Journal: Signal Transduction and Targeted Therapy

Article Title: Gut dysbiosis conveys psychological stress to activate LRP5/β-catenin pathway promoting cancer stemness

doi: 10.1038/s41392-025-02159-1

Figure Lengend Snippet: Blockage of LRP5-mediated Wnt/β-catenin signaling via butyrate inhibits cancer stemness. a Heatmap showing differentially expressed genes (DEGs) between untreated SK-BR-3 cells and SK-BR-3 cells treated with 4 mM NaBu for 48 hours. The number of significant variant genes (FC > 2) are shown (FC, fold change) ( n = 3 biological replicates). b Venn diagram showing the overlap of downregulated genes from NaBu-treated SK-BR-3 cells (FC > 2, P < 0.05, 1165 genes) and upregulated genes in MDA-MB-231 spheres (FC > 2, P < 0.05, 2571 genes) and the subsequent GO analysis. c Venn diagram showing the overlap of downregulated genes from NaBu-treated SK-BR-3 cells, upregulated genes in MDA-MB-231 spheres and stemness genes. d Expression of mRNA for the indicated genes in SK-BR-3 Ctrl cells and spheres ( n = 3 biological replicates). e Relative LRP5 mRNA expression in SK-BR-3 cells treated with NaBu for 48 h in dose-dependent manner ( n = 3 biological replicates). f Relative mRNA expression of LRP5 , SOX2 , and NANOG in Ctrl and LRP5-forced expression SK-BR-3 cells treated with NaBu (4 mM) for 48 h or vehicle ( n = 3 biological replicates). g ALDH + cells in Ctrl and LRP5-forced expression SK-BR-3 cells treated with NaBu (4 mM) for 48 h or vehicle ( n = 3 biological replicates). h ELDA was performed in Ctrl and LRP5-forced expression SK-BR-3 cells treated with NaBu (4 mM) for 48 h or vehicle, and representative sphere images are shown (Scale bars, 50 μm). Stemness frequency with the upper and lower 95% confidence intervals that indicate the frequency of one stem cell in tumors. Spheres were counted from 24 replicate wells. i Representative images of spheroids formed by single cells in the 4 groups (Scale bars, 100 μm) (up), number of spheres per 1000 cells (d > 50 μm) (middle), and distribution pattern of sphere diameter (down). j Relative LRP5, p-GSK3β, GSK3β and β-catenin protein levels in Ctrl and LRP5-forced expression SK-BR-3 cells treated with NaBu (4 mM) for 48 h or vehicle. k Representative immunofluorescence images of β-catenin protein (green) in the 4 groups, with nuclear staining with DAPI (blue) (Scale bars, 25 μm). l Western blot detects the expression of nuclear and cytoplasmic protein extracts from Ctrl and LRP5 forced expression SK-BR-3 cells treated with NaBu (4 mM) for 48 h or vehicle. Actin was used as a cytoplasmic internal loading control and Lamin B1 as the nuclear internal control. Results are presented as mean ± s.d. ( a, d, e ) by a two-tailed, unpaired Student’s t- test, ( f , g , i ) by one-way ANOVA, h by the likelihood ratio test. For multiple comparisons, p-values were adjusted using the FDR correction. P values are as indicated

Article Snippet: The number of significant variant genes (FC > 2, P < 0.05) are shown. e Relative ZFP36 and pre- ZFP36 mRNA levels of SK-BR-3 and MDA-MB-231 cells treated with NaBu for 48 h in dose-dependent manner ( n = 3 biological replicates). f Relative ZFP36, LRP5, and β-catenin protein levels in SK-BR-3 cells after transfection with the siRNA targeting these three GPRs and treated with NaBu (4 mM). g Relative ZFP36, LRP5, β-catenin, Histone H3-K9, and Histone H3 protein levels in SK-BR-3 cells treated with NaBu (4 mM) and TSA (1 μM; MCE, HY-15144). h Genome browser images showing ChIP-seq signals for the ZFP36 promoter using data from the Cistrome Data Browse (GSM810671). i Relative fold change of the ZFP36 promoter binding motif in SK-BR-3 and MDA-MB-231 cells treated with NaBu (4 mM) for 48 h or vehicle as analyzed by ChIP-qPCR ( n = 3 biological replicates). j Enrichment of endogenous LRP5 3’UTR in SK-BR-3 and MDA-MB-231 cells treated with NaBu (4 mM) for 48 h or vehicle was analyzed by RNA immunoprecipitation (RIP) ( n = 3 biological replicates).

Techniques: Variant Assay, Expressing, Immunofluorescence, Staining, Western Blot, Control, Two Tailed Test

Journal: Signal Transduction and Targeted Therapy

Article Title: Gut dysbiosis conveys psychological stress to activate LRP5/β-catenin pathway promoting cancer stemness

doi: 10.1038/s41392-025-02159-1

Figure Lengend Snippet: Butyrate accelerates LRP5 mRNA decay by transactivating ARE binding protein ZFP36. a Stability of LRP5 mRNA in SK-BR-3 cells treated with NaBu (4 mM) or vehicle ( n = 3 biological replicates). b Stability of LRP5 mRNA in MDA-MB-231 cells treated with 4 mM NaBu or vehicle ( n = 3 biological replicates). c Relative luciferase activity of psiCHECK2-LRP5-3’UTR-WT, Mut1, Mut2, and Mut1 + 2 in HEK293T cells treated with NaBu (4 mM) or vehicle ( n = 3 biological replicates). d Volcano plots displaying DEGs comparing SK-BR-3 cells treated with NaBu (4 mM) or vehicle. The number of significant variant genes (FC > 2, P < 0.05) are shown. e Relative ZFP36 and pre- ZFP36 mRNA levels of SK-BR-3 and MDA-MB-231 cells treated with NaBu for 48 h in dose-dependent manner ( n = 3 biological replicates). f Relative ZFP36, LRP5, and β-catenin protein levels in SK-BR-3 cells after transfection with the siRNA targeting these three GPRs and treated with NaBu (4 mM). g Relative ZFP36, LRP5, β-catenin, Histone H3-K9, and Histone H3 protein levels in SK-BR-3 cells treated with NaBu (4 mM) and TSA (1 μM; MCE, HY-15144). h Genome browser images showing ChIP-seq signals for the ZFP36 promoter using data from the Cistrome Data Browse (GSM810671). i Relative fold change of the ZFP36 promoter binding motif in SK-BR-3 and MDA-MB-231 cells treated with NaBu (4 mM) for 48 h or vehicle as analyzed by ChIP-qPCR ( n = 3 biological replicates). j Enrichment of endogenous LRP5 3’UTR in SK-BR-3 and MDA-MB-231 cells treated with NaBu (4 mM) for 48 h or vehicle was analyzed by RNA immunoprecipitation (RIP) ( n = 3 biological replicates). Relative LRP5 and ZFP36 mRNA expression ( k ) and stability of LRP5 mRNA ( l ) in SK-BR-3 cells treated with knockdown of ZFP36 and NaBu (4 mM), as assessed by RT-qPCR ( n = 3 biological replicates). m Relative luciferase activity of psiCHECK2-LRP5-3’UTR-WT, Mut1, Mut2, and Mut1 + 2 in HEK293T cells treated with ZFP36 knocked down and NaBu (4 mM) ( n = 3 biological replicates). n Relative ZFP36, LRP5, p-GSK3β, GSK3β, and β-catenin protein levels in SK-BR-3 cells treated with ZFP36 knocked down and NaBu (4 mM). o Representative immunofluorescence images of β-catenin protein (green) in SK-BR-3 cells treated with ZFP36 knocked down and NaBu (4 mM). The nucleus was stained with DAPI (blue) (Scale bars, 25 μm). p ALDH + SK-BR-3 cells treated with ZFP36 knocked down and NaBu (4 mM) ( n = 3 biological replicates). Results are presented as mean ± s.d. ( a – e, i , j ) by a two-tailed, unpaired Student’s t- test, ( k , l , m , p ) by one-way ANOVA. For multiple comparisons, p-values were adjusted using the FDR correction. P values are as indicated

Article Snippet: The number of significant variant genes (FC > 2, P < 0.05) are shown. e Relative ZFP36 and pre- ZFP36 mRNA levels of SK-BR-3 and MDA-MB-231 cells treated with NaBu for 48 h in dose-dependent manner ( n = 3 biological replicates). f Relative ZFP36, LRP5, and β-catenin protein levels in SK-BR-3 cells after transfection with the siRNA targeting these three GPRs and treated with NaBu (4 mM). g Relative ZFP36, LRP5, β-catenin, Histone H3-K9, and Histone H3 protein levels in SK-BR-3 cells treated with NaBu (4 mM) and TSA (1 μM; MCE, HY-15144). h Genome browser images showing ChIP-seq signals for the ZFP36 promoter using data from the Cistrome Data Browse (GSM810671). i Relative fold change of the ZFP36 promoter binding motif in SK-BR-3 and MDA-MB-231 cells treated with NaBu (4 mM) for 48 h or vehicle as analyzed by ChIP-qPCR ( n = 3 biological replicates). j Enrichment of endogenous LRP5 3’UTR in SK-BR-3 and MDA-MB-231 cells treated with NaBu (4 mM) for 48 h or vehicle was analyzed by RNA immunoprecipitation (RIP) ( n = 3 biological replicates).

Techniques: Binding Assay, Luciferase, Activity Assay, Variant Assay, Transfection, ChIP-sequencing, ChIP-qPCR, RNA Immunoprecipitation, Expressing, Knockdown, Quantitative RT-PCR, Immunofluorescence, Staining, Two Tailed Test

Journal: Signal Transduction and Targeted Therapy

Article Title: Gut dysbiosis conveys psychological stress to activate LRP5/β-catenin pathway promoting cancer stemness

doi: 10.1038/s41392-025-02159-1

Figure Lengend Snippet: Clinical relevance of A. muciniphila , butyric acid and LRP5/β-catenin expression in breast cancer patients with negative mood. a Alpha diversity of gut microbiota between the HADS low ( n = 24) and high ( n = 33) groups of cohort 1 breast cancer patients, as indicated by Chao1 indices. b PCoA of fecal microbiota from cohort 1 breast cancer patients in the HADS low ( n = 24) and high ( n = 33) groups. c Volcano plots of the species level in the patients’ feces between the HADS low ( n = 24) and high ( n = 33) groups. The number of significant variant genes ( P < 0.05) are shown. Levels of fecal butyric acid ( d ) and serum butyric acid ( e ) in cohort 1 breast cancer patients in the HADS low ( n = 24) and HADS high ( n = 33) groups. f Representative ZFP36, LRP5, β-catenin, and NANOG IHC staining images of tumors in patients in the low and high serum butyric acid groups (Scale bar, 50 μm). Pearson correlation between butyric acid concentrations of feces and ZFP36 ( g ), LRP5 ( h ), β-catenin ( i ) and NANOG ( j ) scores in breast cancer patients ( n = 57). Statistical significance was determined by Pearson’s rank test. Relative ZFP36 ( k ) and LRP5 ( l ) mRNA expression between butyric acid in serum low ( n = 19) and high ( n = 20) groups in cohort 1 breast cancer patients. m Immunoblot analysis of proteins in breast tumor tissues (T) and adjacent normal breast tissues (N) ( n = 5). Kaplan-Meier estimates of overall survival (OS) based on the expression of ZFP36 ( n ), LRP5 ( o ) and CTNNB1 ( p ). Patients were stratified into high-expression and low-expression groups using the median gene expression value as the cutoff in breast cancer patients. Results are presented as mean ± s.d. ( a , c , d , e , k , l ) by a two-tailed, unpaired Student’s t- test, ( b ) by weighted UniFrac ANOSIM analysis, g – j by Pearson’s rank test. For multiple comparisons, p-values were adjusted using the FDR correction. P values are as indicated

Article Snippet: The number of significant variant genes (FC > 2, P < 0.05) are shown. e Relative ZFP36 and pre- ZFP36 mRNA levels of SK-BR-3 and MDA-MB-231 cells treated with NaBu for 48 h in dose-dependent manner ( n = 3 biological replicates). f Relative ZFP36, LRP5, and β-catenin protein levels in SK-BR-3 cells after transfection with the siRNA targeting these three GPRs and treated with NaBu (4 mM). g Relative ZFP36, LRP5, β-catenin, Histone H3-K9, and Histone H3 protein levels in SK-BR-3 cells treated with NaBu (4 mM) and TSA (1 μM; MCE, HY-15144). h Genome browser images showing ChIP-seq signals for the ZFP36 promoter using data from the Cistrome Data Browse (GSM810671). i Relative fold change of the ZFP36 promoter binding motif in SK-BR-3 and MDA-MB-231 cells treated with NaBu (4 mM) for 48 h or vehicle as analyzed by ChIP-qPCR ( n = 3 biological replicates). j Enrichment of endogenous LRP5 3’UTR in SK-BR-3 and MDA-MB-231 cells treated with NaBu (4 mM) for 48 h or vehicle was analyzed by RNA immunoprecipitation (RIP) ( n = 3 biological replicates).

Techniques: Expressing, Variant Assay, Immunohistochemistry, Western Blot, Gene Expression, Two Tailed Test

Journal: Signal Transduction and Targeted Therapy

Article Title: Gut dysbiosis conveys psychological stress to activate LRP5/β-catenin pathway promoting cancer stemness

doi: 10.1038/s41392-025-02159-1

Figure Lengend Snippet: Working model. Psychological stress alters gut microbial community and SCFA profiles during tumor development. Administration of Akkermansia muciniphila , butyrate or high-fiber diet markedly reverses stress-caused cancer stemness and anxiety-like behavior. Butyrate transactivates ZFP36 to accelerate LRP5 mRNA decay, thereby blocking Wnt/β-catenin signaling pathway to attenuate cancer stem-like traits. Part of the elements in this figure generated from BioRender (BioRender.com)

Article Snippet: The number of significant variant genes (FC > 2, P < 0.05) are shown. e Relative ZFP36 and pre- ZFP36 mRNA levels of SK-BR-3 and MDA-MB-231 cells treated with NaBu for 48 h in dose-dependent manner ( n = 3 biological replicates). f Relative ZFP36, LRP5, and β-catenin protein levels in SK-BR-3 cells after transfection with the siRNA targeting these three GPRs and treated with NaBu (4 mM). g Relative ZFP36, LRP5, β-catenin, Histone H3-K9, and Histone H3 protein levels in SK-BR-3 cells treated with NaBu (4 mM) and TSA (1 μM; MCE, HY-15144). h Genome browser images showing ChIP-seq signals for the ZFP36 promoter using data from the Cistrome Data Browse (GSM810671). i Relative fold change of the ZFP36 promoter binding motif in SK-BR-3 and MDA-MB-231 cells treated with NaBu (4 mM) for 48 h or vehicle as analyzed by ChIP-qPCR ( n = 3 biological replicates). j Enrichment of endogenous LRP5 3’UTR in SK-BR-3 and MDA-MB-231 cells treated with NaBu (4 mM) for 48 h or vehicle was analyzed by RNA immunoprecipitation (RIP) ( n = 3 biological replicates).

Techniques: Blocking Assay, Generated

Journal: eLife

Article Title: Fan cells in lateral entorhinal cortex directly influence medial entorhinal cortex through synaptic connections in layer 1

doi: 10.7554/elife.83008

Figure Lengend Snippet: Figure 1. Fan cells in lateral entorhinal cortex send projections to medial entorhinal cortex that terminate in layer 1. (A) Schematic of strategy for targeting AAV-Retro-mCherry to the medial entorhinal cortex (MEC) of wild-type mice (top) and a horizontal brain section (bottom) showing retrograde labelling of neurons with mCherry in layers (L) 2a and 5a of lateral entorhinal cortex (LEC). Scale bar represents 500 µm. (B) Schematic of strategy for targeting AAV-FLEX-GFP to fan cells in Sim1Cre mice (top) and horizontal brain section (bottom) showing labelling of fan cell axons with GFP (green) in the outer molecular layer (oml) of the dentate gyrus (DG) and L1 of the MEC. Neurons are counterstained with Neurotrace (blue). Scale bar represents 500 µm. (C) Schematic of strategy for targeting AAV-FLEX-GFP-2A-Synaptophysin-mRuby to fan cells in Sim1Cre mice (top) and horizontal brain section (bottom) showing fan cell axons (green) and their terminals (red) in L1 and L2 of the MEC. Insets show synaptic puncta, indicated by white arrows. Scale bar represents 50 µm. (D) Line plots showing the volumetric density of axon terminals across L1-5b (left) and the mediolateral axis of the MEC (right). The mediolateral (M-L) axis of MEC was binned in 150 µm steps from the parasubiculum border. Black line shows population averages, and grey circles are values for single brain slices. Error bars represent SEM. There was a significant effect of MEC layer (Χ2 (4)=32.8, p=0.000001, Kendall W=0.745, Friedman test) and location (Χ2 (5)=21.6, p=0.0006, Kendall W=0.394) on puncta density. There was a higher density of puncta in L1 (4790±545 mm3) compared to all other layers (L2: W=66, p=0.01; L3: W=66, p=0.01; L5a: W=66, p=0.01; L5b: W=66, p=0.01, pairwise Wilcoxon tests with Bonferroni correction) and a

Article Snippet: Reagent type (species) or resource Designation Source or reference Identifiers Additional information Strain, strain background (mouse) Sim1Cre Gen- Sat, MMRC RRID: MMRRC_034614-UCD NA Chemical compound, drug Biocytin Sigma- Aldrich B4261 Biotin- lysine compound used for neuron reconstruction Chemical compound, drug Gabazine Hello Bio HB0901 GABAA receptor antagonist, diluted to 10 μM Chemical compound, drug CGP55845 Hello Bio HB0960 GABAB receptor antagonist,diluted to 100 μM Chemical compound, drug D- AP5 Hello Bio HB0225 NMDA receptor antagonist, diluted to 50 μM Chemical compound, drug NBQX disodium salt Hello Bio HB0442 AMPA receptor antagonist, diluted to 10 μM Chemical compound, drug Tetrodotoxin Hello Bio HB1034 Na +channel- blocker, diluted to 500 nM Chemical compound, drug 4- Aminopyridine (AP) Hello Bio HB1073 Kv channel- blocker, diluted to 200 μM Software, algorithm R NA Version: 3.6.0 https://www.r-project.org/ Software, algorithm Matlab Mathworks Version: 2013 a https://www.mathworks.com Software, algorithm Igor Pro Wavemetrics Version: 6.3 https://www.wavemetrics.com/ Software, algorithm AxoGraph AxoGraph Version 1.7.6 https://www.axograph.com Software, algorithm ImageJ Fiji NA https://fiji.sc Other (Stains) AlexaFluor Streptavidin 647 Invitrogen Cat #: S21374 1:500 dilution, biotin- binding Other (Stains) Neurotrace 435/55 (Nissl stain) Invitrogen Cat#: N21479 1: 500 Other (Stains) Neurotrace 640/660 (Nissl stain) Invitrogen Cat#: N21479 1:500 Other (AAV) AAV1/2- Retro- Ef1a- Cre- WPRE Addgene Plasmid #: 51502 titer: 2.4x1011 Other (AAV) AAV1/2- FLEX- GFP Addgene Plasmid #: 28304 titer: 2.44x1013 Other (AAV) AAV1/2- FLEX- GFP- 2A- SynmRuby Addgene Plasmid #: 71760 titer: 2.96x1012 Other (AAV) AAV2- FLEX- EF1a- DIOhChR2(H134R)- EYFP UNC Vector Core; Zhang et al., 2006 titer: 4x1012 Other (AAV) AAV2- Retro- Syn- mCherry Addgene Plasmid #: 114472 titer: 1.9x1013 Other (AAV) pENN- AAV1- hSyn- Cre- WPRE Addgene Plasmid #: 105553 titer: 1.8x1013

Techniques:

Journal: eLife

Article Title: Fan cells in lateral entorhinal cortex directly influence medial entorhinal cortex through synaptic connections in layer 1

doi: 10.7554/elife.83008

Figure Lengend Snippet: Figure 2. Projections to the medial entorhinal cortex from lateral entorhinal cortex arise from fan cells that also send projections to the hippocampus. (A) Schematic of strategy for targeting GFP specifically to fan cells in the lateral entorhinal cortex (LEC) that project to the hippocampal dentate gyrus (DG). In a wild-type mouse, adeno-associated virus (AAV) encoding retrograde Cre (AAV-Retro-Cre, grey) was injected into the DG and Cre-dependent AAV encoding GFP (AAV-FLEX-GFP, green) was injected into the LEC. (B) Horizontal brain section from a wild-type mouse showing GFP labelling of fan cell axons (green) in the outer molecular layer (oml) of the DG and layer (L) 1 of the medial entorhinal cortex (MEC). Neurons are counterstained with Neurotrace (red). Scale bar represents 500 µm. (C) Horizontal brain section from the same mouse shown in B, showing retrograde labelling of fan cell bodies with GFP (green) in L2a of the LEC. Scale bar represents 100 µm. (D) Fluorescence intensity of fan cell axons in L1 of MEC and the oml of DG. Intensity was quantified as the mean gray value of pixels in regions of interest (ROIs), where the possible range of values was 1–255 and a value of 255 corresponds to white. Mean intensity values were quantified for ROIs in MEC and DG, and were adjusted to baseline by subtracting the intensity value of an ROI from the same slice that did not contain fan cell axons. Fluorescence intensities were similar for DG and MEC (W=4, p=1, Mann Whitney U test). Black line shows the population average and grey circles are values for single brain slices. Error bars are SEM. (E) Fluorescence intensity as a function of distance across layers of MEC (top) and DG (bottom). For MEC, intensity was sampled from the edge of the slice (L1) towards the hippocampus. For DG, intensity was sampled from the outer edge of the oml towards the hilus. Plots are normalised to the minimum and maximum intensity values. Black line is a polynomial fit to the population data, and gray lines are values for single brain slices. (F) The dorsal-ventral position of the horizontal sections was estimated relative to bregma (top). Epifluorescent images of horizontal brain sections show GFP expression at dorsoventral locations containing the MEC and extending to the injection site in LEC. Zoomed in images of the ROIs highlighted by boxes in the left panels are shown in the panels to the right. Numbers indicate depth of slice from bregma in mm. Scale bar represents 500 µm. (G) Confocal images of a horizontal

Article Snippet: Reagent type (species) or resource Designation Source or reference Identifiers Additional information Strain, strain background (mouse) Sim1Cre Gen- Sat, MMRC RRID: MMRRC_034614-UCD NA Chemical compound, drug Biocytin Sigma- Aldrich B4261 Biotin- lysine compound used for neuron reconstruction Chemical compound, drug Gabazine Hello Bio HB0901 GABAA receptor antagonist, diluted to 10 μM Chemical compound, drug CGP55845 Hello Bio HB0960 GABAB receptor antagonist,diluted to 100 μM Chemical compound, drug D- AP5 Hello Bio HB0225 NMDA receptor antagonist, diluted to 50 μM Chemical compound, drug NBQX disodium salt Hello Bio HB0442 AMPA receptor antagonist, diluted to 10 μM Chemical compound, drug Tetrodotoxin Hello Bio HB1034 Na +channel- blocker, diluted to 500 nM Chemical compound, drug 4- Aminopyridine (AP) Hello Bio HB1073 Kv channel- blocker, diluted to 200 μM Software, algorithm R NA Version: 3.6.0 https://www.r-project.org/ Software, algorithm Matlab Mathworks Version: 2013 a https://www.mathworks.com Software, algorithm Igor Pro Wavemetrics Version: 6.3 https://www.wavemetrics.com/ Software, algorithm AxoGraph AxoGraph Version 1.7.6 https://www.axograph.com Software, algorithm ImageJ Fiji NA https://fiji.sc Other (Stains) AlexaFluor Streptavidin 647 Invitrogen Cat #: S21374 1:500 dilution, biotin- binding Other (Stains) Neurotrace 435/55 (Nissl stain) Invitrogen Cat#: N21479 1: 500 Other (Stains) Neurotrace 640/660 (Nissl stain) Invitrogen Cat#: N21479 1:500 Other (AAV) AAV1/2- Retro- Ef1a- Cre- WPRE Addgene Plasmid #: 51502 titer: 2.4x1011 Other (AAV) AAV1/2- FLEX- GFP Addgene Plasmid #: 28304 titer: 2.44x1013 Other (AAV) AAV1/2- FLEX- GFP- 2A- SynmRuby Addgene Plasmid #: 71760 titer: 2.96x1012 Other (AAV) AAV2- FLEX- EF1a- DIOhChR2(H134R)- EYFP UNC Vector Core; Zhang et al., 2006 titer: 4x1012 Other (AAV) AAV2- Retro- Syn- mCherry Addgene Plasmid #: 114472 titer: 1.9x1013 Other (AAV) pENN- AAV1- hSyn- Cre- WPRE Addgene Plasmid #: 105553 titer: 1.8x1013

Techniques: Virus, Injection, Fluorescence, MANN-WHITNEY, Expressing

Journal: eLife

Article Title: Fan cells in lateral entorhinal cortex directly influence medial entorhinal cortex through synaptic connections in layer 1

doi: 10.7554/elife.83008

Figure Lengend Snippet: Figure 3. Fan cells that project to medial entorhinal cortex receive inputs from piriform and prefrontal cortices (A-B) (Top). Schematic of strategy for targeting GFP specifically to fan cells in the lateral entorhinal cortex (LEC) that receive projections from piriform cortex (PIR, A) or medial prefrontal cortex (mPFC, B). AAV that anterogradely transports Cre (pENN-AAV-hSyn-Cre-WPRE, grey) was injected into the PIR or mPFC of a wild-type mouse. Cre-dependent AAV encoding GFP (AAV-FLEX-GFP, green) was injected into the LEC of the same mouse to label Cre-expressing fan cells and their axons. Horizontal brain section showing labelling of fan cell axons with GFP in the outer molecular layer (oml) of the dentate gyrus (DG) and layer (L) 1 of the MEC (right panels show zoomed in images of the regions of interest highlighted in the left panels). Scale bar is 500 µm. (C) Expression of GFP in fan cells at the injection site in the same animal as A. Neurons are counterstained with Neurotrace (red). (D) Fluorescence intensity in L1 of MEC and the oml of DG of axons from fan cells receiving inputs from PIR. The fluorescence intensity of fan cell axons was higher in the DG (W=16, p=0.029, Mann Whitney U test). Black line shows population average and grey circles are values for single brain slices. Error bars are SEM. (E) Fluorescence intensity of axons from fan cells that receive PIR inputs as a function of distance across layers of MEC (left) and DG (right). For MEC, intensity was sampled from the edge of the slice (L1) towards the hippocampus. For DG, intensity was sampled from the outer edge of the oml towards the hilus. ROIs were oriented perpendicular to the layers of MEC and DG. Plots are normalised to the minimum and maximum intensity values. Black line is population data fit with a polynomial model and gray lines are values for single brain slices. (F) Same as C but for fan cells labelled on the basis of receiving projections from medial prefrontal cortex (mPFC). (G-H) Same as (D-E), but for axons from fan cells that receive inputs for mPFC. Fluorescence intensities of axons from fan cells that receive inputs from mPFC was similar for DG and MEC (W=12, p=0.343).

Article Snippet: Reagent type (species) or resource Designation Source or reference Identifiers Additional information Strain, strain background (mouse) Sim1Cre Gen- Sat, MMRC RRID: MMRRC_034614-UCD NA Chemical compound, drug Biocytin Sigma- Aldrich B4261 Biotin- lysine compound used for neuron reconstruction Chemical compound, drug Gabazine Hello Bio HB0901 GABAA receptor antagonist, diluted to 10 μM Chemical compound, drug CGP55845 Hello Bio HB0960 GABAB receptor antagonist,diluted to 100 μM Chemical compound, drug D- AP5 Hello Bio HB0225 NMDA receptor antagonist, diluted to 50 μM Chemical compound, drug NBQX disodium salt Hello Bio HB0442 AMPA receptor antagonist, diluted to 10 μM Chemical compound, drug Tetrodotoxin Hello Bio HB1034 Na +channel- blocker, diluted to 500 nM Chemical compound, drug 4- Aminopyridine (AP) Hello Bio HB1073 Kv channel- blocker, diluted to 200 μM Software, algorithm R NA Version: 3.6.0 https://www.r-project.org/ Software, algorithm Matlab Mathworks Version: 2013 a https://www.mathworks.com Software, algorithm Igor Pro Wavemetrics Version: 6.3 https://www.wavemetrics.com/ Software, algorithm AxoGraph AxoGraph Version 1.7.6 https://www.axograph.com Software, algorithm ImageJ Fiji NA https://fiji.sc Other (Stains) AlexaFluor Streptavidin 647 Invitrogen Cat #: S21374 1:500 dilution, biotin- binding Other (Stains) Neurotrace 435/55 (Nissl stain) Invitrogen Cat#: N21479 1: 500 Other (Stains) Neurotrace 640/660 (Nissl stain) Invitrogen Cat#: N21479 1:500 Other (AAV) AAV1/2- Retro- Ef1a- Cre- WPRE Addgene Plasmid #: 51502 titer: 2.4x1011 Other (AAV) AAV1/2- FLEX- GFP Addgene Plasmid #: 28304 titer: 2.44x1013 Other (AAV) AAV1/2- FLEX- GFP- 2A- SynmRuby Addgene Plasmid #: 71760 titer: 2.96x1012 Other (AAV) AAV2- FLEX- EF1a- DIOhChR2(H134R)- EYFP UNC Vector Core; Zhang et al., 2006 titer: 4x1012 Other (AAV) AAV2- Retro- Syn- mCherry Addgene Plasmid #: 114472 titer: 1.9x1013 Other (AAV) pENN- AAV1- hSyn- Cre- WPRE Addgene Plasmid #: 105553 titer: 1.8x1013

Techniques: Injection, Expressing, Fluorescence, MANN-WHITNEY

Journal: BMC Biology

Article Title: A systematic sequencing-based approach for microbial contaminant detection and functional inference

doi: 10.1186/s12915-019-0690-0

Figure Lengend Snippet: Overall structure of the proposed pipeline and results of the performance assessment. a Schematic representation of the proposed pipeline that executes rigorous read alignment with a large-scale genome database. b FDR distribution in the reversion tests considering falsely mapped reads to other species or to other genera. Particular genera, including Raoultella , Shigella , and Kluyvera , are difficult to distinguish genomically. c Comparative analysis for the effects of uniq-genus-hits and weighted multi-genera-hits in quantification. “Total mapped” represents the sum of uniq-genus-hits (Unique and Unambiguous) and multi-genera-hits (Multiple and Ambiguous). “Weighted” represents the adjusted “Total mapped” by our scoring scheme. d Correlations between the detection quantification and spike-in concentration assayed by DNA-seq (0-day cultured hPDL-MSCs with antibiotics). e RPMH differences among three NGS protocols in Mycoplasma spike-in detections (3-day cultured hPDL-MSCs)

Article Snippet: We performed the same analysis by incorporating the Myco(+) human bone marrow MSCs (hBM-MSCs) used in Fig. a and Myco(−) hBM-MSCs (GSE90273).

Techniques: Concentration Assay, DNA Sequencing, Cell Culture

Journal: BMC Biology

Article Title: A systematic sequencing-based approach for microbial contaminant detection and functional inference

doi: 10.1186/s12915-019-0690-0

Figure Lengend Snippet: Results of the Mycoplasma prevalence analysis and the functional impacts on host cells. a Twenty-two out of 432 public RNA-seq datasets contained significant Mycoplasma -mapped reads (red-colored bar) that were normalized to RPMHs (blue-colored line); the x -axis labels are colored black for DRA001846, gray for IHBM2, blue for ENCODE, and red for Mycoplasma -positive samples. b Gene expression correlation plots between Mycoplasma -positive (Myco+) and Mycoplasma -negative (Myco-) MSCs; Myco(+) hPDL-MSCs are Mycoplasm a spike-in cells (2000 CFU × 7 species, 3 days cultured without antibiotics), FPKMs were transformed onto the log 10 scale by adding one, and the black-labeled genes are the 13 genes listed in d . c Highly enriched Gene Ontology terms and Reactome pathways ( q value after Bonferroni correction < 0.001). d Venn diagram showing unique or shared differentially upregulated genes (DUGs) in MSCs, including 13 out of 967 DUGs unique to Myco(+) MSCs. e Expression levels of the 13 genes in Myco(+) ESCs and MSCs; the values are expressed as relative TPM (transcripts per million)

Article Snippet: We performed the same analysis by incorporating the Myco(+) human bone marrow MSCs (hBM-MSCs) used in Fig. a and Myco(−) hBM-MSCs (GSE90273).

Techniques: Functional Assay, RNA Sequencing, Gene Expression, Cell Culture, Transformation Assay, Labeling, Expressing

Journal: BMC Biology

Article Title: A systematic sequencing-based approach for microbial contaminant detection and functional inference

doi: 10.1186/s12915-019-0690-0

Figure Lengend Snippet: Inference of DUGs associated with multiple contaminants in Myco(+) DG75 samples. a Expression profile of 967 DUGs unique to Myco(+) MSCs. b Contamination profile with MSC, ESC, and DG-75 samples; the x -axis labels are colored black for Myco(−) and red for Myco(+). c Schematic representation of module identification from two input profiles by the jNMF algorithm. d An example showing the module that captured genes and contaminants co-elevated in a DG-75 sample. e Network representation of the association between genes and contaminants co-elevated in the seven DG-75 samples; GO:0010941 is the enriched GO term in the genes found in at least four DG-75 samples ( p = 3.76e−3). f Expression profiles of the 33 genes involved in the biological process “regulation of cell death”, DG75_1 (GSM1197380), DG75_2 (GSM1197385), DG75_3 (GSM1197386), DG75_4 (GSM1197381), DG75_5 (GSM1197382), DG75_6 (GSM1197383), DG75_7 (GSM1197384), NB_1 (GSM2225743), and NB_2 (GSM2225744)

Article Snippet: We performed the same analysis by incorporating the Myco(+) human bone marrow MSCs (hBM-MSCs) used in Fig. a and Myco(−) hBM-MSCs (GSE90273).

Techniques: Expressing

Journal: Scientific Reports

Article Title: Urine stabilization and normalization strategies favor unbiased analysis of urinary EV content

doi: 10.1038/s41598-022-22577-3

Figure Lengend Snippet: Detection urine of EV-miRNAs by RT-qPCR in longitudinal urine samples. Following ultracentrifugation, total RNA has been extracted from EV and MV pellets and RT-qPCR amplification and quantification of a 24 miRNA Taqman Custom Array performed as described. The results are expressed both as Ct values ( B ) and as relative RNA expression (Table A , and panel B on the right) calculated as delta Ct versus reference sample (RNA extracted from Prostate Cancer LnCAP cell line exosomes). The correlation index was determined between the trend observed for miRNA expression levels across the samples and the respective values for selected EV parameters (particle concentration and protein content) as well as biochemical parameters measured in the same urine samples (creatinine, albumin and total protein content) ( C ). Finally, the normalization of miRNA expression levels at longitudinal points is attempted using best correlating EV and urine parameters ( D ). EVs (UC) were purified by differential (ultra)centrifugation-based protocol from urine obtained from two healthy volunteers, one female (marked as 1) and one male (marked as 2), 45 and 43 years old, in 3 subsequent days. Experimental points are shown on relative x axes. For each sample two aliquots of urine were processed independently and averaged.

Article Snippet: Assay was calibrated using a standard curve prepared with linear dilutions of exosomes purified from human urine (HBM-PEU; HansaBioMed Life Sciences) embedded in the ELISA kit as standards.

Techniques: Quantitative RT-PCR, Amplification, RNA Expression, Expressing, Concentration Assay, Purification, Centrifugation