Journal: The Journal of Experimental Medicine

Article Title: Sarco/endoplasmic reticulum Ca 2+ -ATPase (SERCA) activity is required for V(D)J recombination

doi: 10.1084/jem.20201708

Figure Lengend Snippet: SERCA3 supports V(D)J recombination in SERCA2-deficient cells. (A) Quantitative RT-PCR analysis of Atp2a3 mRNA in WT abl preB cells without (−) gAtp2a2 and bulk Atp2a2 inactivated with (+) gAtp2a2 normalized to β-actin mRNA (mean ± SD; n = 3). **, P < 0.01. (B) Western blot analysis of SERCA2, SERCA3, and β-actin in WT and bulk Atp2a2 inactivated abl preB cells performed as in ( n = 1 for Dox 2 d; n = 4 for Dox 4 d or more). (C) Western blot analysis of SERCA3 and β-actin in WT and Atp2a2 −/− abl preB cells without (−) gAtp2a3 and bulk Atp2a3 inactivated with (+) gAtp2a3 ( n = 3). (D) Flow-cytometric analysis of pMGINV rearrangement in WT and Atp2a2 −/− abl preB cells (−) and these cells after bulk Atp2a3 inactivation with (+) gAtp2a3 (mean ± SD; n = 3). ***, P < 0.001. (E) Mouse Igk locus schematic showing multiple Vk and Jk gene segments, relative positions of SacI (S) and EcoRI (E) restriction sites, and Jk probe. The 3-kb Jk probe-hybridizing SacI–EcoRI fragment is shown, as are several different Vk to Jk rearrangements that will give rise to Jk probe-hybridizing SacI–EcoRI fragments of different sizes. (F) Southern blot of SacI and EcoRI digested genomic DNA probed with the Jk probe from WT and Atp2a2 −/− abl preB cells (−) and the same cells bulk Atp2a2 ( gAtp2a2 ) or Atp2a3 ( gAtp2a3 ) inactivated and treated with imatinib for the indicated number of days. The fragment generated by the unrearranged (UR) Igk locus is indicated. The percentage of lane hybridization that corresponds to the UR Igk locus is shown with 0-d imatinib set at 100%. Quantification described in Materials and methods. (G) Mean ± SD of the percentage of UR Igk locus quantified as described in F in imatinib-treated WT, Atp2a2 −/− , and bulk Atp2a3 inactivated Atp2a2 −/− abl preB cells ( n = 3). *, P < 0.05; **, P < 0.01; ***, P < 0.001. Dox, doxycycline; expo., exposure; n.s., not significant.

Article Snippet: Primary antibodies used were anti-SERCA2, rabbit polyclonal, 1:2,000 (Cell Signaling Technology; 4388); anti-SERCA3, rabbit polyclonal, 1:5,000 (GeneTex; GTX102381); anti-RAG1, rabbit monoclonal, 1:1,000 (Abcam; ab172637); anti-XBP1s, rabbit monoclonal, 1:1,000 (Cell Signaling Technology; 12782); anti–β-actin, mouse monoclonal, 1:20,000 (Sigma-Aldrich; A2228); and anti-GAPDH, mouse monoclonal, 1:5,000 (Sigma-Aldrich; G8795).

Techniques: Quantitative RT-PCR, Western Blot, Southern Blot, Generated, Hybridization

Journal: The Journal of Experimental Medicine

Article Title: Sarco/endoplasmic reticulum Ca 2+ -ATPase (SERCA) activity is required for V(D)J recombination

doi: 10.1084/jem.20201708

Figure Lengend Snippet: Loss of SERCA2 and SERCA3 disrupts Ca 2+ homeostasis. (A–D) Flow-cytometric analysis of WT abl preB cells expressing ER-GCaMP6-150 (A) or incubated with Fluo-3 AM Ca 2+ indicator (C) without further treatment (black histogram), treated with thapsigargin (TG; red histogram), or after bulk inactivation of Atp2a2 and Atp2a3 genes (green histogram). Median fluorescence intensity was calculated and normalized to that of the black histogram in each experiment to derive the relative intensity of ER-GCaMP6-150 (B; mean ± SD; n = 4) and Fluo-3 dye (D; mean ± SD; n = 3). *, P < 0.05; **, P < 0.01; ***, P < 0.001 compared with the untreated sample. (E) Western blot analysis of XBP1s in imatinib-treated WT abl preB cells bulk Hspa5 ( gHspa5 ) inactivated or Atp2a2 −/− cells bulk Atp2a3 ( gAtp2a3 ) inactivated ( n = 3). (F) Flow-cytometric analysis of GFP expression from pMGINV rearrangement in cells from E treated with imatinib for 0, 2, or 4 d. (G) Mean ± SD of three pMGINV experiments performed as in F. **, P < 0.01; ***, P < 0.001. n.s., not significant.

Article Snippet: Primary antibodies used were anti-SERCA2, rabbit polyclonal, 1:2,000 (Cell Signaling Technology; 4388); anti-SERCA3, rabbit polyclonal, 1:5,000 (GeneTex; GTX102381); anti-RAG1, rabbit monoclonal, 1:1,000 (Abcam; ab172637); anti-XBP1s, rabbit monoclonal, 1:1,000 (Cell Signaling Technology; 12782); anti–β-actin, mouse monoclonal, 1:20,000 (Sigma-Aldrich; A2228); and anti-GAPDH, mouse monoclonal, 1:5,000 (Sigma-Aldrich; G8795).

Techniques: Expressing, Incubation, Fluorescence, Western Blot

Journal: The Journal of Experimental Medicine

Article Title: Sarco/endoplasmic reticulum Ca 2+ -ATPase (SERCA) activity is required for V(D)J recombination

doi: 10.1084/jem.20201708

Figure Lengend Snippet: Loss of SERCA2 and SERCA3 leads to diminished Rag expression. (A) Schematic of pMGINV with locations of PCR primers (arrows) used to amplify CJ and the plasmid backbone (Amp R ). (B) Quantitative PCR analysis of pMGINV CJ formation in plasmids recovered from WT and Atp2a2 −/− abl preB cells treated with imatinib (mean ± SD; n = 3). ***, P < 0.001. (C and D) Levels of Rag1 (C) and Rag2 (D) mRNA assayed by quantitative RT-PCR in abl preB cells treated with imatinib for 1 or 2 d. WT and Atp2a2 −/− abl preB clones (−) were assayed, as were Atp2a2 −/− abl preB clones with bulk Atp2a3 inactivation (+). Rag1 and Rag2 mRNA levels were normalized to β-actin mRNA and relative to the WT value, which was set at 1. Rag1 and Rag2 mRNAs were not detected at significant levels in cells not treated with imatinib (mean ± SD; n = 3). **, P < 0.01; ***, P < 0.001 compared with WT. (E) Western blot analysis for RAG1 and actin in WT and Atp2a2 −/− abl preB cell clones (−) and cells bulk Atp2a2 ( gAtp2a2 ) or Atp2a3 ( gAtp2a3 ) inactivated and treated with imatinib for 1 or 2 d ( n = 1 for 1 d, n = 2 for 2 d).

Article Snippet: Primary antibodies used were anti-SERCA2, rabbit polyclonal, 1:2,000 (Cell Signaling Technology; 4388); anti-SERCA3, rabbit polyclonal, 1:5,000 (GeneTex; GTX102381); anti-RAG1, rabbit monoclonal, 1:1,000 (Abcam; ab172637); anti-XBP1s, rabbit monoclonal, 1:1,000 (Cell Signaling Technology; 12782); anti–β-actin, mouse monoclonal, 1:20,000 (Sigma-Aldrich; A2228); and anti-GAPDH, mouse monoclonal, 1:5,000 (Sigma-Aldrich; G8795).

Techniques: Expressing, Plasmid Preparation, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Clone Assay, Western Blot

Journal: The Journal of Experimental Medicine

Article Title: Sarco/endoplasmic reticulum Ca 2+ -ATPase (SERCA) activity is required for V(D)J recombination

doi: 10.1084/jem.20201708

Figure Lengend Snippet: Analysis of SERCA2-deficient primary B cells. (A) PCR strategy used to distinguish the Atp2a2a and Atp2a2b mRNA isoforms. (B) RT-PCR analysis of mRNA representing all Atp2a2 isoforms and Atp2a2b or Atp2a2a isoforms in addition to Atp2a3 transcripts in proB (B220 + CD43 + ), preB (B220 + CD43 − ), and immature B (B220 + IgM + ) cells purified from bone marrow of WT B6 mice ( n = 2). (C) Gating strategies for the flow-cytometric analysis of B cell development in mouse bone marrow and spleen shown in . The analyses shown are of 4-wk-old littermates of Atp2a2 flox/flox and Atp2a2 flox/flox :mb1-cre mice. SSC, side scatter; FSC, forward scatter.

Article Snippet: Primary antibodies used were anti-SERCA2, rabbit polyclonal, 1:2,000 (Cell Signaling Technology; 4388); anti-SERCA3, rabbit polyclonal, 1:5,000 (GeneTex; GTX102381); anti-RAG1, rabbit monoclonal, 1:1,000 (Abcam; ab172637); anti-XBP1s, rabbit monoclonal, 1:1,000 (Cell Signaling Technology; 12782); anti–β-actin, mouse monoclonal, 1:20,000 (Sigma-Aldrich; A2228); and anti-GAPDH, mouse monoclonal, 1:5,000 (Sigma-Aldrich; G8795).

Techniques: Reverse Transcription Polymerase Chain Reaction, Purification

Journal: Neural Regeneration Research

Article Title: FUBP3 mediates the amyloid-β-induced neuronal NLRP3 expression

doi: 10.4103/NRR.NRR-D-23-01799

Figure Lengend Snippet: Study design flowchart. Aβ: Amyloid-β; FUBP3: far upstream binding protein 3; NLRP3: NOD-, LRR- and pyrin domain-containing protein 3.

Article Snippet: The slices were then pretreated with 10% normal donkey serum (Solarbio, Beijing, China) for 30 minutes at room temperature, followed by incubation with the appropriate primary antibodies, including: anti-FUBP3 (rabbit polyclonal, 1:500, PAG312Mu01, RRID: AB_3083686, Cloud-clone), anti-NLRP3 (mouse monoclonal, 1:500, AG-20B-0014-C100, RRID: AB_2885199, AdipoGen, San Diego, CA, USA), anti-Iba1 (rabbit polyclonal, 1:500, 019-19741, RRID: AB_839504, Wako, Shanghai, China), and anti-NeuN (rabbit monoclonal, 1:500, SAB5700017, RRID: AB_3083690, Millipore or mouse polyclonal, 1:250, 94403, RRID: AB_2904530, Cell Signaling).

Techniques: Binding Assay

Journal: Neural Regeneration Research

Article Title: FUBP3 mediates the amyloid-β-induced neuronal NLRP3 expression

doi: 10.4103/NRR.NRR-D-23-01799

Figure Lengend Snippet: Primers for plasmids constructed in this study

Article Snippet: The slices were then pretreated with 10% normal donkey serum (Solarbio, Beijing, China) for 30 minutes at room temperature, followed by incubation with the appropriate primary antibodies, including: anti-FUBP3 (rabbit polyclonal, 1:500, PAG312Mu01, RRID: AB_3083686, Cloud-clone), anti-NLRP3 (mouse monoclonal, 1:500, AG-20B-0014-C100, RRID: AB_2885199, AdipoGen, San Diego, CA, USA), anti-Iba1 (rabbit polyclonal, 1:500, 019-19741, RRID: AB_839504, Wako, Shanghai, China), and anti-NeuN (rabbit monoclonal, 1:500, SAB5700017, RRID: AB_3083690, Millipore or mouse polyclonal, 1:250, 94403, RRID: AB_2904530, Cell Signaling).

Techniques: Construct, Sequencing

Journal: Neural Regeneration Research

Article Title: FUBP3 mediates the amyloid-β-induced neuronal NLRP3 expression

doi: 10.4103/NRR.NRR-D-23-01799

Figure Lengend Snippet: FUBP3 binds the minimal NLRP3 promoter in neurons. (A) The indicated DNA fragments from the promoter region of NLRP3 were biotin-labeled and used to pull down bound proteins from Neuro2A cell nuclear extracts. The –21 to +217 fragment, which has no promoter activity, was used as a negative control. The precipitated proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis, and the gel was stained with Coomassie Blue. The ~ 70 kDa band was excised and analyzed by mass spectrometry. Transcription factors represented by high amounts of peptide in the mass spectrometry results are listed. (B) 5′ and 3’ biotin-labeled Del5, the minimal promoter, were used to pull down FUBP3 from Neuro2A nuclear extracts. A 5′ labeled irrelevant DNA (ir. DNA) was used as the control. Western blot analysis of FUBP3 in the precipitates, and equal loading of the bait DNAs, (input probes), as assessed by EMSA. (C) Neuro2A nuclear extracts were mixed with the 5′ labeled probe (Del5), and the mixtures were loaded onto native gels and transferred to nylon membranes for regular EMSA or to nitrocellulose membranes for EMSA–western blot using an anti-FUBP3 antibody. (D) Neuro2A nuclear extracts were mixed with the 5′ labeled single-stranded probe. After incubation for 30 minutes at room temperature, the mixtures were loaded onto native gels, and transferred to nylon membranes for regular EMSA or to nitrocellulose membranes for EMSA–western blot using an anti-FUBP1 antibody. (E) The listed components were mixed, and the mixtures were subjected to regular EMSA. a.s.: Antisense strand; ds: double strand probes; EMSA: electrophoretic mobility shift assay; FUBP1: far upstream binding protein 1; NLRP3: NOD-, LRR- and pyrin domain-containing protein 3; sen: sense strand; ss: single strand; WB: western blot.

Article Snippet: The slices were then pretreated with 10% normal donkey serum (Solarbio, Beijing, China) for 30 minutes at room temperature, followed by incubation with the appropriate primary antibodies, including: anti-FUBP3 (rabbit polyclonal, 1:500, PAG312Mu01, RRID: AB_3083686, Cloud-clone), anti-NLRP3 (mouse monoclonal, 1:500, AG-20B-0014-C100, RRID: AB_2885199, AdipoGen, San Diego, CA, USA), anti-Iba1 (rabbit polyclonal, 1:500, 019-19741, RRID: AB_839504, Wako, Shanghai, China), and anti-NeuN (rabbit monoclonal, 1:500, SAB5700017, RRID: AB_3083690, Millipore or mouse polyclonal, 1:250, 94403, RRID: AB_2904530, Cell Signaling).

Techniques: Labeling, Activity Assay, Negative Control, Polyacrylamide Gel Electrophoresis, Staining, Mass Spectrometry, Control, Western Blot, Incubation, Electrophoretic Mobility Shift Assay, Binding Assay

Journal: Neural Regeneration Research

Article Title: FUBP3 mediates the amyloid-β-induced neuronal NLRP3 expression

doi: 10.4103/NRR.NRR-D-23-01799

Figure Lengend Snippet: FUBP3 is required for NLRP3 expression in neurons in the presence of Aβ. (A) Control siRNA or mouse FUBP3 -specific siRNA and pNLRP3-Del5 were transfected into Neu2A cells for the luciferase assay. The promoter activities after normalization to the pGL3-basic vector were plotted. (B) Neuro2A cells were transfected with control siRNA or FUBP3-specific siRNA, and treated with DMSO as a control or with 1 μM Aβ overnight. The NRKP3 mRNA levels in these cells were determined by quantitative PCR, and the proteins were examined by western blot. FUBP3 and NLRP3 expression levels were quantified. (C) Primary neurons from wild-type mice (PN WT ) were infected with viruses expressing control shRNA or FUBP3 -targeting shRNA under the syn1 promoter, and treated with or without Aβ. FUBP3 and NLRP3 expression levels were compared among the different treatment groups. (D) PN WT were infected with viruses expressing control shRNA or shFUBP3, and the cells were treated with cerebrospinal fluid from patients with viral encephalitis. FUBP3 and NLRP3 were detected by western blot, and their expression levels were quantified. (E) PN APP were infected with viruses expressing control shRNA or shFUBP3, and FUBP3 and NLRP3 were detected by western blot, and their expression levels were quantified. (F) PN WT and primary neurons from APP Swedish mutant transgenic mice (PN APP ) growing on coverslips were stained with antibodies to FUBP3 (green, Alexa Fluor 488) and NeuN (red, Alexa Fluor 564), a neuronal cell marker. There was no discernable difference in FUBP3 expression levels between PN WT and PN APP . Asterisks indicate contaminating cells in the neuronal culture. (G) Brains sections from young wild-type mice, APP/PS1ΔE9 AD model mice, and aged wild-type mice were stained for FUBP3 (green, Alexa Fluor 488) and NeuN (red, Alexa Fluor 564), a neuronal marker. (H) FUBP3 was also expressed in the microglial cell line BV2. All data are expressed as mean ± SEM ( n = 3 independent repeats). * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 (two-tailed Student’s t -test). Aβ: Amyloid-β; B.E.: brightness enhanced; CSF: cerebrospinal fluid; DAPI: 4′,6-diamidino-2-phenylindole; DMSO: dimethyl sulfoxide; DNA: nuclear stained by DAPI that labels DNA; FUBP3: far upstream binding protein 3; NLRP3: NOD-, LRR- and pyrin domain-containing protein 3; ns: not significant; PCR: polymerase chain reaction; shRNA: short hairpin RNA.

Article Snippet: The slices were then pretreated with 10% normal donkey serum (Solarbio, Beijing, China) for 30 minutes at room temperature, followed by incubation with the appropriate primary antibodies, including: anti-FUBP3 (rabbit polyclonal, 1:500, PAG312Mu01, RRID: AB_3083686, Cloud-clone), anti-NLRP3 (mouse monoclonal, 1:500, AG-20B-0014-C100, RRID: AB_2885199, AdipoGen, San Diego, CA, USA), anti-Iba1 (rabbit polyclonal, 1:500, 019-19741, RRID: AB_839504, Wako, Shanghai, China), and anti-NeuN (rabbit monoclonal, 1:500, SAB5700017, RRID: AB_3083690, Millipore or mouse polyclonal, 1:250, 94403, RRID: AB_2904530, Cell Signaling).

Techniques: Expressing, Control, Transfection, Luciferase, Plasmid Preparation, Real-time Polymerase Chain Reaction, Western Blot, Infection, shRNA, Mutagenesis, Transgenic Assay, Staining, Marker, Two Tailed Test, Binding Assay, Polymerase Chain Reaction

Journal: Neural Regeneration Research

Article Title: FUBP3 mediates the amyloid-β-induced neuronal NLRP3 expression

doi: 10.4103/NRR.NRR-D-23-01799

Figure Lengend Snippet: Nuclear enrichment of FUBP3 is unlikely required for NLRP3 expression in the presence of Aβ. (A) FUBP3-FLAG was overexpressed in Neuro2A cells at low or high levels relative to endogenous FUBP3. Total FUBP3, overexpressed FUBP3-FLAG, and endogenous NLRP3 expression levels were assessed by western blot to evaluate the effect of FUBP3 overexpression on NLRP3 expression. (B) FUBP3-FLAG was overexpressed in Neuro2A cells at low or high levels, and nuclear fractionation was performed to separate nuclear FUBP3 from cytosolic FUBP3. GAPDH was used as a marker of the cytosolic fraction, and histone H3 was used as a marker of the nuclear fraction. (C) The effect of Aβ exposure on FUBP3 and NLRP3 expression levels was examined in Neuro2A cells and primary neurons from wild-type mice (PN WT ). After Aβ treatment, the cell lysates were subjected to western blotting for FUBP3 and NLRP3. (D) The effect of Aβ exposure on FUBP3 nuclear translocation in Neuro2A cells. After Aβ treatment, the cell lysates were subjected to western blotting for FUBP3, NLRP3, GAPDH, and histone H3. All numeric data are expressed as mean ± SEM ( n = 3 independent repeats). * P < 0.05 (two-tailed Student’s t -test). (E) Aβ induced nuclear accumulation of FUBP3 in PN WT . PN WT with or without overnight exposure to 1 μM Aβ were stained with an anti-FUBP3 antibody (red, Alexa Fluor 564). The nuclei were stained with DAPI. Upon exposure to Aβ, FUBP3 became highly concentrated in the nucleus in a portion of neurons. (F) FUBP3 did not accumulate in the nucleus of PN APP . PN APP were stained with an anti-FUBP3 antibody (red, Alexa Fluor 564). The nuclei were stained with DAPI. Aβ: Amyloid-β; DAPI: 4′,6-diamidino-2-phenylindole; DMSO: dimethyl sulfoxide; DNA: nuclear stained by DAPI that labels DNA; FUBP3: FUBP3: far upstream binding protein 3; NLRP3: NOD-, LRR- and pyrin domain-containing protein 3; ns: not significant.

Article Snippet: The slices were then pretreated with 10% normal donkey serum (Solarbio, Beijing, China) for 30 minutes at room temperature, followed by incubation with the appropriate primary antibodies, including: anti-FUBP3 (rabbit polyclonal, 1:500, PAG312Mu01, RRID: AB_3083686, Cloud-clone), anti-NLRP3 (mouse monoclonal, 1:500, AG-20B-0014-C100, RRID: AB_2885199, AdipoGen, San Diego, CA, USA), anti-Iba1 (rabbit polyclonal, 1:500, 019-19741, RRID: AB_839504, Wako, Shanghai, China), and anti-NeuN (rabbit monoclonal, 1:500, SAB5700017, RRID: AB_3083690, Millipore or mouse polyclonal, 1:250, 94403, RRID: AB_2904530, Cell Signaling).

Techniques: Expressing, Western Blot, Over Expression, Fractionation, Marker, Translocation Assay, Two Tailed Test, Staining, Binding Assay

Journal: Neural Regeneration Research

Article Title: FUBP3 mediates the amyloid-β-induced neuronal NLRP3 expression

doi: 10.4103/NRR.NRR-D-23-01799

Figure Lengend Snippet: FUBP3 is required for IL1β secretion and tau phosphorylation in PN APP . (A) PN APP were infected with AAV expressing shFUBP3 shRNA to suppress FUBP3 expression. IL1β levels in the conditioned media were determined by ELISA (PN APP from 10 different embryos). (B) PN APP were infected with AAV expressing shFUBP3 or control shRNAs. The cell lysates were subjected to western blot for indicated proteins ( n = 6). Data are expressed as mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001 (two-tailed Student’s t -test). AAV: Adeno-associated virus; CaMK2α: calmodulin-dependent kinase IIα; ELISA: enzyme-linked immunosorbent assay; FUBP3: far upstream binding protein 3; IL1β: interleukin-1β; NLRP3: NOD-, LRR- and pyrin domain-containing protein 3; PME1: phosphatase methylesterase 1; PN APP : primary neurons of APP Swedish mutant single transgenic mouse; shRNA: short hairpin RNA.

Article Snippet: The slices were then pretreated with 10% normal donkey serum (Solarbio, Beijing, China) for 30 minutes at room temperature, followed by incubation with the appropriate primary antibodies, including: anti-FUBP3 (rabbit polyclonal, 1:500, PAG312Mu01, RRID: AB_3083686, Cloud-clone), anti-NLRP3 (mouse monoclonal, 1:500, AG-20B-0014-C100, RRID: AB_2885199, AdipoGen, San Diego, CA, USA), anti-Iba1 (rabbit polyclonal, 1:500, 019-19741, RRID: AB_839504, Wako, Shanghai, China), and anti-NeuN (rabbit monoclonal, 1:500, SAB5700017, RRID: AB_3083690, Millipore or mouse polyclonal, 1:250, 94403, RRID: AB_2904530, Cell Signaling).

Techniques: Infection, Expressing, shRNA, Enzyme-linked Immunosorbent Assay, Control, Western Blot, Two Tailed Test, Virus, Binding Assay, Mutagenesis, Transgenic Assay

Journal: Neural Regeneration Research

Article Title: FUBP3 mediates the amyloid-β-induced neuronal NLRP3 expression

doi: 10.4103/NRR.NRR-D-23-01799

Figure Lengend Snippet: FUBP3 is involved in the immune response in Neuro2A cells. RNA was extracted from three paired Neuro2A cell lines expressing control- or siFUBP3-siRNA. (A) FPKM analysis of the effects of FUBP3 inhibition on gene expression compared with control cells. (B) Venn diagram showing the number of differentially expressed genes in each group. (C) PCR validation of the RNA-seq results. The expression levels of NLRP6 , GSDMD , PPP1R1A , and IL1b were examined. (D) PPP1R1A expression in primary neurons from APP Swedish mutant transgenic mice (PN APP ) in which FUBP3 was knocked down was examined by western blot ( N = 3 independent repeats, n = 5 (PN APP from five different embryos)). Data are expressed as mean ± SEM. ** P < 0.01 (two-tailed Student’s t -test). (E) KEGG analysis of key pathways highly enriched in differentially expressed genes. The red arrows indicate that genes involved in the immune response, as well as human diseases-infectious diseases viral/bacteria pathways, were highly enriched. (F) Histogram showing the number of differentially expressed genes in each KEGG pathway classification. The red arrows indicate immune system and infectious diseases (viral), and the green arrows indicate the endocrine system. (G) Gene set enrichment analysis (GSEA) using the Reactome database showed that the ATR and HDR pathways were negatively regulated in the NAF group compared with the NAC group. Aβ: Amyloid-β; DMSO: dimethyl sulfoxide; FPKM: fragments per kilobase of transcript per million fragments mapped; FUBP3: far upstream binding protein 3; GSDMD: gasdermin D; IL1b: interleukin-1b; KEGG: Kyoto Encyclopedia of Genes and Genomes; NAC: Neuro2A expressing control siRNA treated with Aβ; NAF: Neuro2A expressing siFUBP3 siRNA treated with Aβ; NDC: Neuro2A expressing control siRNA treated with DMSO; NDF: Neuro2A expressing siFUBP3 siRNA treated with DMSO; NLRP6: NOD-, LRR- and pyrin domain-containing protein 6; ns: not significant; PCR: polymerase chain reaction; PPP1R1A: protein phosphatase 1 regulatory inhibitor subunit 1A; siRNA: small interfering RNA.

Article Snippet: The slices were then pretreated with 10% normal donkey serum (Solarbio, Beijing, China) for 30 minutes at room temperature, followed by incubation with the appropriate primary antibodies, including: anti-FUBP3 (rabbit polyclonal, 1:500, PAG312Mu01, RRID: AB_3083686, Cloud-clone), anti-NLRP3 (mouse monoclonal, 1:500, AG-20B-0014-C100, RRID: AB_2885199, AdipoGen, San Diego, CA, USA), anti-Iba1 (rabbit polyclonal, 1:500, 019-19741, RRID: AB_839504, Wako, Shanghai, China), and anti-NeuN (rabbit monoclonal, 1:500, SAB5700017, RRID: AB_3083690, Millipore or mouse polyclonal, 1:250, 94403, RRID: AB_2904530, Cell Signaling).

Techniques: Expressing, Control, Inhibition, RNA Sequencing Assay, Mutagenesis, Transgenic Assay, Western Blot, Two Tailed Test, Bacteria, Binding Assay, Polymerase Chain Reaction, Small Interfering RNA

Journal: PLoS ONE

Article Title: M30 Antagonizes Indoleamine 2,3-Dioxygenase Activation and Neurodegeneration Induced by Corticosterone in the Hippocampus

doi: 10.1371/journal.pone.0166966

Figure Lengend Snippet: Levels of protein expression of (A) Bcl-2, (B) Cleaved Caspase 3 and (C) Cleaved PARP-1 in the hippocampus of the control, CORT-treated (CORT), M30-treated (M30), CORT and M30 co-treated (CORT+M30) or vehicle groups are summarized. β-actin was an internal control. Data from each group were expressed as mean ± SEM (n = 8). Statistical comparisons between groups were performed using the One way Anova followed by Tukey post hoc test to detect differences in all groups. For Bcl-2, *p < 0.01 when compared with Control, # p < 0.01 when compared with M30, $ p < 0.01 when compared with CORT + M30 groups, ! p < 0.01 when compared with Vehicle. For cleaved caspase 3 and cleaved PARP-1, *p < 0.001 when compared with Control, # p < 0.001 when compared with M30, $ p < 0.001 when compared with CORT + M30 groups, ! p < 0.001 when compared with Vehicle.

Article Snippet: Primary antibodies of SOD-2 (rabbit polyclonal, 1:1000), GPx-1 (goat polyclonal, 1:500), NFκB p65 (rabbit polyclonal, 1:250) and p50 (mouse monoclonal, 1:250), IκBα (mouse monoclonal, 1:500), TNF α (goat polyclonal, 1:80), IL-1β (rabbit polyclonal 1:100), IL-6 (goat polyclonal, 1:1000) and COX-2 (goat polyclonal, 1:100) were purchased from Santa Cruz Biotechnology, CA, USA; Synapsin 1 (rabbit polyclonal, 1:500) and Synaptophysin (rabbit polyclonal, 1:2000) were purchased from Novus Biologicals, USA; PSD95 (rabbit polyclonal 1:500), Cleaved Caspase 3 (rabbit polyclonal 1:500) was purchased from Cell Signaling Technology; Cleaved PARP-1 (rabbit polyclonal, 1:2000) was purchased from Bioworld Technology; IDO-1 (rabbit polyclonal, 1:250) was purchased from antibodies-online (ABIN1714836).

Techniques: Expressing