EMBO/soda-nel huggingface dataset:
Commensal microbiota divergently affect myeloid subsets in the mammalian central nervous system during homeostasis and disease (2021)
Figure 4

Figure 4: Perivascular macrophages show microbiota-dependent transcriptional alterations under steady-state conditions.

(A) UMAP plot illustrating the color-coded aggregated transcript counts of published core signature genes for CAMs. The scale bar represents color coded sequencing depth-corrected log transformed transcript counts.

(B) UMAP representation of 885 individual cells from SPF (green dots) and GF (yellow dots) mice.

(C) UMAP plot of the perivascular macrophages from SPF and GF mice color-coded for the results of Seurat clustering. The mappings between colors and clusters are shown on the right. The cell type annotations are based on published gene expression profiles.

(D) Stacked bar plot (Marimekko chart) color-coded for the relative contribution of perivascular macrophages derived from SPF (green) and GF (yellow) mice. P-value ranges are provided for significantly enriched regions per cluster, based on hypergeometric tests. Benjamini-Hochberg correction was applied to correct for multiple testing (*P < 0.05; ***P < 0.001).

(E) Volcano plot of the differential gene expression analysis of n=182 SPF and 228 GF pvMΦ based on Wilcoxon Rank Sum tests. The shown genes are filtered based on an average log2 fold change (avg. log2FC) > 0.2 and adjusted p-value < 0.05.

(F) Heatmap of the top 10 differentially expressed genes per cluster (adjusted p-value of <0.05 based on Wilcoxon Rank Sum tests). The scale bar represents color-coded Pearson residuals. (G) UMAP maps color-coded for transcript counts of genes enriched in subsets of the captured cells. The scale bars represent color coded sequencing depth-corrected log transformed transcript counts.

(H) Representative Iba1+ immunohistochemistry in the perivascular space of SPF, GF, ABX-treated, ASF, recolonized ASF, sDMDMm2, TLR2,3,4,7,9-deficient, SCFA-treated GF mice or FFAR2-deficient mice. Arrows indicate Iba1+ perivascular macrophages. Scale bar = 50 µm.

(I-O) Quantification thereof. Each symbol represents one mouse. Three to four sections per mouse were examined. Means ± s.e.m. are indicated. No significant differences were determined by unpaired t-test or one-way ANOVA followed by Tukey's post-hoc comparison test. Data are representative of at least two independent experiments.
Commensal microbiota divergently affect myeloid subsets in the mammalian central nervous system during homeostasis and disease (2021)
Figure 5

Figure 5: Meningeal macrophages display subtle changes through the absence of the microbiota under steady-state conditions.

(A) UMAP plot illustrating the color-coded aggregated transcript counts of published core signature genes for CAMs. The scale bar represents color coded sequencing depth-corrected log transformed transcript counts.

(B) UMAP representation of 545 individual cells of SPF (green dots) and GF (yellow dots) mice.

(C) UMAP representation of the meningeal macrophages of SPF and GF mice color-coded for the clustering results of the Seurat algorithm. The mappings between colors and clusters are shown on the right. The cell type annotations are based on published gene expression profiles.

(D) Stacked bar plot (Marimekko chart) color-coded for relative contribution of meningeal macrophages derived from SPF (green) and GF (yellow) mice. P-value ranges are provided for significantly enriched regions per cluster, based on hypergeometric tests. Benjamini-Hochberg correction was applied to correct for multiple testing (*P < 0.05; ***P < 0.001).

(E) Volcano plot of the differential gene expression analysis of of n=144 SPF and 87 GF mMΦs based on Wilcoxon Rank Sum tests. The shown genes are filtered based on an average log2 fold change (avg. log2FC) > 0.2 and adjusted p-value < 0.05.

(F) Heatmap of the top 10 differentially expressed genes per cluster (adjusted p-value of <0.05 based on Wilcoxon Rank Sum tests). The scale bar represents color-coded Pearson residuals.

(G) UMAP maps color-coded for transcript counts of genes enriched in subsets of the captured cells. The scale bars represent color coded sequencing depth-corrected log transformed transcript counts.

(H) Representative Iba1+ immunohistochemistry in the leptomeninges of SPF, GF, ABX-treated, ASF, recolonized ASF, sDMDMm2, TLR2,3,4,7,9-deficient, SCFA-treated GF mice or FFAR2-deficient mice. Arrows indicate Iba1+ meningeal macrophages. Scale bar = 50 μm.

(I-O) Quantification thereof. Each symbol represents one mouse. Three to four sections per mouse were examined. Means ± s.e.m. are indicated. No significant differences were determined by unpaired t-test or one-way ANOVA followed by Tukey's post-hoc comparison test. Data are representative of at least two independent experiments.
Commensal microbiota divergently affect myeloid subsets in the mammalian central nervous system during homeostasis and disease (2021)
Figure 6

Figure 6: Reduced immune response of myeloid cells to LCMV infection under GF conditions.

(A) Iba1 immunohistochemistry depicting cortical microglia, meningeal macrophages, perivascular macrophages and choroid plexus macrophages 4 days after i.c. challenge with LCMV or vehicle controls. Arrows indicate Iba1+ meningeal or perivascular macrophages, respectively.

(B) Quantification of Iba1+ microglia, choroid plexus macrophages, meningeal macrophages and perivascular macrophages. Each symbol represents one mouse. Three to four sections per mouse were examined. Significant differences were evaluated by two-way ANOVA followed by Tukey's post-hoc comparison test and marked with asterisks (*P < 0.05, **P < 0.01, ***P < 0.001). Data are representative of two independent experiments.

(C, D) Representative immunofluorescence images (left) of (C) Iba1 (green) and KI67 (red) or (D) CD74 (red) on cortical sections from SPF and GF mice. Nuclei were stained with DAPI (blue). Scale bars: 25 μm. White arrows indicate KI67+/Iba1+ or CD74+/Iba1+ macrophages and arrowheads show KI67-/Iba1+ or CD74-/Iba1+ macrophages. Quantification thereof (right). Each symbol represents one mouse. At least three slides were examined per individual mouse. Data are presented as mean ± s.e.m. Significant differences were evaluated by two-way ANOVA followed by Tukey's post-hoc comparison test and marked with asterisks (*P < 0.05, **P < 0.01, ***P < 0.001). Data are representative of two independent experiments.
CIB1 and CO interact to mediate CRY2-dependent regulation of flowering (2018)
Figure 1

Figure 1. CIB1 promotes FT transcription and flowering in a CO-dependent manner and CO acts independently of CIBs.

A, F. 52-day-old (A), or 25-day-old plants (F) of the genotypes indicated grown in LDs.

Scale bars: 5 cm.

B-C, G-H. The number of rosette leaves and the time to flowering at the time of flowering of the genotypes shown in A (for B and C), F (for G and H); the numbers indicate independent transgenic lines. The letters "a" to "c" indicate statistically significant differences between flowering time or leaf numbers of the indicated genotypes.

D-E, I-J. qPCR results showing mRNA expression of CIB1, CO, FT in the genotypes indicated. The numbers indicate independent transgenic lines. The transcript levels of CIB1 or CO or FT in Col were set to 1.0. Samples were collected from 7-day-old LD grown seedlings at ZT16.

Data information: In (B-C, G-H), data are presented as mean ± SD, n ≥ 15 per genotypes indicated. P≤0.05 (Tukey's HSD test). For data in (D-E, I-J), error bars indicate standard error within three biological replicates and for each of these two technical replicates were performed.
CIB1 and CO interact to mediate CRY2-dependent regulation of flowering (2018)
Figure 2

Figure 2. CIB1 and CO are Co-expressed.

A, B. Immunoblots showing CIB1 (A) and CO (B) protein levels in WT and co-1 backgrounds (A) or WT and cibh backgrounds (B) over the course of 20 hours under LDs. Samples were separated on 10% SDS-PAGE, blotted, probed with the anti-Myc antibody, stripped and re-probed with the anti-actin antibody (ACT).

C, D. The relative levels of CIB1 (A) and CO proteins (B) in WT and co-1 backgrounds (A) or WT and cibh backgrounds (B) over the course of 20 hours under LDs were calculated as described (see Supplemental Materials and Methods).

Data information: In (C, D), Error bars represent the standard deviation of three biological replicates of the Western blots.
CIB1 and CO interact to mediate CRY2-dependent regulation of flowering (2018)
Figure 3

Figure 3. CIB1 and CO interact directly.

A. Fluorescent microscopy images showing that CIB1 co-localizes with CO in nuclear speckles. Scale bars: 2 μm.

B. BiFC assays to show in vivo protein interactions. Epidermal cells of N. benthamiana leaves were co-transformated with cCFP-CO and nYFP-CIB1, with cCFP-CO and nYFP, or with cCFP and nYFP-CIB1. BF, bright field. Merge, overlay of the YFP and bright field images. Scale bars: 50 μm.

C. Split-LUC assay showing that CIB1 interacts with CO. Leaf epidermal cells of N. benthamiana were co-transformed with CO-nLUC and cLUC-CIB1 or cLUC or nLUC with cLUC-CIB or cLUC.

D. Co-IP assays of samples prepared from 10-day-old 35S:Myc-CIB1, 35S:CO-Flag or 35S:Myc-CIB1/35S:CO-Flag seedlings grown in LDs.

E. Schematic representation of CO used in this work was showing. CO contains two B-Box domains and a CCT domain.

F. In vitro pull-down assays showing the interaction between CIBC and CO, or CIBC and COΔN and the lack of interaction between CIB1C and COΔC. His-TF tagged CO, COΔN, COΔC or His-TF tagged UVR8 were mixed with GST-CIB1C purified from E. coli, and His antibody was used for the in vitro pull-down assay. The products were analyzed by immunoblots probed with the anti-CIB1C antibody or the anti-His antibody (UVR8, CO, COΔC, COΔN).

Data information: For each experiment, two or more biological replicates were performed.
CIB1 and CO interact to mediate CRY2-dependent regulation of flowering (2018)
Figure 4

Figure 4. CO and CIB1 promote flowering and FT transcription together.

A. 28-day-old plants of the genotypes indicated grown in LDs. Scale bars: 5 cm.

B, C. The number of rosette leaves and the time to flowering at the time of flowering of the genotypes shown in A. The letters "a" to "c" indicate statistically significant differences between flowering time or leaf numbers of the indicated genotypes.

D. Immunoblots showing Myc-CIB1 and CO-Flag protein levels in the genotypes indicated. Samples were separated on 10% SDS-PAGE, blotted, probed with the anti-Myc antibody, stripped and re-probed with the anti-Flag antibody.

E. qPCR results showing mRNA expression of FT in the genotypes indicated. The transcript levels in Col are set to 1.0. Samples were collected from 7-day-old seedlings of the genotypes indicated at ZT16.

F. Structure of the FT promoter-driven dual-Luc reporter gene. 35S promoter, FT promoter (-1034bp to -1bp) (proPFT), FT promoter with mutated E-boxes (ProFTm1), FT promoter without CO binding site (ProFTm2), FT promoter without CO binding site plus with mutated E-boxes (ProFTm3). REN luciferase (REN), firefly luciferase (LUC), are indicated.

G. Box plots (the minimum, first quartile, median, third quartile, and maximum are shown, with data >1.5 interquartile ranges denoted with circles) for relative reporter activity (LUC/REN) in plants expressing different reportors and effectors. The relative LUC activities normalized to the REN activity are shown. The LUC/REN in plants expressing ProFT alone is set to 1.0.

Data information: In (B, C), data are presented as mean ± SD, n ≥ 15 per genotypes indicated. P≤0.05 (Tukey's HSD test). For data in (E), error bars indicate standard error within three biological replicates and for each of these two technical replicates were performed. In (G), data are presented as mean ± SD, n=4. The letters "a" to "g" indicate statistically significant differences determined by Tukey's HSD test (P≤0.05). Two biological replicates were performed.
CIB1 and CO interact to mediate CRY2-dependent regulation of flowering (2018)
Figure 5

Figure 5. CRY2 forms a complex with CO and CIB1 in response to blue light to activate FT transcription.

A. Fluorescent microscopy images showing that CRY2, CIB1 and CO co-localized to the nucleus. Scale bars: 2 μm.

B. Co-IP assays showing that CRY2, CIB1 and CO form a protein complex. Samples were prepared from 10-day-old WT or 35S:Myc-CIB1/35S:CO-Flag or 35S:Myc-CIB1/35S:CO-Flag /cry2-1 seedlings grown in LDs (16L/8D), and samples were collected at ZT16.

C. Co-IP assays of samples prepared from 10-day-old 35S:Myc-CIB1/35S:CO-Flag 35S:Myc-CIB1/35S:CO-Flag /cry2-1 seedlings grown in LDs (16L/8D). Samples were collected at the times indicated over the course of one day.

D. Diagram depicting the putative promoter and genomic region of FT. The diamond symbol indicates the position of CORE. The white circles indicate positions of E boxes (CANNTG) located at proximal promoter of FT.

E. Representative results of the ChIP-qPCR assays. Chromatin fragments (~500 bp) were prepared from 10-day-old 35S:Myc-CIB1/35S:CO-Flag seedlings, immunoprecipitated by the anti-CRY2, anti-Flag (CO-Flag) , anti-Myc (Myc-CIB1) antibody or anti-preimmune IgG, and the precipitated DNA was analyzed by qPCR using the primer pairs indicated. The IP/input ratio of anti-preimmune is set to 1.0.

F. Box plots (the minimum, first quartile, median, third quartile, and maximum are shown, with data >1.5 interquartile ranges denoted with circles) for relative reporter activity (LUC/REN) in plants expressing different reportors and effectors. The LUC/REN in plants expressing ProFT alone is set to 1.0. Immunoblots were done to show GFP-CRY2W374A, Myc-CIB1 and Myc-CO protein levels in the transient Dual-LUC assay. Samples were separated on 10% SDS-PAGE, blotted, probed with the anti-Myc antibody, and with the anti-GFP antibody.

G. Model representing the action of CIB1 and CO on FT expression under long day condition.

Data information: For data in (E), the relative IP/input ratios are shown with their standard deviation (n = 3). Two biological replicates were performed. For data in (F), the relative LUC activities normalized to the REN activity are shown (LUC/REN) with their standard deviation (n =6). The letters "a" to "f" indicate statistically significant differences determined by Tukey's HSD test (P≤0.05). Two biological replicates were performed.

Expanded View figure legends
Sodium Butyrate Induces Endoplasmic Reticulum Stress and Autophagy in Colorectal Cells: Implications for Apoptosis. (2016)
Fig 1

Sodium butyrate inhibited proliferation and induced apoptosis in colorectal cancer cells.

HCT-116 (A) and HT-29 (B) cells were treated with the indicated concentrations of sodium butyrate (NaB) for 24 (grey bars), 48 (pink bars), or 72 (yellow bars) hours and cell proliferation was assessed. HCT-116 (C, E) and HT-29 (D, F) cells were treated with the indicated concentrations of NaB for 24 h. C.D. The percentage of apoptotic cells in HCT-116 (C) and HT-29 (D) cells was quantified in three independent experiments using an annexinV/PI assay (annexinV+/PI-) by flow cytometry and expressed as mean ± SD. One-way ANOVA was used to compare between the control cells and NaB treatments. *p<0.05, ** p<0.01 compared to control. E.F. Representative Western blots of full-length and cleaved PARP products in HCT-116 (E) and HT-29 (F) cells.
Sodium Butyrate Induces Endoplasmic Reticulum Stress and Autophagy in Colorectal Cells: Implications for Apoptosis. (2016)
Fig 3

Sodium butyrate increased expression of LC3-II.

HCT-116 (A) or HT-29 (B) cells were treated with the indicated concentrations of sodium butyrate (NaB) for 24 h. Representative blots showed expression of Beclin1, ATG3, and LC3 as well as GAPDH, the loading control. C. Electron microscopy images of control and NaB (2mM) treated HCT-116 cells. ER, endoplasmic reticulum; Mt, mitochondrion; AL.
Sodium Butyrate Induces Endoplasmic Reticulum Stress and Autophagy in Colorectal Cells: Implications for Apoptosis. (2016)
Fig 4

3-MA and chloroquine blocked sodium butyrate induced autophagy in colorectal cancer cells.

HCT-116 or HT-29 cells were treated with 5mM 3-MA or 5μM chloroquine (CQ) for 30 min, and then with 2mM sodium butyrate (NaB) for 24 h. Representative Western blots of LC3-I and LC3-II expression were quantified by densitometry and normalized to GAPDH (ratio of LC3:GAPDH). The fold change from control is indicated as the mean ± SD of three independent experiments. One-way ANOVA was used for statistical analysis. ** p<0.01, compared to the respective control group. (A) HCT-116 cells treated with 3-MA and NaB; (B) HT-29 cells treated with 3-MA and NaB; (C) HCT-116 cells treated with CQ and NaB; (D) HT-20 cells treated with CQ and NaB.
Sodium Butyrate Induces Endoplasmic Reticulum Stress and Autophagy in Colorectal Cells: Implications for Apoptosis. (2016)
Fig 5

Sodium butyrate induced endoplasmic reticulum stress in colorectal cancer cells.

HCT-116 (A) and HT-29 (B) cells were treated with the indicated concentrations of sodium butyrate (NaB) for 24 h. Representative Western blots show the expression of IRE-1a, BIP, PDI, and CHOP. GAPDH was used as the loading control.
Sodium Butyrate Induces Endoplasmic Reticulum Stress and Autophagy in Colorectal Cells: Implications for Apoptosis. (2016)
Fig 6

Cycloheximide blocked sodium butyrate induced endoplasmic reticulum stress in colorectal cancer cells.

HCT-116 or HT-29 cells were treated with 10 μg/mL cycloheximide for 30 min followed by sodium butyrate (NaB) for 24 h. Representative Western blots showed the expression of IRE-1a, BIP, PDI, and GADPH (loading control) in HCT-116 (A) or HT-29 (C) cells. Protein expression was quantified by densitometry and normalized to GAPDH (ratio of protein:GAPDH). The fold change from control for each protein is expressed as mean ± SD of three independent experiments for HCT-116 (B) and HT-29 (D) cells. are shown. One-way ANOVA was used for statistical analysis. * P<0.05, ** p<0.01, compared to the respective control group.
Sodium Butyrate Induces Endoplasmic Reticulum Stress and Autophagy in Colorectal Cells: Implications for Apoptosis. (2016)
Fig 7

Mithramycin blocked sodium butyrate induced endoplasmic reticulum stress in colorectal cancer cells.

HCT-116 (A, B) or HT-29 (C,D) cells were treated with 0.1μM mithramycin for 30 min followed by sodium butyrate (NaB) for 24 h. Representative Western blots showing the expression of IRE-1a, BIP, and PDI in HCT-116 (A) or HT-29 (C) cells are shown. GAPDH was used as loading control. Protein expression (IRE-1a, BIP, and PDI) was quantified by densitometry and normalized to GAPDH (ratio of protein:GAPDH). The fold change from control for each protein is expressed as mean ± SD of three independent experiments for HCT-116 (B) and HT-29 (D) cells. One-way ANOVA was used for statistical analysis. * P<0.05, ** p<0.01, compared to the respective control group.
Sodium Butyrate Induces Endoplasmic Reticulum Stress and Autophagy in Colorectal Cells: Implications for Apoptosis. (2016)
Fig 8

Cyclohexamide and mithramycin blocked sodium butyrate induced autophagy in colorectal cancer cells.

HCT-116 or HT-29 cells were treated with 10 μg/mL cycloheximide or 0.1μM mithramycin for 30 min and then with 2mM sodium butyrate (NaB) for 24 h. Representative Western blots of the expression of LC3-II are shown. The level of LC3-II expression was quantified by densitometry and normalized to GAPDH (ratio of LC3-II:GAPDH). The fold change from control cells is shown. Means and standard deviation (SD) of three independent experiments are shown. One-way ANOVA was used for statistical analysis. * P<0.05, ** p<0.01, compared to the control group. (A-B) HCT-116 cells treated with cyclohexamide; (C-D) HCT-116 cells treated with mithramycin; (E-F) HT-29 cells treated with cyclohexamide; (G-H) HT-29 cells treated with mithramycin.
Sodium Butyrate Induces Endoplasmic Reticulum Stress and Autophagy in Colorectal Cells: Implications for Apoptosis. (2016)
Fig 9

RNA interference targeting BIP or CHOP blocked sodium butyrate induced autophagy in colorectal cancer cells.

HCT-116 cells were transfected with BIP or CHOP specific siRNAs for 48 h and then treated with or without 2mM sodium butyrate (NaB) for 24 h. Negative control (NC) scramble siRNA was used the negative control for the transfection. Representative Western blots are shown for BIP siRNA (A) and CHOP siRNA (C). The expression level of each protein was determined by densitometry and normalized to GAPDH (ratio of protein:GAPDH). (B) Normalized expression levels of BIP and LC3-II in HCT-116 cells treated with BIP specific siRNA. (C) Normalized expression levels of CHOP and LC3-II in HCT-116 cells treated with CHOP specific siRNA. Means and standard deviation (SD) of three independent experiments are shown. One-way ANOVA was used for statistical analysis. * P<0.05, ** p<0.01, compared to the respective control group.
Sodium Butyrate Induces Endoplasmic Reticulum Stress and Autophagy in Colorectal Cells: Implications for Apoptosis. (2016)
Fig 10

Inhibition of autophagy potentiates sodium butyrate-induced apoptotic cell death.

HCT-116 (A) and HT-29 (B) cells were exposed to 5mM 3-MA or 5μM chloroquine (CQ) for 30 min and then treated with 2mM sodium butyrate (NaB) for 24 h. The percentage of apoptotic cells (annexinV+/PI-) was quantified by flow cytometry using an annexinV/PI assay and expressed as mean ± SD of three independent experiments. One-way ANOVA was used for statistical analysis to compare control cells and NaB treatments. *p<0.05, ** p<0.01 compared to control. (C.D.) Cells were treated as described above and the proteins were harvested for Western blot analysis. Expression of full-length and cleaved PARP in HCT-116 (C) or HT-29 (D) cells was compared to GAPDH, the loading control. The ratio of cleaved PARP to total PARP was calculated and the fold change in normalized PARP expression relative to cells treated with 2mM NaB is shown as the mean ± SD of three independent experiments. One-way ANOVA was used for statistical analysis. *P<0.05, ** p<0.01, compared to the 2mM NaB group.
Morphology of the archaellar motor and associated cytoplasmic cone in Thermococcus kodakaraensis (2017)
Figure 4

Figure 4. Structure of the T. kodakaraensis archaellum. (A) A sub-tomogram average of the archaellum reveals structural features, including four layers of density in the cytoplasm (L1-L3, cone). CM, cytoplasmic membrane. The speculated identity of densities in the archaellum is proposed: archaellum fiber = FlaA/B flagellins; integral membrane density = FlaJ; L1 = FlaI; L2/L3/cone = FlaH/FlaC/D/E. (B) For comparison, a subtomogram average of the type IVa pilus machine from Myxococcus xanthus is shown (adapted with permission from [8]). Arrows indicate components with recognized homology. OM, outer membrane; IM, inner membrane. Scale bar 10 nm (applies to A, B).