EMBO/soda-nel huggingface dataset:
Coordinated removal of repressive epigenetic modifications during induced reversal of cell identity (2019)
Figure 1

Figure 1: Kdm3b plays an important role in reprogramming to pluripotency

a. Quantification of NANOG-positive iPSC colonies obtained after 14 days of OSKM induction in MEFs treated with either non-target or Kdm3b siRNA in FBS media. Values represent fold change over non-target control providing relative quantitation. Experimental values are provided in Appendix Fig S1. Error bars represent the standard deviation of three biological replicates. ** p-value < 0.01 by paired t-test

b. Quantification of NANOG-positive iPSC colonies obtained after 12 or 14 days of OSKM induction in MEFs treated with either non-targeting or Kdm3b siRNA in KSR media. Values represent fold change over non-target control providing relative quantitation. Experimental values are provided in Appendix Fig S1. Error bars represent the standard deviation of three biological replicates. ** p-value < 0.01 by paired t-test

c. Quantification of NANOG positive colonies after retroviral expression of OSKM (Left) or OSK (Right) in MEF treated with non-targeting or siRNA targeting Kdm3. Nanog counts were performed on Day 16 for OSKM or Day 21 for OSK reprogramming. Values represent fold change over non-target control providing relative quantitation. Experimental values are provided in Appendix Fig S1. Error bars represent the standard deviation of three biological replicates. * p-value < 0.05 by paired t-test

d. Immunoblot of H3K9me1/2 histone demethylase in WT or Kdm3b CRISPR targeted pre-iPSC clones. These pre-iPSCs carry a Nanog-GFP reporter. Parent line (WT), and two Kdm3b-KO clones using gRNA targeting either exon 1 (KO#1) or exon 2 (KO#2) are shown. RNA Polymerase II levels are the loading control.

e. Quantification of Nanog-GFP positive cells after 10 days of AA+2i treatment of WT or Kdm3b-deleted pre-iPSCs. Error bars represent the standard deviation of three or five biological replicates. *** p-value<0.001 by paired t-test.

f. Immunoblot of H3K9 methylation levels upon addition of H3K9 methyltransferase inhibitor UNC0638 in WT or Kdm3b-KO pre-iPSCs.

g. Quantification of percent Nanog-GFP positive cells after 10 days of AA+2i or AA+2i with 5µM UNC0638 in WT or Kdm3b deleted pre-iPSCs. Error bars represent a standard deviation of three biological replicates. * p-value < 0.05 by paired t-test.
Coordinated removal of repressive epigenetic modifications during induced reversal of cell identity (2019)
Figure 2

Figure 2: Tet1 upregulation is a mediator of Kdm3b function during reprogramming

a. Pearson correlation of transcripts per million (TPM) of WT and Kdm3b-KO pre-iPSC upon DMSO or AA+2i treatment for 2 or 10 days and mESC, reprogramming populations from Chronis et. al (2017), (labelled as CC).

b. k-means clustering of the union of DEG (FDR > 0.99) between day 2 and DMSO in WT and Kdm3b-KO pre-iPSCs. Values represent a log2-fold change of day 2 over DMSO. Key pluripotency genes are shown on the right.

c. k-means clustering of the union of DEG (FDR > 0.99) between day 10 and DMSO in WT and Kdm3b-KO pre-iPSCs. Values represent a log2-fold change of day 10 over DMSO. Key pluripotency genes or epigenetic regulators are shown on the right.

d. Expression of Tet enzymes and Tdg during AA+2i treatment in WT or Kdm3b-KO pre-iPSCs. Error bars represent the standard deviation of two biological replicates from the RNA-Seq sample.

e. Quantification of percent Nanog-GFP cells of WT, Kdm3b-KO and Kdm3b-KO pre-iPSC clones expressing Tet1 catalytic domain (Tet1CD), Nanog, or both. Error bars represent a standard deviation of three biological replicates. * p-value < 0.05 by paired t-test.
Coordinated removal of repressive epigenetic modifications during induced reversal of cell identity (2019)
Figure 4

Figure 4: Active DNA demethylation is important for pluripotency acquisition

a. Representative profiles of regions that undergo demethylation through a 5hmC intermediate. Top - 5hmC profile during AA+2i treatment. Middle - Percent methylation via bisulfite sequencing in MEFs and iPSCs, data from Rais et al 2013. Bottom - Percent 5hmC via Tet-assisted bisulfite sequencing of MEFs expressing OSKM for 0 or 5 days, data from Hu et al 2014.

b. Left - Quantification of percent Nanog-GFP positive colonies. Right - Total Nanog-GFP positive colonies obtained after ten days of AA+2i treatment in Tdg-depleted pre-iPSC (Tdg) or control (Empty). Error bars represent a standard deviation of three biological replicates. * p-value < 0.05- paired t-test.
Coordinated removal of repressive epigenetic modifications during induced reversal of cell identity (2019)
Figure 5

Figure 5: Kdm3b-KO cells retain 5hmC at pluripotency-associated locations where it should be resolved in reprogramming

a. Quantification of percent Nanog-GFP positive cells of WT or Kdm3b-KO pre-IPSC clones expressing Nanog, Tet1CD, or Nanog+Tet1CD after 15 days of AA+2i or AA+2i with 4 or 5µM of UNC0638. Error bars represent standard deviation of 3 biological replicates. ** p < 0.01 by paired t-test.

b. Left - Heatmap of K-mean clustered 5hmC patterns in WT pre-iPSC with 5hmC profile of Kdm3b-KO pre-iPSC treated with AA+2i for 0, 2 or 10 days. Middle - Metaplot of 5hmC signal on day 10 between WT (orange) and Kdm3b-KO (black) for each cluster. Y-axis scale is in normalized coverage data (1x106/Total Count). Right - Illustration of 5hmC pattern in WT and Kdm3b-KO during AA+2i treatment.

c. Representative 5hmC profiles during AA+2i conversion of WT and Kdm3b-KO pre-iPSC.

d. Scheme for identifying genes with both misregulated 5hmC and expression. Heatmap of two-fold misregulated 5hmC and two-fold differential expression between WT and Kdm3b-KO after 10 days of AA+2i treatment (607 genes). Peaks are sorted by 5hmC signal in WT. RNA seq values represent a log2-fold change of day 10 over DMSO for either WT or Kdm3b KO pre-iPSCs.

e. Box plot of expression for genes that are two-fold misregulated 5hmC and expression between WT and Kdm3b-KO on day 10 (607 genes). Middle line designates median value while box ranges between 25th and 75th percentile and whiskers indicate minimum and maximum values. ****p-value<0.0001- paired t-test

f. Gene ontology for genes that are two-fold misregulated 5hmC and expression between WT and Kdm3b-KO on day 10.
Coordinated removal of repressive epigenetic modifications during induced reversal of cell identity (2019)
Figure 6

Figure 6: Ectopic 5hmC retention is incompatible with POU5F1 chromatin association

a. Heatmap of POU5F1 bound peaks in both mESC and pre-IPSC (Cluster 1) and mESC alone (Cluster 2) with 5hmC levels during Day 0, 2 and 10 of AA+2i treatment.

b. Metaplots of POU5F1 binding for Cluster 1 (Left) and Cluster 2 (Right) regions in pre-iPSCs and mESCs.

c. Metaplots of 5hmC Enrichment for Cluster 1 (Left) and Cluster 2 (Right) regions for Day 0, 2 and 10 of AA+2i treatment.

d. Left - Scheme for identifying sites that lose 5hmC and their association with POU5F1 binding. Right - Heatmap of 5hmC profile during AA+2i treatment of WT pre-iPSC and POU5F1 binding in pre-iPSCs or mESCs at the loci identified in the left panel.

e. Representative 5hmC profiles during AA+2i conversion of WT and Kdm3b-KO pre-iPSC with POU5F1 binding in pre-iPSCs and mESCs. Loci were assigned to the nearest gene.

f. ChIP-PCR for POU5F1 during AA+2i reprogramming of WT and Kdm3b-KO pre-iPSCs. Values are fold change of enrichment between WT and Kdm3b KO (WT/Kdm3bKO). Error bars represent a standard deviation of two technical replicates. Three independent reprogramming experiments are shown.
Coordinated removal of repressive epigenetic modifications during induced reversal of cell identity (2019)
Figure 7

Figure 7: Tet1 activation is important at later stages of pluripotency acquisition

a. tSNE of Tet1, Tet2, Nanog, and Cdh1 expression in single cell RNA sequencing of MEF undergoing OSKM reprogramming from Tran et al 2019b. MEF, Reprogramming (REPROG), and mESCs cells are highlighted in red dotted lines. Specific reprogramming timepoints are highlighted within the reprogramming cluster in arrows. Color corresponds to log10 of transcript counts for each individual cell.

b. Bar graphs indicating number of cells expressing Tet1, Tet2, Cdh1 or Nanog from the reprograming population (Day 3, 6, 9 and 12). Number of cells per reprogramming population is highlighted within legend.

c. Scheme for deriving Tet1 and Tet2 double knockout pre-iPSC lines. Tet1KO/KO Tet2fl/fl or Tet1WT/WT Tet2fl/fl MEFs were treated with retroviruses containing OSKM. Tet1KO/KO Tet2fl/fl or Tet1WT/WT Tet2fl/fl pre-iPSC clones were isolated and treated with either Adenoviral expressed Cre recombinase or Empty vector to generate a population of pre-iPSCs with Tet1KO/KO Tet2fl/fl, Tet1KO/KO Tet2KO/KO, Tet1WT/WT Tet2fl/fl, and Tet1WT/WT, and Tet2KO/KO. Scale bar, 50 μm.

d. Quantification of percent Nanog positive cells by intracellular flow cytometry in each genotype as indicated of Tet1WT/WT Tet2fl/fl, Tet1WT/WT Tet2KO/KO, Tet1KO/KO Tet2fl/fl, and Tet1KO/KO Tet2KO/KO after 10 days of AA+2i treatment. Error bars represent a standard deviation of three to six different pre-iPSC biological replicates. p-values were calculated by unpaired t-test. ** p < 0.01.

e. Top - Model for action of Kdm3b and 5hmC during pre-iPSC to iPSC conversion. Kdm3b mediated demethylation of H3K9me2 and 5hmC processing allows recruitment of POU5F1 and pluripotency gene upregulation. In Kdm3b deleted pre-iPSCs, 5hmC is maintained and prevents POU5F1 binding at specific genes. Bottom - Distinct temporal expression of Tet1 and Tet2 during reprogramming.
Targeted delivery of a phosphoinositide 3-kinase inhibitor to restore organ function in sepsis (2021)
Figure 1

Figure 1 Cell-type-specific expression pattern of PI3Kγ in the liver, induction, and function under inflammatory conditions.

(A) PI3Kγ is expressed by human hepatocytes and infiltrating immune cells in biopsies from patients with minimal to mild inflammatory activity. Triangles point to immune cells (clusters), including some neutrophils known to express PI3Kγ highly. In the negative control, the primary antibody was replaced by an equal volume buffer. The number of included patients, gender, and diagnosis are summarized in Appendix Table S1.

(B) PI3Kγ expression in human primary hepatocytes from 20 male (♂) or female (♀) donor pools (HEP, DP20), but not non-parenchymal cells (NPC). Isolated human leukocytes from healthy volunteers (LEU) served as positive controls. The characterization of the donor pools is summarized in Appendix Table S2.

(C) Primary murine hepatocytes and HepG2 cells expressed PI3Kγ under basal conditions; expression was increased at 24 h after stimulation with LPS, IFN-γ, IL-1β, and TNF-α (CM).

(D) Survival of WT, PI3Kγ null (left) and liver-specific PI3Kγ knockout mice (PI3Kγ floxtg/tg x AlbCre(tg)/tg, middle) or systemic application of the PI3Kγ inhibitor, AS605240 (right) in a model of polymicrobial sepsis induced by peritoneal contamination with a human stool suspension. Animal numbers: B6: 86 (%male: 52, %female: 48), B6-Pi3kcgko/ko: 59 (%male: 51, %female 49), B6-Pi3kcgflox/flox x AlbCre(tg)/tg: 25 (%male: 68, %female 32), FVB/N Vehicle: 25 (%male: 44, %female 56), FVB/N AS: 43 (%male: 44, %female 56)
Targeted delivery of a phosphoinositide 3-kinase inhibitor to restore organ function in sepsis (2021)
Figure 2

Figure 2. Formulation of targeted liposomes, in vitro uptake characteristics, and in vivo pharmacokinetic and pharmacodynamic properties.

(A) Scheme for the preparation of DY-635 targeted liposomes containing the PI3Kγ inhibitor AS605240 (T-LipoAS). LC-MS determined concentrations of AS605240 and DY-635 individually prepared batches used for all subsequent experiments. The graph depicts the mean ± SD from five different batches.

(B) Hydrodynamic diameters of different liposomes in histidine buffer were analyzed by dynamic light scattering. The graph depicts representative plots obtained from dynamic light scattering in Histidine buffer. Further, all batches in this study have been analyzed for size by the manufacturer using DLS and were confirmed to be stable in solution for at least one year.

(C) Uptake characteristics of T-LipoAS by primary human hepatocytes demonstrate competitive inhibition by cyclosporin A- and energy dependence, confirming active uptake by organic anion transporters (OATPs). The data points are depicted mean ± SD from two batches of primary human hepatocytes in quadruples.

(D) T-LipoAS plasma disappearance rate in murine ear veins and estimated half-life obtained by intravital microscopy. The mean fluorescence intensity averaged between the remaining 24 regions of interest (from n = 4 FVB/N mice) and the positive standard error depicted in the figure.

(E) Quantification of AS60524060 min after injection of 1 mg kg-1 AS605240 (i.p. in DMSO, n=4 animals) or 2.5 mg kg-1 T-LipoAS (i.v. n=5 animals) in tissue lysates quantified by LC-MS. Bars depict the mean + SD for each group. *p<0.05 for Wilcoxon test between T-LipoAS and AS605240 in each tissue.

(F) T-LipoAS effectively inhibits fMLP-stimulated PI3Kγ, reflected by inhibiting AKT S473 phosphorylation activity in hepatocytes from three different batchtes. The mean pAKT/AKT ratio and standard deviation (SD) were calculated for all experiments separately.

(G) Episcopic imaging of mice treated with T-LipoAS for 15 or 60 min and relative concentration of the targeting moiety DY-635 in the different compartments.

(H) Intravital microscopy to monitor T-LipoAS kinetics in the liver. Liposomes appear in the sinusoidal vasculature after 5-15 minutes and accumulate within hepatocytes. After 35-60 min, the bile canalicular system appears stained, indicating the processing and elimination of the targeting moiety (DY-635) from T-LipoAS. Similar distribution patterns had been observed in all repetitions (n=7).
Targeted delivery of a phosphoinositide 3-kinase inhibitor to restore organ function in sepsis (2021)
Figure 4

Figure 4. Nanoformulated PI3Kγ inhibition restores excretory liver function in vivo.

(A) Intravital microscopy of elimination of CDFDA, a fluorescent substrate subject to excretory elimination by hepatocytes via Mrp-2, allows direct visualization of excretory function in sham or septic (PCI) animals treated with vehicle or a hepatocyte-directed inhibitor of PI3Kγ (T-LipoAS).

(B) Normalized CDFDA intensities quantified from intravital images depict plasma disappearance curves (A, B) Representative intravital images and analysis (mean ±SD) from 5 (sham), 4 (PCI T-LipoAS) and 3 (PCI Veh) animals. Sham and PCI animals treated with T-LipoAS showed a classical biphasic decay curve, while PCI vehicles showed a relatively linear decay and a higher area under the curve indicative of excretory liver failure.

(C) Similar results were obtained when the liver excretory function was tested by indocyanine green clearance. Indocyanine green was detected non-invasively by multispectral optoacoustic tomography. The ability to clear ICG was quantitatively analyzed by comparing areas under the curve (AUC). Groups of 9 (sham vehicle), 8 (sham LipoAS), 12 (PCI vehicle) and 15 (PCI LipoAS) animals were analyzed. Bars depict mean + SD.

(D) Representative transmission electron micrographs from bile canaliculi of healthy control (sham) and septic animals (PCI) treated with vehicle or the PI3Kγ inhibitors, free AS, or T-LipoAS. The asterisk marks a bile canaliculus where typical brush borders are lost due to sepsis.
Bulk RNA degradation by nitrogen starvation‐induced autophagy in yeast (2014)
Figure 1

Dynamics of intracellular nucleosides under starvation

A Electron microscopic analyses to see autophagic bodies in the vacuoles under both growth and nitrogen starvation conditions. Autophagic bodies typically contain cytosolic ribosomes. pep4∆prb1∆ and pep4∆prb1∆atg2∆ cells were grown in SD to mid‐log phase (OD600 = 1) and transferred to SD(‐N) for 5 h. The cells were examined by transmission electron microscopy as described in . Scale bar, 500 nm.
B–D Time‐dependent changes in nucleoside contents under nitrogen starvation (B), rapamycin treatment (C), and carbon starvation (D) conditions. Wild‐type and atg2Δ cells were grown in SD and transferred to SD(‐N), SD medium with 0.2 μM rapamycin, or SD(‐C) at time 0. At the indicated time points, nucleosides were analyzed by LC/MS as described in . Results are presented as normalized intensities on the basis of peak height of each metabolite in wild‐type cells. All data are means of triplicate samples. The error bars represent the standard deviation.
Bulk RNA degradation by nitrogen starvation‐induced autophagy in yeast (2014)
Figure 2

Changes in intracellular nucleoside levels under nitrogen starvation

Time‐dependent changes in nucleoside contents under nitrogen starvation. Nucleosides in wild‐type, atg2Δ, atg7Δ, atg11Δ, atg17Δ, atg19Δ, atg32Δ, nvj1Δ, and pep4prb1Δ cells under nitrogen starvation. All data are means of triplicates. The error bars represent the standard deviation.
Alkaline phosphatase (ALP) assay (Pho8∆60). Wild‐type, atg2∆, atg11Δ, atg17Δ, and atg19Δ cells expressing Pho8∆60 were grown in SD to mid‐log phase and transferred to SD(‐N) at time 0. At the indicated time points, lysates were prepared and subjected to the ALP assay. The bars represent the standard deviation of three independent experiments. *P 0.05; **P 0.005; ***P 0.0005 (paired t‐test, two‐tailed)
Bulk RNA degradation by nitrogen starvation‐induced autophagy in yeast (2014)
Figure 3

Changes in intracellular nucleotide and nucleoside levels under nitrogen starvation

A–C Wild‐type, atg2Δ, pho8Δ, and rny1Δ cells were grown in SD to mid‐log phase and transferred to SD(‐N) at time 0. At the indicated time points, nucleosides (A), 5′‐NMPs (B), and 3′‐NMPs (C) were analyzed by LC/MS as described in . The results are presented as normalized intensities on the basis of the peak height of each metabolite in wild‐type cells. Note that 3′‐NMPs in pho8Δ are presented in a different scale. All data are means of triplicates. The error bars represent the standard deviation.
Bulk RNA degradation by nitrogen starvation‐induced autophagy in yeast (2014)
Figure 4

Accumulation of RNA in the vacuole in rny1∆ cells under nitrogen starvation

A, B Expression and localization of Rny1 and Pho8. Wild‐type cells and atg2∆ cells expressing Rny1‐GFP or Pho8‐GFP were grown in SD to mid‐log phase and transferred to SD(‐N). After 2 h of starvation, GFP‐tagged proteins were observed by fluorescence microscopy (A) or analyzed by Western blot with anti‐GFP antibody (B). Scale bar, 5 μm.
C Detection of free RNA. Wild‐type, atg2∆, rny1∆, and rny1∆atg2∆ cells grown in SD (left) or SD(‐N) for 2 h (right) were stained with FM4‐64 and GR Green and observed under a fluorescence microscope. Scale bar, 5 μm.
Bulk RNA degradation by nitrogen starvation‐induced autophagy in yeast (2014)
Figure 5

Evaluation of ribophagy on RNA degradation under nitrogen starvation

Time‐dependent change in nucleoside levels after nitrogen starvation. Wild‐type, atg2∆, ubp3∆, and bre5∆ cells were grown in SD to mid‐log phase and transferred to SD(‐N) at time 0. Metabolites were analyzed by LC/MS as described in . The results are presented as normalized intensities on the basis of the peak height of each metabolite in wild‐type cells. All data are means of triplicates. The error bars represent the standard deviation.
Detection of free RNA within cells. rny1∆, rny1∆atg2∆, rny1∆ubp3∆, and rny1∆bre5∆ cells grown in SD(‐N) for 2 h were stained with FM4‐64 and GR Green and observed under a fluorescence microscope. Scale bar, 5 μm.
Bulk RNA degradation by nitrogen starvation‐induced autophagy in yeast (2014)
Figure 6

Quantitative analysis of nucleotides, nucleosides, and bases at their destinations

A Schematic representation of reactions of the nucleotide degradation pathway.
B, C Nucleosides (B) and nucleobases (C) within the cells. Wild‐type and atg2∆ were grown in SD to mid‐log phase and transferred to SD(‐N) at time 0. At the indicated time points, the cells were collected; their metabolites were extracted, and the nucleosides and nucleobases were analyzed by LC/MS. The level of each nucleoside and base is presented as its absolute concentration, as described in and Supplementary Methods. All data are means of duplicates except the wild‐type strain at 1 h.
D, E Nucleosides (D) and nucleobases (E) in the medium. The culture medium of the above cultures was filtered, and nucleosides and nucleobases were directly analyzed by LC/MS as described in and Supplementary Methods. All data are means of duplicates.
F, G Destinations of nucleosides and bases. Based on the data from (B–E), the level of each nucleoside and base in cells (open circle) and in cells and medium (closed circle) of wild‐type strain were estimated. See Supplementary Methods in detail (flow chart of the calculation procedure).
Bulk RNA degradation by nitrogen starvation‐induced autophagy in yeast (2014)
Figure 7

RNA degradation via autophagy in S. pombe

Wild‐type and atg1Δ of S. pombe were grown in SD to mid‐log phase and transferred to SD(‐N) at time 0. At the indicated time points, the cells were collected; the metabolites in these cells were extracted and analyzed by LC/MS as described in . The results are presented as normalized intensities on the basis of peak height of each metabolite in wild‐type cells. All data are means of triplicates. The error bars represent the standard deviation.
Niacin-mediated Tace activation ameliorates CMT neuropathies with focal hypermyelination (2016)
Figure 1

Figure 1. Expression levels of Tace and phosphorylation of ErbB2 in Mtmr2-/- sciatic nerves and Schwann cell/DRG neuron co-cultures.

A. Western blot analysis of Tace using lysates from WT and TaceFl/Fl;P0-Cre mouse sciatic nerves at P10 (65ug of total protein lysate loaded). The arrowhead indicates the pro-protein and the arrow shows the cleaved active form of Tace. Representative of two independent experiments.

B. Western blot analysis (50ug of total protein lysate loaded) shows that Tace expression in axons is maximal at P5-P10 and then progressively declines in the adult. Representative of two independent experiments.

C. Western blot analysis of Tace in Mtmr2-/- sciatic nerves at P5, P10, and P20. Representative of two independent experiments.

D. Western blot analysis of Tace using lysates from Mtmr2-/- mouse sciatic nerves at 5 months, with quantification, p=0.10, two-tailed Mann Whitney U test. Representative of two independent experiments.

E. Phosphorylation of ErbB2 in Mtmr2-/- sciatic nerves at P2, P10 and P15. At P2, each lane is a pool of n=7 animals per genotype, representative of six independent experiments using n=6 different nerve pools per genotype, and quantification using WT values arbitrary set to1, p=0.0313, Wilcoxon Rank Sum test. At P10 and P15, mean values of two samples are shown. At P10, each lane is a pool of n=3/4 animals per genotype, representative of three independent experiments using n=6 different pools per genotype. Nf-l is the neurofilament light chain and Clx is calnexin.

F. Phosphorylation of ErbB2 in Mtmr2-/- Schwann cell/DRG neuron co-cultures after 4 days of ascorbic acid treatment. Each lane is a pool from at least 10 coverslips/DRG per genotype. Representative of two independent experiments using n=4 different pools of coverslips/DRG per genotype, p=0.20, two-tailed Mann Whitney U test.