Journal: Molecular Cancer

Article Title: Integrated single-cell and spatial transcriptomic profiling decodes lineage plasticity and immune microenvironment remodeling in prostate cancer progression

doi: 10.1186/s12943-026-02617-6

Figure Lengend Snippet: Functional characterization of FOSL1 as a key regulator in double-negative prostate cancer subtype. A Transcription factor activity in malignant epithelial subtypes. Heatmap displaying the top transcription factors with the highest inferred activity (based on SCENIC analysis) in each of the four malignant epithelial subtypes. Color scale (blue to red) represents low to high regulon activity. B Spatial activity of FOSL1 across subtypes. UMAP of malignant epithelial cells colored by the SCENIC-derived regulon activity score of FOSL1, showing its specific enrichment in Subtype 4. Gradient from white to blue indicates low to high activity. C Effect of FOSL1 knockdown on cell proliferation (CCK-8 assay). Growth curves of PC-3 cells transfected with control siRNA or three independent siRNAs targeting FOSL1. Data are presented as mean ± SD; *** P < 0.001, ** P < 0.01 (two-way ANOVA). D Effect of FOSL1 knockdown on clonogenic survival. Representative images of colony formation assays for PC-3 cells treated as in (C). E Effect of FOSL1 knockdown on apoptosis. (Left) Representative flow cytometry plots of Annexin V/PI staining. Quadrants: viable cells (Annexin V⁻/PI⁻), early apoptotic (Annexin V⁺/PI⁻), late apoptotic (Annexin V⁺/PI⁺), and necrotic (Annexin V⁻/PI⁺). (Right) Quantification of total apoptotic cells (early + late apoptosis). Data are mean ± SD; *** P < 0.001, ** P < 0.01 (one-way ANOVA). F Identification of FOSL1-regulated DNPC genes. Venn diagram showing the overlap between predicted FOSL1 target genes (from SCENIC) and the DNPC gene signature, yielding 8 candidate genes. G FOSL1 binding to candidate gene promoters (ChIP-qPCR). ChIP-qPCR analysis showing enrichment of FOSL1 at the promoters of the 8 candidate genes in control vs. FOSL1-knockdown PC-3 cells. Results are presented as % input. Data are mean ± SD; *** P < 0.001, ** P < 0.01, ns = not significant (two-tailed Student’s t-test). H Expression of candidate genes upon FOSL1 knockdown (qRT-PCR). mRNA expression levels of four selected candidate genes in PC-3 cells after FOSL1 knockdown. Data are normalized to control and presented as mean ± SD; *** P < 0.001, ** P < 0.01, ns = not significant (two-tailed Student’s t-test). I Correlation between FOSL1 and HMGA1 expression. Scatter plot showing a significant positive correlation between FOSL1 and HMGA1 expression across all malignant epithelial cells (n = 152,872). The red line indicates the linear regression fit (R² = 0.212, P < 0.0001, Pearson correlation). J Spatial co-expression of FOSL1 and HMGA1. (Left three panels) Spatial mapping of FOSL1 and HMGA1 expression and their co-localization in a representative section (Patient 01). Red spots indicate high co-expression, yellow indicates high FOSL1 alone, green indicates high HMGA1 alone, and gray indicates low expression of both. (Right panel) Correlation analysis of FOSL1 and HMGA1 expression specifically within the co-expressing spots (red spots, left panel), showing a strong positive correlation (R = 0.923, P = 1.28e-10, Pearson correlation). K - M Western blot analysis of downstream pathways. Protein levels of (K) stemness markers (CD44, OCT4, SOX2, NANOG), (L) EMT markers (ZO-1, N-cadherin, E-cadherin, Vimentin, SNAIL), and (M) DNPC markers (DSG3, KRT6A, KRT5), along with FOSL1 and HMGA1, in PC-3 cells under four conditions: Control, FOSL1 knockdown (siFOSL1), HMGA1 overexpression (oeHMGA1), and FOSL1 knockdown combined with HMGA1 rescue (siFOSL1 + oeHMGA1). GAPDH served as a loading control. N Rescue of proliferation by HMGA1 overexpression. CCK-8 proliferation assay of PC-3 cells under the four conditions described in (K-M). Data are mean ± SD; *** P < 0.001, ** P < 0.01, ns = not significant (two-way ANOVA). O Rescue of apoptosis by HMGA1 overexpression. Quantification of total apoptosis (Annexin V⁺ cells) by flow cytometry under the four conditions described in (K-M). Data are mean ± SD; *** P < 0.001, ** P < 0.01, ns = not significant (one-way ANOVA). P Pharmacological inhibition of FOSL1 reduces HMGA1. Western blot showing dose-dependent decrease of FOSL1 and its downstream target HMGA1 in PC-3 cells treated with increasing concentrations (0, 0.2, 2, 5, 10 µM) of the FOSL1 degrader T-5224. Q In vivo combination therapy schematic. Workflow of the xenograft study. Nude mice bearing PC-3 subcutaneous tumors were treated starting at day 13 post-inoculation with vehicle, Docetaxel (1 mg/kg), FOSL1 degrader T-5224 (10 mg/kg), or the combination via intraperitoneal injection every 48 h. Treatment continued until day 28. R Representative images of resected tumors. Photographs of excised tumors from each treatment group at the study endpoint. S Tumor growth curves. Tumor volume (mm³) was measured over time for each treatment group. Data are presented as mean ± SEM; *** P < 0.001 (two-way ANOVA). T Tumor weight at endpoint. Final tumor weights (g) for each group. Data are mean ± SEM; *** P < 0.001, ** P < 0.01 (one-way ANOVA). U Assessment of tumor cell proliferation (IHC). Representative immunohistochemistry images showing Ki67 expression in tumor sections from each treatment group. Scale bar, 100 μm

Article Snippet: HMGA1 overexpression plasmid (Cat: HG11613-NF), and the empty vector plasmid pCMV3-N-FLAG were purchased from SinoBiological.

Techniques: Functional Assay, Activity Assay, Derivative Assay, Knockdown, CCK-8 Assay, Transfection, Control, Flow Cytometry, Staining, Binding Assay, ChIP-qPCR, Two Tailed Test, Expressing, Quantitative RT-PCR, Western Blot, Over Expression, Proliferation Assay, Inhibition, In Vivo, Injection, Immunohistochemistry

Journal: Science Advances

Article Title: NOTCH1-driven UBR7 stimulates nucleotide biosynthesis to promote T cell acute lymphoblastic leukemia

doi: 10.1126/sciadv.abc9781

Figure Lengend Snippet: ( A and B ) Co-IP from the whole-cell extracts of HEK293T parental or stably expressing UBR7-FLAG showing the interaction of UBR7-FLAG with endogenous PRPS1 (A) or PRPS2 (B). ( C and D ) FLAG-PRPS1 (C) or FLAG-PRPS2 (D) interaction with endogenous UBR7 in HEK293T cells transfected with either empty vector, FLAG-PRPS1, or FLAG-PRPS2. ( E and F ) Co-IP showing the interaction of endogenous UBR7 with PRPS1 (E) or PRPS2 (F) in HEK293T cells. ( G ) Schematic representing N-terminally FLAG-tagged UBR7 full length and various truncation mutants. ( H to J ) A Co-IP assay from the whole-cell extracts of HEK293T cells transiently transfected with FLAG-UBR7 wild-type, various truncation (H and I), or point mutants (J), with endogenous PRPS1 and PRPS2. The numbers below the IP blots in (J) represent the band intensities of PRPS relative to FLAG-UBR7.

Article Snippet: FLAG-PRPS1(HG17214-NF) and PRPS2(HG15128-NF) were purchased from Sino Biological.

Techniques: Co-Immunoprecipitation Assay, Stable Transfection, Expressing, Transfection, Plasmid Preparation

Journal: Science Advances

Article Title: NOTCH1-driven UBR7 stimulates nucleotide biosynthesis to promote T cell acute lymphoblastic leukemia

doi: 10.1126/sciadv.abc9781

Figure Lengend Snippet: ( A and B ) Immunoblot showing the PRPS1 (A) and PRPS2 (B) levels in HEK293T cells transduced with control (Ctrl.) or UBR7 shRNA. ( C ) UBR7, PRPS1, and PRPS2 mRNA fold change in UBR7 knockdown cells normalized to control as measured from qPCR. ( D and E ) PRPS1 and PRPS2 levels in doxycycline-inducible UBR7 shRNA stable HEK293T cells treated with doxycycline (1 μg/ml) or DMSO for 48 hours. ( F ) UBR7, PRPS1, and PRPS2 mRNA fold change in doxycycline-inducible UBR7 knockdown cells normalized to control as measured from qPCR. ( G and H ) Co-IP demonstrating the interaction of FLAG-tagged PRPS1 (G) or UBR7 (H) with endogenous PRPSAP1 in HEK293T cells 1 and 2, represented by arrows, indicate two isoforms of PRPSAP1. ( I ) PRPS1 and PRPSAP1 levels in control or UBR7 shRNA HEK293T cells. ( J ) Ubiquitination of transiently transfected vector or FLAG-PRPSAP1 in control or UBR7 shRNA–transduced HEK293T cells treated with 10 μM MG132 for 6 hours before anti-FLAG IP. ( K ) PRPS1 levels in control or PRPSAP1 shRNA transduced HEK293T cells. Data points in (C) and (F) represent independent biological replicates. Each biological replicate is the mean of at least three technical replicates. Error bars are means ± SD, and P values were computed from Student’s t test and shown with respect to control shRNA ( n.s. P > 0.05, * P ≤ 0.05, ** P ≤ 0.01). Numbers below the immunoblots represent the relative band intensities.

Article Snippet: FLAG-PRPS1(HG17214-NF) and PRPS2(HG15128-NF) were purchased from Sino Biological.

Techniques: Western Blot, Transduction, shRNA, Co-Immunoprecipitation Assay, Transfection, Plasmid Preparation

Journal: Science Advances

Article Title: NOTCH1-driven UBR7 stimulates nucleotide biosynthesis to promote T cell acute lymphoblastic leukemia

doi: 10.1126/sciadv.abc9781

Figure Lengend Snippet: ( A ) UBR7 mRNA expression from CCLE dataset. ( B ) Tracks showing NOTCH1 ChIP-seq signal enrichment at the UBR7 locus in CUTLL1 cells treated with GSI (+) or GSI washed off (−). These data are accessible through GSE51800 . ( C ) Immunoblot showing UBR7, PRPS1, and PRPS2 levels in two NICD1-negative cell lines, T-ALL1 and Loucy, and various NICD1-positive cell lines. The band intensities (arbitrary unit) of UBR7, PRPS1, and PRPS2 normalized to β-actin and correlation curve with 95% confidence interval and Pearson correlation value ( R ) are shown below immunoblot. ( D to G ) UBR7, PRPS1, and PRPS2 levels in DND41 or ALL-SIL cells treated with increasing doses of GSI, DAPT, for 24 hours. The table below immunoblots shows the relative levels of UBR7, PRPS1, and PRPS2 normalized to β-actin.

Article Snippet: FLAG-PRPS1(HG17214-NF) and PRPS2(HG15128-NF) were purchased from Sino Biological.

Techniques: Expressing, ChIP-sequencing, Western Blot

Journal: Science Advances

Article Title: NOTCH1-driven UBR7 stimulates nucleotide biosynthesis to promote T cell acute lymphoblastic leukemia

doi: 10.1126/sciadv.abc9781

Figure Lengend Snippet: ( A and B ) Co-IP demonstrating the interaction of endogenous UBR7 with PRPS1 or PRPS2 in CUTLL1 (A) and Jurkat (B) cells. ( C and D ) Immunoblot showing the PRPS1 and PRPS2 levels in CUTLL1 (C) and Jurkat (D) cells transduced with control or UBR7 shRNA. ( E and F ) Fold change in UBR7, PRPS1, and PRPS2 mRNA in UBR7 shRNA CUTLL1 (E) or Jurkat (F) normalized to their control counterparts as measured from qPCR. ( G ) Schematic representing the involvement of PRPP in nucleotide synthesis by de novo and salvage pathway. 14 C-glycine and 3 H-hypoxanthine are highlighted as they are used in (J) and (K) to show their involvement in de novo and salvage purine synthesis pathway. Dashed lines represent the multiple steps. PPP, pentose phosphate pathway. ( H and I ) PRPP levels per unit total protein in control or UBR7 shRNA–treated CUTLL1 (H) and Jurkat (I) cells as measured from LC-MS. ( J and K ) De novo and salvage purine synthesis measured from 14 C-glycine or 3 H-hypoxanthine incorporation, respectively, in total RNA in CUTLL1 (J) and Jurkat (K) vector or UBR7-FLAG–expressing cells transduced with control or UBR7 shRNA. Data points represent independent biological replicate. Error bars are means ± SD, and P values were computed from Student’s t test and shown only with respect to control or vector + control shRNA as indicated ( n.s. P > 0.05, * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, and **** P ≤ 0.0001).

Article Snippet: FLAG-PRPS1(HG17214-NF) and PRPS2(HG15128-NF) were purchased from Sino Biological.

Techniques: Co-Immunoprecipitation Assay, Western Blot, Transduction, shRNA, Liquid Chromatography with Mass Spectroscopy, Plasmid Preparation, Expressing

Journal: Science Advances

Article Title: NOTCH1-driven UBR7 stimulates nucleotide biosynthesis to promote T cell acute lymphoblastic leukemia

doi: 10.1126/sciadv.abc9781

Figure Lengend Snippet: List of primers used in the study.

Article Snippet: FLAG-PRPS1(HG17214-NF) and PRPS2(HG15128-NF) were purchased from Sino Biological.

Techniques:

Journal: Nature neuroscience

Article Title: Activated microglia cause metabolic disruptions in developmental cortical interneuron that persist in schizophrenia-patient-derived interneurons

doi: 10.1038/s41593-020-00724-1

Figure Lengend Snippet: A. Scheme for coculturing developmental cINs with activated microglia. Microglial cells were activated in the tissue culture insert and co-cultured with cINs via insert without direct contact. B. qPCR analysis of inflammatory gene expression before and after activating microglial cells. Data were normalized by GAPDH expression and are presented as mean±SEM. Two-tailed paired t-test was used for analysis (n=4 batches of independent activation and harvest, IL-1β : t=7.770, df=3, IL-6 : t=9.542, df=3, iNOS : t=11.07, df=3, TNF α : t=12.05, df=3). C. Various phenotype marker expression in cINs with or without co-culture with activated microglia, analyzed by RNA-seq. Gene expression is shown as RPKM (Reads Per Kilobase of transcript), obtained from STAR-featureCount. Differentially expressed genes were analyzed by Kallisto-Sleuth (Wald test for two-sided significance testing, n=24 batches from 4 HC lines and 4 SCZ lines, each line with 3 independent differentiations). Error bars are SEM. D. Inflammatory response gene expression in cINs with or without activated microglia co-culture, analyzed by RNA-seq. Gene expression is shown as RPKM, obtained from STAR-featureCount. Differentially expressed genes were analyzed by Kallisto-Sleuth (Wald test for two-sided significance testing, n=24 batches from 4 HC lines and 4 SCZ lines, each line with 3 independent differentiations). Error bars are SEM. E. Pathway analysis of enriched genes with differential expression using DAVID ( https://david.ncifcrf.gov/summary.jsp ). Significant enrichment was tested by the Fisher’s Exact Test. FDR: false discovery rate with Benjamini-Hochberg multiple testing. F. RNA-seq analysis of cINs with or without activated microglia co-culture. Gene expression is shown as RPKM, obtained from STAR-featureCount. Differentially expressed genes were analyzed by Kallisto-Sleuth (Wald test for two-sided significance testing, n=24 batches from 4 HC lines and 4 SCZ lines, each line with 3 independent differentiations). Error bars are SEM. G. qPCR analysis of CTGF and THBS1 expression in cINs with or without activated microglia co-culture. Data were normalized by GAPDH expression and are presented as mean±SEM. Two-Level Hierarchical Linear Mixed Effect Model after log-transformation was used for analysis (n=24 batches from 4 HC lines and 4 SCZ lines, each line with 3 independent differentiations). H. qPCR analysis of replication cohort cINs with or without activated microglia co-culture. Data were normalized by GAPDH expression and are presented as mean±SEM. Two-Level Hierarchical Linear Mixed Effect Model after log-transformation was used for analysis (n=36 batches from 6 HC lines and 6 SCZ lines, each line with 3 independent differentiations). The lines used in these experiments are summarized in and .

Article Snippet: The next day, cINs were transfected with pMax-GFP (Lonza) as a control vector or CTGF -expressing vector (Cat# HG13612-NF, Sino Biological) and THBS1 -expresing vector (Cat# HG10508-NF, Sino Biological) using 1mg/ml PEI (Polysciences, Inc.).

Techniques: Cell Culture, Expressing, Two Tailed Test, Activation Assay, Marker, Co-Culture Assay, RNA Sequencing Assay, Transformation Assay

Journal: Nature neuroscience

Article Title: Activated microglia cause metabolic disruptions in developmental cortical interneuron that persist in schizophrenia-patient-derived interneurons

doi: 10.1038/s41593-020-00724-1

Figure Lengend Snippet: A. Expression of CTGF in HC cINs and SCZ cINs with or without co-culture with activated microglia, analyzed by qPCR analysis. Paired data from the same line are connected by a solid line. B. Expression of THBS1 in HC cINs and SCZ cINs with or without co-culture with activated microglia, analyzed by qPCR analysis. Paired data from the same line are connected by a solid line. C. Expression of KLF5 in HC cINs and SCZ cINs with or without co-culture with activated microglia in the pilot cohort, analyzed by qPCR analysis. Paired data from the same line are connected by a solid line. D. Expression of KLF5 in HC cINs and SCZ cINs with or without co-culture with activated microglia in the replication cohort, analyzed by qPCR analysis. Paired data from the same line are connected by a solid line.

Article Snippet: The next day, cINs were transfected with pMax-GFP (Lonza) as a control vector or CTGF -expressing vector (Cat# HG13612-NF, Sino Biological) and THBS1 -expresing vector (Cat# HG10508-NF, Sino Biological) using 1mg/ml PEI (Polysciences, Inc.).

Techniques: Expressing, Co-Culture Assay

Journal: Nature neuroscience

Article Title: Activated microglia cause metabolic disruptions in developmental cortical interneuron that persist in schizophrenia-patient-derived interneurons

doi: 10.1038/s41593-020-00724-1

Figure Lengend Snippet: A. Schematic diagram of Seahorse analysis OCR data. Basal Respiration=baseline OCR-Rotenone/antimycin A OCR, ATP production=Baseline OCR-Oligomycin OCR and Maximum Respiration=FCCP OCR-Rotenone/antimycin A OCR. B. Schematic diagram of Seahorse analysis of ECAR data. Glycolytic Reserve= Oligomycin ECAR- Baseline ECAR. C. Overexpression and siRNA knock down of CTGF and THBS1 , probed by qPCR analysis. Data were normalized by GAPDH expression and are presented as mean±SEM. Two-tailed Ratio paired t-test was used for analysis (n=3 batches, O.E. of CTGF : t=19.35, df=2, O.E. of THBS1 : t=47.92, df=2, siRNA of CTGF : t=18.89, df=2, siRNA of THBS1 : t=17.86, df=2). D. Analysis of oxidative phosphorylation (ATP Production) and glycolysis (Baseline Glycolysis and Glycolytic Reserve) of cINs cultured with or without activated microglia-conditioned media using a Seahorse Analyzer. Data are presented as mean±SEM. Two-tailed paired t-test after log transformation was used for analysis of ATP Production and Baseline Glycolysis and Two-tailed paired t-test was used for analysis of Glycolytic Reserve (n=15 lines, ATP Production: t=3.978, df=14, Baseline Glycolysis: t=0.06106, df=14, Glycolytic Reserve: t=1.534, df=14). E. Analysis of oxidative phosphorylation (ATP Production) and glycolysis (Baseline Glycolysis and Glycolytic Reserve) with or without overexpression of CTGF and THBS1 using a Seahorse Analyzer. Data are presented as mean±SEM. Two-tailed paired t-test was used for analysis of ATP Production and Two-tailed paired t-test after log transformation was used for analysis of Baseline Glycolysis and Glycolytic Reserve (n=12 lines, ATP Production: t=4.186, df=11, Baseline Glycolysis: t=0.9984, df=11, Glycolytic Reserve: t=0.9361, df=11). F. Analysis of oxidative phosphorylation (ATP Production) and glycolysis (Baseline Glycolysis and Glycolytic Reserve) with or without siRNA-mediated knockdown of CTGF and THBS1 using a Seahorse Analyzer. Data are presented as mean±SEM. Two-tailed paired t-test was used for analysis of ATP Production and Two-tailed paired t-test after log transformation was used for analysis of Baseline Glycolysis and Glycolytic Reserve (n=12 lines, ATP Production: t=3.164, df=11, Baseline Glycolysis: t=1.293, df=11, Glycolytic Reserve: t=1.300, df=11). G. Analysis of oxidative phosphorylation (Basal Respiration and ATP Production) and glycolysis (Baseline Glycolysis and Glycolytic Reserve) in HC cINs using a Seahorse Analyzer one week after removal of activated microglia-conditioned media. Data are presented as mean±SEM. Two-tailed paired t-test was used for analysis of Basal Respiration, ATP Production and Baseline Glycolysis. Two-tailed paired t-test after log transformation was used for analysis of Glycolytic Reserve (n=8 lines; Each data point is averaged from 1-4 independent differentiations, Basal Respiration: t=8.404, df=2, ATP Production: t=0.7116, df=7, Baseline Glycolysis: t=0.6444, df=7, Glycolytic Reserve: t=1.495, df=7). H. Analysis of oxidative phosphorylation (Basal respiration and ATP production) and glycolysis (Baseline glycolysis and glycolysis reserve) in SCZ cINs using a Seahorse Analyzer one week after removal of activated microglia-conditioned media. Data are presented as mean±SEM. Two-tailed paired t-test was used for analysis (n=7 lines, Each data point is averaged from 1-2 independent differentiations, Basal Respiration: t=5.622, df=6, ATP Production: t=3.750, df=6, Baseline Glycolysis: t=0.2902, df=6, Glycolytic Reserve: t=0.1759, df=6).

Article Snippet: The next day, cINs were transfected with pMax-GFP (Lonza) as a control vector or CTGF -expressing vector (Cat# HG13612-NF, Sino Biological) and THBS1 -expresing vector (Cat# HG10508-NF, Sino Biological) using 1mg/ml PEI (Polysciences, Inc.).

Techniques: Over Expression, Expressing, Two Tailed Test, Cell Culture, Transformation Assay

Journal: Nature neuroscience

Article Title: Activated microglia cause metabolic disruptions in developmental cortical interneuron that persist in schizophrenia-patient-derived interneurons

doi: 10.1038/s41593-020-00724-1

Figure Lengend Snippet: A. Analysis of mitochondrial function using a Seahorse Analyzer, showing significant decreases in mitochondrial function in cINs cultured in activated microglia-conditioned media. Data are presented as mean±SEM. Two-tailed paired t-test after log transformation was used for analysis of Basal Respiration and Two-tailed paired t-test was used for analysis of Maximum Respiration (n=15 lines, Basal Respiration: t=5.129, df=14, Maximum Respiration: t=5.295, df=14). B. Analysis of mitochondrial function of cINs with overexpression of CTGF and THBS1 using Seahorse Analyzer. Data are presented as mean±SEM. Two-tailed paired t-test was used for analysis (n=12 lines, Basal Respiration: t=5.030, df=11, Maximum Respiration: t=3.076, df=11). C. Analysis of mitochondrial function of cINs cultured in activated microglia-conditioned media with CTGF and THBS1 knock down using Seahorse Analyzer. Data are presented as mean±SEM. Two-tailed paired t-test after log transformation was used for analysis (n=12 lines, Basal Respiration: t=2.721, df=11, Maximum Respiration: t=2.612, df=11). D. Scheme of arborization analysis of cINs infected with a limiting titer of GFP-expressing lentivirus cultured with or without activated microglia-conditioned media. E. Representative images of arborization analysis of cINs cultured without activated microglia-conditioned media, with activated microglia-conditioned media or with activated microglia-conditioned media + ALA/ALC treatment. Scale bar= 50 μm. Analysis was repeated at least three times with comparable results. F. Arborization analysis of cINs without activated microglia-conditioned media, with activated microglia-conditioned media or with activated microglia-conditioned media + ALA/ALC treatment. Images were analyzed using ImageJ with the Neuron J plugin. Center and error bars show mean ± SEM. Data were collected from 4 HC lines and 4 SCZ lines, each line with 10 neurons (n=80 neurons). Two-Level Hierarchical Linear Mixed Effect Model after log-transformation was used for analysis of neurite length and Two-Level Hierarchical Mixed Effect Log-Linear Model was used for analysis of branch number, followed by Dunnett’s test as a post hoc analysis. (n=80 neurons from 4 HC lines and 4 SCZ lines, each line with 10 neurons). G. Scheme of cIN organoid synapse analysis with or without activated microglia-conditioned media. H. Analyses of inhibitory synapses in cIN organoids with or without treatment with activated microglia-conditioned media using Imaris software. The data are presented as the number of inhibitory synapses (juxtaposed GFP + VGAT + puncta and Gephyrin + puncta) per 100 μm 2 of GAD1 + cINs. Scale bar= 5 μm. Data are presented as mean ±SEM (n = 30 116 μm x 116 μm images per group). Two-Level Hierarchical Linear Mixed Effect Model after log-transformation was used for analysis. The lines used in these experiments are summarized in .

Article Snippet: The next day, cINs were transfected with pMax-GFP (Lonza) as a control vector or CTGF -expressing vector (Cat# HG13612-NF, Sino Biological) and THBS1 -expresing vector (Cat# HG10508-NF, Sino Biological) using 1mg/ml PEI (Polysciences, Inc.).

Techniques: Cell Culture, Two Tailed Test, Transformation Assay, Over Expression, Infection, Expressing, Software

Journal: Nature neuroscience

Article Title: Activated microglia cause metabolic disruptions in developmental cortical interneuron that persist in schizophrenia-patient-derived interneurons

doi: 10.1038/s41593-020-00724-1

Figure Lengend Snippet: A. qPCR analysis of CTGF and THBS1 expression in HC cINs vs. SCZ cINs with or without activated microglia co-culture. Data were normalized by GAPDH expression and are presented as mean±SEM. Two-tailed Unpaired t-test after log transformation was used for analysis of CTGF and THBS1 for control conditions. Two-tailed Unpaired t-test was used for analysis of CTGF and THBS1 for those with activated microglia co-culture (n=10 lines for HC and n=10 lines for SCZ, Each data point is averaged from 3 independent differentiations, CTGF of Control: t=0.7972, df=18, THBS1 of Control: t=0.7871, df=18, CTGF of Act.MG: t=0.3137, df=8, THBS1 of Act.MG: t=0.1880, df=18). B. Analysis of oxidative phosphorylation in HC cINs vs. SCZ cINs cultured with or without activated microglia-conditioned media. Data are presented as mean±SEM. Two-tailed Unpaired t-test was used for analysis of Basal Respiration and Maximum Respiration for control conditions. Two-tailed Unpaired t-test after log transformation was used for analysis of Basal Respiration for those cultured in activated microglia-conditioned media. Two-tailed Unpaired t-test was used for analysis of Maximum Respiration for those cultured in activated microglia-conditioned media (n=7 lines for HC and n=7 lines for SCZ, Basal Respiration of Control: t=2.428, df=12, Maximum Respiration of Control: t=2.517, df=12, Basal Respiration of Act.MG: t=5.424, df=12, Maximum Respiration of Act.MG: t=4.262, df=12). C. qPCR analysis of KLF5 expression in HC cINs vs. SCZ cINs with or without co-culture with activated microglia one week after the removal of activated microglia co-culture. Data were normalized by GAPDH expression and are presented as mean±SEM. Two-tailed Unpaired t-test was used for analysis of KLF5 qPCR for control conditions and Two-tailed Unpaired t-test after log transformation was used for analysis of KLF5 qPCR for those with activated microglia co-culture (n=10 lines for HC and n=10 lines for SCZ; Each data point is averaged from 3 independent differentiations, Control: t=0.1021, df=18, Act.MG: t=1.435, df=18). D. Analysis of oxidative phosphorylation in HC cINs vs. SCZ cINs cultured with or without activated microglia-conditioned media one week after removal of activated microglia-conditioned media. Data are presented as mean±SEM. Two-tailed Unpaired t-test after log transformation was used for analysis of Maximum Respiration for control conditions and Two-tailed Unpaired t-test was used for analysis of Maximum Respiration for those cultured in activated microglia-conditioned media (n=7 lines for HC and n=7 lines for SCZ; Each data point is averaged from 1-4 independent differentiations, Maximum Respiration of Control: t=4.646, df=12, Maximum Respiration of Act.MG: t=3.256, df=12). E. Arborization analysis of cINs infected with a limiting titer of GFP-expressing lentivirus and cultured without activated microglia-conditioned media, with activated microglia-conditioned media or with activated microglia-conditioned media + ALA/ALC treatment. Data are presented as mean ± SEM. One-way ANOVA, followed by posthoc analysis using Dunnett’s multiple comparisons test was used for analysis (n=8 lines consisting of 4 HC lines and 4 SCZ lines, Neurite Length: f=6.116, df=2, Branch Number: f=6.465, df=2).

Article Snippet: The next day, cINs were transfected with pMax-GFP (Lonza) as a control vector or CTGF -expressing vector (Cat# HG13612-NF, Sino Biological) and THBS1 -expresing vector (Cat# HG10508-NF, Sino Biological) using 1mg/ml PEI (Polysciences, Inc.).

Techniques: Expressing, Co-Culture Assay, Two Tailed Test, Transformation Assay, Cell Culture, Infection

Journal: Nature neuroscience

Article Title: Activated microglia cause metabolic disruptions in developmental cortical interneuron that persist in schizophrenia-patient-derived interneurons

doi: 10.1038/s41593-020-00724-1

Figure Lengend Snippet: A. Venn diagram of acute phase DE genes with or without activated microglia co-culture in HC cINs and SCZ cINs. B. Pathway analysis of acute phase DE genes with or without activated microglia co-culture in HC cINs and SCZ cINs using DAVID ( https://david.ncifcrf.gov/summary.jsp ). Significant enrichment was tested by the Fisher’s Exact Test. C. qPCR analysis of CTGF and THBS1 expression with or without activated microglia coculture in HC cINs or SCZ cINs. Data were normalized by GAPDH expression and are presented as mean±SEM. Two-Level Hierarchical Linear Mixed Effect Model after log-transformation was used for analysis (n=30 batches from 10 lines, each line with 3 independent differentiations). D. Analysis of oxidative phosphorylation using a Seahorse Analyzer, showing significant decreases in mitochondrial function in cINs cultured in activated microglia-conditioned media both in HC cINs and SCZ cINs. Data are presented as mean±SEM. Two-tailed paired t-test was used for analysis (n=8 lines for HC and n=7 lines for SCZ, Basal Respiration of HC: t=3.248, df=7, Maximum Respiration of HC: t=6.266, df=7, Basal Respiration of SCZ: t=2.884, df=6, Maximum Respiration of SCZ: t=2.744, df=6). E. Arborization analysis of HC cINs or SCZ cINs infected with a limiting titer of GFP-expressing lentivirus and cultured with or without activated microglia-conditioned media. Images were analyzed using ImageJ with the Neuron J plugin. Center and error bars show mean ± SEM. Two-Level Hierarchical Linear Mixed Effect Model after log-transformation was used for analysis of Neurite Length and Two-Level Hierarchical Mixed Effect Log-Linear Model was used for analysis of Branch Number (n=40 neurons for 4 HC lines and n=40 neurons for 4 SCZ lines, each line with 10 neurons). The lines used in these experiments are summarized in .

Article Snippet: The next day, cINs were transfected with pMax-GFP (Lonza) as a control vector or CTGF -expressing vector (Cat# HG13612-NF, Sino Biological) and THBS1 -expresing vector (Cat# HG10508-NF, Sino Biological) using 1mg/ml PEI (Polysciences, Inc.).

Techniques: Co-Culture Assay, Expressing, Transformation Assay, Cell Culture, Two Tailed Test, Infection

Journal: Nature neuroscience

Article Title: Activated microglia cause metabolic disruptions in developmental cortical interneuron that persist in schizophrenia-patient-derived interneurons

doi: 10.1038/s41593-020-00724-1

Figure Lengend Snippet: A. qPCR analysis of inflammatory gene expression in cINs with and without culturing in activated microglia HMC3-conditioned media. Data were normalized by GAPDH expression and are presented as mean±SEM. Two-tailed paired t-test was used for analysis of NF-κBIZ and TNFRSF12A and Two-tailed paired t-test after log transformation was used for analysis of TNFAIP3 (n=7 lines, Each data point is averaged from 3 independent differentiations, NF-κBIZ : t=4.406, df=6, TNFRSF12A : t=3.351, df=6, TNFAIP3 : t=7.189, df=6). B. qPCR analysis of CTGF and THBS1 expression in HC cINs or SCZ cINs cultured with or without activated microglia HMC3-conditioned media. Data were normalized by GAPDH expression and are presented as mean±SEM. Two-tailed Ratio paired t-test was used for analysis (n=5 lines for HC and n=5 lines for SCZ; Each data point is averaged from 3 independent differentiations, CTGF of HC: t=4.018, df=4, THBS1 of HC: t=3.027, df=4, CTGF of SCZ: t=4.131, df=4, THBS1 of SCZ: t=3.638, df=4). C. qPCR analysis of KLF5 expression in HC cINs or SCZ cINs cultured with or without activated microglia HMC3-conditioned media one week after the removal of activated microglia conditioned media. Data were normalized by GAPDH expression and are presented as mean±SEM. Two-tailed paired t-test after log transformation was used for analysis of HC KLF5 qPCR and Two-tailed paired t-test was used for analysis of SCZ KLF5 qPCR (n=4 lines from HC and n=4 lines from SCZ; Each data point is averaged from 3 independent differentiations, HC: t=0.6992, df=3, SCZ: t=4.364, df=3).

Article Snippet: The next day, cINs were transfected with pMax-GFP (Lonza) as a control vector or CTGF -expressing vector (Cat# HG13612-NF, Sino Biological) and THBS1 -expresing vector (Cat# HG10508-NF, Sino Biological) using 1mg/ml PEI (Polysciences, Inc.).

Techniques: Expressing, Two Tailed Test, Transformation Assay, Cell Culture

Journal: Nature neuroscience

Article Title: Activated microglia cause metabolic disruptions in developmental cortical interneuron that persist in schizophrenia-patient-derived interneurons

doi: 10.1038/s41593-020-00724-1

Figure Lengend Snippet: A. Scheme for generating developmental glutamatergic neurons. B. Immunocytochemistry analysis of differentiated glutamatergic neurons from HC vs SCZ iPSCs. Scale bar= 25 μm. Analysis was repeated at least three times with comparable results. C. qPCR analysis of CTGF and THBS1 expression with or without activated HMC3 microglia-conditioned media in HC glutamatergic neurons or SCZ glutamatergic neurons. Data were normalized by GAPDH expression and are presented as mean±SEM. Two-Level Hierarchical Linear Mixed Effect Model after log-transformation was used for analysis of HC CTGF and THBS1 and Two-Level Hierarchical Linear Mixed Effect Model was used for analysis of SCZ CTGF and THBS1 (n=4 batches from 2 lines, each line with 2 independent differentiations). D. Analysis of oxidative phosphorylation using a Seahorse Analyzer, showing no changes in mitochondrial function either in HC glutamatergic neurons or SCZ glutamatergic neurons cultured in activated HMC3 microglia-conditioned media. Data are presented as mean±SEM. Two-Level Hierarchical Linear Mixed Effect Model after log transformation was used for analysis of HC Basal Respiration and Two-Level Hierarchical Linear Mixed Effect Model was used for analysis of HC Maximum Respiration, SCZ Basal Respiration and SCZ Maximum Respiration (n=4 batches from 2 lines, each line with 2 independent differentiations). E. Analysis of oxidative phosphorylation in iPSCs using a Seahorse Analyzer. Data are presented as mean±SEM. Two-tailed paired t-test was used for analysis (n=3 batches from 1 line for HC and n=3 batches from 1 line for SCZ, Basal Respiration of HC: t=8.404, df=2, Maximum Respiration of HC: t=28.98, df=2, Basal Respiration of SCZ: t=3.960, df=2, Maximum Respiration of SCZ: t=4.190, df=2). The lines used in these experiments are summarized in .

Article Snippet: The next day, cINs were transfected with pMax-GFP (Lonza) as a control vector or CTGF -expressing vector (Cat# HG13612-NF, Sino Biological) and THBS1 -expresing vector (Cat# HG10508-NF, Sino Biological) using 1mg/ml PEI (Polysciences, Inc.).

Techniques: Immunocytochemistry, Expressing, Transformation Assay, Cell Culture, Two Tailed Test

Journal: Nature Communications

Article Title: Hypothalamic astrocyte NAD + salvage pathway mediates the coupling of dietary fat overconsumption in a mouse model of obesity

doi: 10.1038/s41467-024-46009-0

Figure Lengend Snippet: a, b Effects of CD38 blockade via 78c treatment (100 nM, 48 h) ( a ) and Cd38 siRNA (siCd38) ( b) on Nampt O/E-induced proinflammatory cytokine/chemokine expression in primary hypothalamic astrocytes derived from C57 male mice ( n = 3 wells). c Effects of palmitate treatment with or without cotreatment of 78c and Nampt O/E on CD38 activity in primary hypothalamic astrocytes ( n = 3 wells). d CD38 enzyme activity in hypothalamic astrocytes isolated from the MBH of 16-week-old C57 male mice fed a CD or HFD for 4 weeks or from the MBH of 16-week-old Nampt-AKO and Nampt-WT males on a 4-week HFD ( n = 3 mice). e Effect of 4 week-intracerebroventricular (i.c.v.) infusion of 78c on the hypothalamic inflammatory gene expression in 16-week-old C57 male mice during HFD feeding ( n = 5). f Effects of 78c-induced hypothalamic CD38 inhibition on reactive astrogliosis and activated microgliosis in the ARH of 16-week-old C57 male mice fed an HFD for 4 weeks ( n = 4 mice). Scale bars: 50 μm. g – i Comparison of weight gain, fat mass, and lean mass between 16-week-old C57 male mice that received i.c.v. infusion of 78c and those that received i.c.v. vehicle infusion (vehicle, n = 8 mice; 78c, n = 7 mice). j , k Effects of hypothalamic CD38 inhibition with 78c infusion on EE and RER in HFD-fed, 14-week-old male mice ( n = 5). l Adipose tissue histology in the BAT, iWAT, and gWAT of mice subjected to i.c.v. infusion of either vehicle or 78c in 16-week-old C57 male mice. Scale bars: 50 μm. m – p Effects of ARH astrocyte-specific CD38 knockdown on the body weight gain, body mass, EE, and RER in 14 ~ 16 weeks-old male hGFAP-Cre ERT2 mice that received bilateral iARH injection of Cd38 shRNA (shCd38)-AAV and were subjected to a HFD ( m , n , n = 7 mice; o , p , n = 4 mice). Two-sided unpaired t-test ( d – f , h , i , k , n , p ), one-way ANOVA ( a – c ) and two-way repeated measures ANOVA ( g , m ) followed by Fisher’s LSD test, and one-way ANCOVA using lean mass as covariate ( j, o ). n.s.: not significant. Two independent replicates were performed for all studies and measurement were taken from distinct samples. Results are presented as the mea n ± SEM. Source data are provided as a Source Data file.

Article Snippet: Plasmids expressing Nampt (obtained from Dr. Shin-ichiro Imai at Washing University) and Cd38 (Sino Biological, MG50191-NY) were transfected into cells at the indicated doses using Lipofectamine 3000 (Invitrogen) when they reached 60% ~ 70% confluency.

Techniques: Expressing, Derivative Assay, Activity Assay, Isolation, Inhibition, Comparison, Injection, shRNA

Journal: Nature Communications

Article Title: Hypothalamic astrocyte NAD + salvage pathway mediates the coupling of dietary fat overconsumption in a mouse model of obesity

doi: 10.1038/s41467-024-46009-0

Figure Lengend Snippet: a , b Alterations in intracellular cADPR and ADPR contents in primary hypothalamic astrocytes treated with palmitate (200 μM, 48 h) or those overexpressing Nampt and Cd38 ( n = 3 wells). c , d Effects of pre- (30 min) or co-treatment of 78c (100 μM) ( c ) or gene knockdown of Nampt or Cd38 ( d ) on palmitate (200 μM)-induced increase in intracellular Ca 2+ (i[Ca 2+ ]) levels in primary hypothalamic astrocytes ( n = 30 cells). i[Ca 2+ ] levels were assessed using a FRET-based Ca 2+ sensor. The Ca 2+ response was quantified as the area under the curve (AUC) of FRET/CFP values after palmitate treatment. e Significant increases in basal i[Ca 2+ ] levels observed in primary hypothalamic astrocytes following Nampt or Cd38 overexpression (O/E) ( n = 30 cells). f – h Decreased Ca 2+ responses to treatment with leptin (1 μg/mL), insulin (0.01 unit/mL), or glucagon-like peptide-1 (GLP-1, 1 μg/mL) in primary hypothalamic astrocytes that were pre-exposed to palmitate (200 μM, 48 h) ( n = 30 cells). The Ca 2+ response was quantified as the AUC of the FRET/CFP values after hormone treatment. One-way ANOVA followed by Fisher’s LSD test ( a , b Nampt, Cd38 O/E, d , e ) and two-sided unpaired t-test ( a , b- palmitate, c , f , g ). Three independent replicates were performed for all studies and measurement were taken from distinct samples. Results are presented as the mea n ± SEM. Source data are provided as a Source Data file.

Article Snippet: Plasmids expressing Nampt (obtained from Dr. Shin-ichiro Imai at Washing University) and Cd38 (Sino Biological, MG50191-NY) were transfected into cells at the indicated doses using Lipofectamine 3000 (Invitrogen) when they reached 60% ~ 70% confluency.

Techniques: Over Expression

Journal: PLoS ONE

Article Title: Spink2 Modulates Apoptotic Susceptibility and Is a Candidate Gene in the Rgcs1 QTL That Affects Retinal Ganglion Cell Death after Optic Nerve Damage

doi: 10.1371/journal.pone.0093564

Figure Lengend Snippet: (A–C) Retinal whole mounts stained for the ganglion cell marker BRN3A (A) and SPINK2 (Santa Cruz goat polyclonal) (B). A majority of BRN3A positive cells are positive for SPINK2 staining. Additionally, nerve fiber bundles emanating from ganglion cells are SPINK2 positive (arrow). SPINK2 also stains BRN3A negative cells (asterisks), which may be displaced amacrine cells or some of the 10–15% of ganglion cells that do not stain for this transcription factor . SPINK2 also stains cells with morphology typical of astrocytes (arrowhead). BV = blood vessel. Size bar = 30 μm. (D,E) Frozen sections of a control (D) and crush (E) mouse retina (DBA/2J.BALB Rgcs1 mouse shown) stained for an antibody against the C-terminus of mouse SPINK2 (ProSci Inc., rabbit polyclonal). This antibody predominantly stains the cells in the ganglion cell layer (GCL), although some light staining is evident in the innermost cells of the inner nuclear layer (INL) and the inner plexiform layer (IPL) and outer plexiform layer (OPL). This antibody may also stain putative Müller cell processes (see ). There is no appreciable change in the staining pattern of SPINK2 before and after crush when viewed on sections. The overall pattern of staining shown here is consistent among 3 different SPINK2 antibodies tested. Size bar = 50 μm. (F–I) Retinal whole mounts from DBA/2J (F,G) and DBA/2J.BALB Rgcs1 (H,I). Ganglion cells positive for BRN3A (yielding pink colored nuclei with the DAPI counterstain – arrowheads) show diffuse SPINK2 staining (green, rabbit polyclonal, see ref 18) in control retinas (F,H). At 7 days after optic nerve crush, some cells in the retinas of both strains exhibit intense SPINK2 immunoreactivity (examples marked by arrows) that surrounded nuclei with condensed and fragmented chromatin (asterisks). At this stage, ganglion cells exhibit only limited staining for BRN3A (see text). Size bar = 5 μm.

Article Snippet: The rabbit polyclonal antibody from ProSci (see Methods) also revealed staining in putative Müller cell processes , while a rabbit polyclonal used in previous studies , exhibited strong immunoreactivity in the nerve fibers of the ganglion cell layer.

Techniques: Staining, Marker, Control