Flag-tagged SUMO4, respectively. CDK9 was IP with anti-HA beads, followed by IB with antibodies against HA-tag, Flag-tag and GAPDH in total samples (lower panel), and IB with antibody against HA-tag in IP samples (upper panel). ( J ) HA-tagged TRIM28 was co-overexpressed with Flag-tagged SUMO1, Flag-tagged SUMO2 and Flag-tagged SUMO4, respectively. TRIM28 was IP with anti-HA beads, followed by IB with antibodies against HA-tag, Flag-tag and GAPDH in total samples (lower panel), and IB with antibody against HA-tag in IP samples (upper panel).

Article Snippet: After blocking, primary antibodies against TRIM28 (Proteintech, 66630–1-Ig), SUMO4 (Abcam, ab126606) and CDK9 (CST, 2316) were incubated with cells for 60 min at RT in 5% NDS/0.05% Triton X-100.

Techniques: Knockdown, Expressing, Cell Culture, Isolation, ChIP-chip, ChIP-qPCR, FLAG-tag

Journal: eLife

Article Title: TRIM28 promotes HIV-1 latency by SUMOylating CDK9 and inhibiting P-TEFb

doi: 10.7554/eLife.42426

Figure Lengend Snippet: ( A ) HA-tagged CDK9 was co-overexpressed with Flag-tagged SUMO4, UBC9 or TRIM28. CDK9 was IP with anti-HA-tag beads, followed by IB with anti-HA and –Flag antibodies. TRIM28, UBC9 and GAPDH in total samples were IB with specific antibodies targeting each proteins. ( B ) HA-tagged CDK9 was co-overexpressed with Flag-tagged SUMO4, Flag-tagged UBC9 and different amount of Flag-tagged TRIM28. Target proteins were IB as in ( A ). ( C ) In vitro purified CDK9, SUMO4, SAE1, UBA2, UBC9 and TRIM28 were co-cultured in SUMO conjugation reaction buffer. Proteins including SUMOylated CDK9 were IB with antibodies against each targets. ( D ) HA-tagged CDK9 was co-overexpressed with Flag-tagged SUMO4, Flag-tagged UBC9 or Flag-tagged TRIM28, and siNC. In the last group, CDK9 was co-overexpressed with SUMO4, UBC9 and siRNA against TRIM28. Target proteins were IB as in ( A ). ( E ) HA-tagged CDK9 was co-overexpressed with Flag-tagged SUMO4, Flag-tagged UBC9, Flag-tagged TRIM28 or two gradients of SENP3. Target proteins were IB as in ( A ).

Article Snippet: After blocking, primary antibodies against TRIM28 (Proteintech, 66630–1-Ig), SUMO4 (Abcam, ab126606) and CDK9 (CST, 2316) were incubated with cells for 60 min at RT in 5% NDS/0.05% Triton X-100.

Techniques: In Vitro, Purification, Cell Culture, Conjugation Assay

Journal: eLife

Article Title: TRIM28 promotes HIV-1 latency by SUMOylating CDK9 and inhibiting P-TEFb

doi: 10.7554/eLife.42426

Figure Lengend Snippet: ( A ) siRNAs targeting six SENPs were transfected into TZM-bl cells. The luciferase from the clarified lysates of each group was quantitated and normalized to siNC. Data represents mean ±SEM in triplicates. p-Values were calculated by Student’s t -test. *p<0.05. ( B ) Model proposed based on and . CDK9 which was subunit of P-TEFb complex was SUMOylated by TRIM28. The SUMO peptides were removed by SENP3-mediated deSUMOylation. ( C ) CD4 + T cells isolated from a healthy donor were transfected with siRNAs targeting negative control and SUMO4, respectively. Forty-eight hours later, the total lysates were immunoblotted with antibody against SUMO4. The lower band indicated free SUMO4. The middle band indicated SUMO4 dimer. The upper bands indicated SUMO4-SUMOylated cellular targets. ( D ) CD4 + T cells isolated from two healthy donors were lysed. The endogenous CDK9 was IP with antibody against CDK9. Both total samples and IP samples were IB with antibodies against CDK9 and GAPDH. Arrows indicated SUMOylated CDK9. ( E ) CD4 + T cells isolated from a heathy donor were transfected with SUMO4, UBC9 and TRIM28 constructs, or left untreated. Forty-eight hours later, endogenous CDK9 of the cell lysates was IP with antibody against CDK9. GAPDH, CDK9, UBC9, TRIM28 and SUMO4 were IB with corresponding antibodies in total samples. CDK9 and SUMO4 in IP samples were IB with antibodies against CDK9 and SUMO4 respectively.

Article Snippet: After blocking, primary antibodies against TRIM28 (Proteintech, 66630–1-Ig), SUMO4 (Abcam, ab126606) and CDK9 (CST, 2316) were incubated with cells for 60 min at RT in 5% NDS/0.05% Triton X-100.

Techniques: Transfection, Luciferase, Isolation, Negative Control, Construct

Journal: eLife

Article Title: TRIM28 promotes HIV-1 latency by SUMOylating CDK9 and inhibiting P-TEFb

doi: 10.7554/eLife.42426

Figure Lengend Snippet: ( A ) cSTORM image of endogenous TRIM28 and SUMO4 in HEK293T cells. The first row: the original whole nucleus; the second row: one of the amplified region of the nucleus; the third row: the 3D-cSTORM image of the amplified region. Merged views of TRIM28 and SUMO4 were shown on the left column. Endogenous TRIM28 was shown in the middle column and colored green. Endogenous SUMO4 was shown in the right column and colored red. Of note, DAPI and Hoechst were not allowed to dye DNA according to cSTORM protocol. ( B ) cSTORM image of endogenous TRIM28 and CDK9 in HEK293T cells. Each row was shown as in ( A ). First column: merged view of TRIM28 and CDK9, yellow indicating co-localization; second column: endogenous TRIM28 which was colored green; third column: endogenous CDK9 which was colored red. ( C–D ) cSTORM-imaged protein molecules and complexes were transformed into small or large spots based on their diameter. The left panel of each figure showed the original transformation. The middle panel showed spots-spots co-localization in compliance with the criterion of maximal distance of 10 nm. The right panel showed complexes-spots co-localization in compliance with the criterion of maximal distance of 100 nm. Green spots indicated TRIM28 molecules. Red spots indicated SUMO4 or CDK9 molecules. ( E ) Quantitation of co-localization of TRIM28 with SUMO4 or CDK9. Both of total proteins-proteins, spots-spots and complexes-spots co-localizations were measured.

Article Snippet: After blocking, primary antibodies against TRIM28 (Proteintech, 66630–1-Ig), SUMO4 (Abcam, ab126606) and CDK9 (CST, 2316) were incubated with cells for 60 min at RT in 5% NDS/0.05% Triton X-100.

Techniques: Amplification, Transformation Assay, Quantitation Assay

Journal: eLife

Article Title: TRIM28 promotes HIV-1 latency by SUMOylating CDK9 and inhibiting P-TEFb

doi: 10.7554/eLife.42426

Figure Lengend Snippet: ( A–B ) cSTORM-imaged protein molecules and complexes were transformed and displayed as in .Data represented amplified views of each transformed co-localization images. Green spots indicated TRIM28 molecules. Red spots indicated SUMO4 or CDK9 molecules.

Article Snippet: After blocking, primary antibodies against TRIM28 (Proteintech, 66630–1-Ig), SUMO4 (Abcam, ab126606) and CDK9 (CST, 2316) were incubated with cells for 60 min at RT in 5% NDS/0.05% Triton X-100.

Techniques: Transformation Assay, Amplification

Journal: eLife

Article Title: TRIM28 promotes HIV-1 latency by SUMOylating CDK9 and inhibiting P-TEFb

doi: 10.7554/eLife.42426

Figure Lengend Snippet: ( A ) Flag-tagged GFP and Flag-tagged TRIM28 were co-overexpressed with HA-tagged CDK9, respectively. Flag-tagged proteins were IP with anti-Flag beads. Flag-tagged proteins from another two similar groups were IP in the presence of RNase. Both total samples and IP samples were IB with antibodies against GAPDH, HA-tag and Flag-tag. ( B ) Flag-tagged full length TRIM28 and Flag-tagged TRIM28 mutants were co-overexpressed with SUMO4 and UBC9 respectively. Flag-tagged proteins were IP with anti-Flag beads followed by IB with antibodies against Flag-tag. Both total samples (lower panel) and IP samples (upper panel) were IB for each group.

Article Snippet: After blocking, primary antibodies against TRIM28 (Proteintech, 66630–1-Ig), SUMO4 (Abcam, ab126606) and CDK9 (CST, 2316) were incubated with cells for 60 min at RT in 5% NDS/0.05% Triton X-100.

Techniques: FLAG-tag

Journal: eLife

Article Title: TRIM28 promotes HIV-1 latency by SUMOylating CDK9 and inhibiting P-TEFb

doi: 10.7554/eLife.42426

Figure Lengend Snippet: ( A ) HA-tagged CDK9 was co-overexpressed with Flag-tagged full length TRIM28 or domain-truncated TRIM28 mutants. Flag-tagged proteins were IP, followed by IB with antibodies against HA-tag, Flag-tag and GAPDH. ( B ) HA-tagged CDK9 was co-overexpressed with Flag-tagged SUMO4, Flag-tagged UBC9, Flag-tagged full length TRIM28 or Flag-tagged domain-truncated TRIM28 mutants. CDK9 was IP with anti-HA-tag beads, followed by IB with antibodies against HA-tag, Flag-tag and GAPDH. ( C ) GFP-tagged TRIM28 or TRIM28-dRING mutant was co-overexpressed with RFP-tagged CDK9 in HEK293T cells. The samples were fixed and dyed according to the immunofluorescence procedure, then visualized in Nikon A1 N-SIM. DAPI was used to dye DNA which was colored into blue. ( D ) Quantitation of co-localization of TRIM28 or TRIM28-dRING with CDK9. The percentage of co-localization was indicated by percentage of target protein voxels above threshold co-localized voxels. Both Pearson’s coefficient and thresholded Mander’s coefficient were used to evaluate co-localization. For Pearson’s coefficient, a value of 1 represents perfect co-localization, 0 no co-localization, and −1 perfect inverse co-localization. For thresholded Mander’s coefficient, a value of 1 represents perfect co-localization and 0 no co-localization.

Article Snippet: After blocking, primary antibodies against TRIM28 (Proteintech, 66630–1-Ig), SUMO4 (Abcam, ab126606) and CDK9 (CST, 2316) were incubated with cells for 60 min at RT in 5% NDS/0.05% Triton X-100.

Techniques: FLAG-tag, Mutagenesis, Immunofluorescence, Quantitation Assay

Journal: eLife

Article Title: TRIM28 promotes HIV-1 latency by SUMOylating CDK9 and inhibiting P-TEFb

doi: 10.7554/eLife.42426

Figure Lengend Snippet: ( A–B ) TRIM28-defective (sgTRIM28) J-Lat 10.6 cell line was generated by CRISPR-CAS9 technique. ATAC-Seq was conducted with sgNT and sgTRIM28 J-Lat 10.6 cell lines, as well as siNC and siTRIM28 TZM-bl cell lines. The tag reads of the HIV-1 pseudotyped virus/minigenome 5’LTR integration sites were counted and normalized to the total mapped reads, and represented as relative tag density. The highest tag density was set as 100. Figures showed 2 kb range centered the 5’LTR integration sites. ( C–D ) ChIP assays with antibodies against CDK9 and Ser2 Pho-Pol II were performed in TZM-bl cell lines which were treated with siNC, siSUMO4 and siTRIM28, respectively. ( E ) Cyclin T1 or GFP was co-overexpressed with CDK9 in the absence or presence of SUMO4, UBC9 and TRIM28. Cyclin T1 and GFP were IP followed by IB. ( F ) Fold change of kinase activity when CDK9 was SUMOylated. Data represents mean ±SEM in triplicates. p-Values were calculated by Student’s t -test. *p<0.05, **p<0.01.

Article Snippet: After blocking, primary antibodies against TRIM28 (Proteintech, 66630–1-Ig), SUMO4 (Abcam, ab126606) and CDK9 (CST, 2316) were incubated with cells for 60 min at RT in 5% NDS/0.05% Triton X-100.

Techniques: Generated, CRISPR, Virus, Activity Assay

Journal: eLife

Article Title: TRIM28 promotes HIV-1 latency by SUMOylating CDK9 and inhibiting P-TEFb

doi: 10.7554/eLife.42426

Figure Lengend Snippet: ( A ) In vitro expressed and purified CDK9 was incubated with SUMO system components (SUMO4, E1, UBC9 and TRIM28) or left untreated. Five groups were set. Group 1 ( G1 ): CDK9 only; Group 2 ( G2 ): CDK9 and SUMO4; Group 3 ( G3 ): CDK9, SUMO4 and E1 (SAE1 and UBA2); Group 4 ( G4 ): CDK9, SUMO4, E1 and E2 (UBC9); Group 5 ( G5 ): CDK9, SUMO4, E1, E2 and E3 (TRIM28). After in vitro SUMOylation, CDK9 substrate PDKtides and ATP were added and incubated for 120 min at room temperature. The ADP which was consumed during CDK9 kinase assay was converted to ATP and quantitated by luciferase assay.

Article Snippet: After blocking, primary antibodies against TRIM28 (Proteintech, 66630–1-Ig), SUMO4 (Abcam, ab126606) and CDK9 (CST, 2316) were incubated with cells for 60 min at RT in 5% NDS/0.05% Triton X-100.

Techniques: In Vitro, Purification, Incubation, Kinase Assay, Luciferase

Journal: eLife

Article Title: TRIM28 promotes HIV-1 latency by SUMOylating CDK9 and inhibiting P-TEFb

doi: 10.7554/eLife.42426

Figure Lengend Snippet: ( A ) Schematic of different CDK9 mutants. CDK9-K0R indicated that all lysines had been mutated to arginines. CDK9-KKR indicated that the third part of CDK9 had mutated all lysines to arginines. The left five clones were mutated similarly. ( B ) HA-tagged wild type CDK9 and different CDK9 mutants were co-overexpressed with Flag-tagged SUMO4, Flag-tagged UBC9 and Flag-tagged TRIM28. HA-tagged proteins were IP with anti-HA beads followed by IB with antibodies against HA-tag and Flag-tag. Both total samples (lower panel) and IP samples (upper panel) were IB for each group. ( C ) Schematic of target-specific SUMO-MS. HA-tagged CDK9 was co-overexpressed with Flag-tagged SUMO4-Q88R or His-tagged SUMO4-Q88R respectively. Anti-HA-tag beads were used to IP CDK9 and SUMO-CDK9 (left panel). The SUMOylation efficiency was determined by IB (middle panel). HA-tagged targets were separated by SDS-PAGE and developed by silver staining (right panel). Red frames indicated that SUMO-CDK9. SUMO-CDK9 was cut out and conducted in-gel digestion. The digested peptides were desalted and proceeded to nanoscale LC-MS/MS. ( D ) Results of the target-specific SUMO-MS of CDK9. Protein sequence coverage was 81% covering nearly all lysines. Three SUMOylation sites which were identified through SUMOylation assay were shown on the right panel. ( E ) Second-order mass spectra of CDK9 SUMOylation sites Lys44, Lys56 and Lys68.

Article Snippet: After blocking, primary antibodies against TRIM28 (Proteintech, 66630–1-Ig), SUMO4 (Abcam, ab126606) and CDK9 (CST, 2316) were incubated with cells for 60 min at RT in 5% NDS/0.05% Triton X-100.

Techniques: Clone Assay, FLAG-tag, SDS Page, Silver Staining, Liquid Chromatography with Mass Spectroscopy, Sequencing

Journal: eLife

Article Title: TRIM28 promotes HIV-1 latency by SUMOylating CDK9 and inhibiting P-TEFb

doi: 10.7554/eLife.42426

Figure Lengend Snippet: ( A ) Different HA-tagged CDK9 reversing mutation constructs or wild type CDK9 were co-overexpressed with SUMO4, UBC9 and TRIM28, respectively. CDK9 and CDK9 mutants were IP with anti-HA-tag beads followed by IB. S4: SUMO4. ( B ) HA-tagged wild type CDK9 and 12 identified SUMOylation site reversing mutation constructs were co-overexpressed with Flag-tagged SUMO4 and Flag-tagged UBC9. The endogenous TRIM28 was knocked down with siRNAs. CDK9 and CDK9 mutants were IP with anti-HA-tag beads followed by IB. Asterisks represented the constructs whose SUMOylation bands disappeared upon TRIM28 knockdown. ( C ) Three angles of co-crystal structure of Cyclin T1 and CDK9 (PDB ID: 4EC8). Three SUMOylation sites Lys44, Lys56 and Lys68 were shown in ball-and-stick models. The two upper panels showed the ribbon models, while two lower panels showed the surface models. The inner six framed figures which numbered from I to VI represented the amplification views of Lys44, Lys56 and Lys68 sites.

Article Snippet: After blocking, primary antibodies against TRIM28 (Proteintech, 66630–1-Ig), SUMO4 (Abcam, ab126606) and CDK9 (CST, 2316) were incubated with cells for 60 min at RT in 5% NDS/0.05% Triton X-100.

Techniques: Mutagenesis, Construct, Knockdown, Amplification

Journal: eLife

Article Title: TRIM28 promotes HIV-1 latency by SUMOylating CDK9 and inhibiting P-TEFb

doi: 10.7554/eLife.42426

Figure Lengend Snippet:

Article Snippet: After blocking, primary antibodies against TRIM28 (Proteintech, 66630–1-Ig), SUMO4 (Abcam, ab126606) and CDK9 (CST, 2316) were incubated with cells for 60 min at RT in 5% NDS/0.05% Triton X-100.

Techniques: Infection, Recombinant, Expressing, Plasmid Preparation, Construct, Mutagenesis, Sequencing, shRNA, Luciferase, Electron Microscopy, Protease Inhibitor, Conjugation Assay, Chromatin Immunoprecipitation, Magnetic Beads, Enzyme-linked Immunosorbent Assay, Silver Staining, Kinase Assay, CCK-8 Assay, Flow Cytometry, Software, Microscopy, Imaging