EMBO/soda-nel huggingface dataset:
Tnfaip2/Exoc3 driven lipid metabolism is essential for stem cell differentiation & organ homeostasis (2020)
Figure 1

Figure 1. Knockdown of Tnfaip2 enhances reprogramming of MEFs into iPSCs. (A) Stable RNAi screening of cancer-related genes was conducted to identify gene knockdowns that enhance reprogramming of MEFs into iPSCs. The graph shows the 10 shRNAs that were most strongly, positively selected in iPSCs compared to non-reprogrammed cells on day 14 of reprogramming (n=3 biological replicates). (B-E) Mouse embryonic fibroblasts (MEFs) were co-infected with shRNAs against Tnfaip2 or a scramble shRNA and reprogramming factors. The co-transduced cells were sorted and were reprogrammed for 14 days: (B) Representative images and (C) quantification of alkaline-phosphatase-positive (AP+) iPSC colonies on day 14 of reprogramming by transfection of 4 reprogramming factors (=4F: Oct4, Sox2, Klf4, c-Myc = OSKM) or 3 factors (=3F: OSK). [n=3 biological replicates per group; data were normally distributed (P>0.05 as per Shapiro-Wilk test) and analyzed by one-sided t-test analyzed by one-sided t-test with Holm-Sidak correction for multiple testing]. (D) Representative FACS profiles and (E) percentage of Oct4-GFP+ iPSCs obtained after 14 days of reprogramming of MEFs from Oct4-eGFP mice using 4F-transduction on day zero that were infected with shRNAs targeting Tnfaip2 or a scrambled shRNA control [n=5 biological replicates; data were normally distributed (P>0.05 as per Shapiro-Wilk test) and analyzed by one-sided t-test with Holm-Sidak correction for multiple testing]. (F) mRNA of Tnfaip2 was measured by RT-qPCR at the indicated days of reprogramming by 4F-transduction on day zero [n=3 biological replicates; log-transformed data were not normally distributed (P>0.05 as per Shapiro-Wilk test), P value for the upregulation of Tnfaip2 on consecutive test days-9,-12 and -14 was calculated starting with the probability of maximum rank-based difference, i.e. having two groups of three data points perfectly separating, p1,b = 0.1 (bidirectional) and p1,u = 0.05 (unidirectional); the probability of finding a triple series of max. difference starting at some timepoint is Ptriple,tp = p1,b · p1,u · p1,u, and finding such a triple somewhere across the 5-step time series is Ptriple = Ptriple,tp + 2 · (1- p1,u) · Ptriple,tp = 0.0007.
Tnfaip2/Exoc3 driven lipid metabolism is essential for stem cell differentiation & organ homeostasis (2020)
Figure 3

Figure 3. Smed-exoc3 repression increases relative number of stem cells in planarians and reduces the expression of progenitor cell markers. (A-F) Planarians were injected with Smed-exoc3 dsRNA = exoc3(RNAi) or with control gfp(RNAi) injections. (A) The representative photographs show staining for phosphorylation of 10th serine residue of Histone 3 (H3S10p, a stem cell marker in planarians) at days-38 of the injection protocol (n=3 experimental replicates with 8-10 biological replicates per experiment, scale bar: 0.5 mm). (B-F) exoc3(RNAi)-treated planarians versus gfp(RNAi)-treated controls were analyzed at the indicated time-points of the dsRNA injection protocol: (B) Quantification of H3S10p+ cells per mm2 in planarians at day-30, -34 and -38 of the injection protocol [n=3 experimental replicates with 5-7 biological replicates per experiment; data were normally distributed (P>0.05 as per Shapiro-Wilk test) and analyzed by multiple t-tests with Holm-Sidak correction for multiple comparisons]. (C,D) FACS-based time-course analysis on changes in the fraction of somatic stem cells (X1-population) at indicated time points. Cells were obtained from whole body-trypsinized planarians of gfp(RNAi)-injected controls and exoc3(RNAi)-treated planarians and stained with the cytoplasmic dye Calcein-AM and the nuclear (DNA) dye Hoechst 33342: (C) Representative FACS profiles and (D) quantification of the relative number of X1 cells [n=3 biological replicates; data were normally distributed (P>0.05 as per Shapiro-Wilk test) and analyzed by multiple t-tests with Holm-Sidak correction for multiple comparisons]. (E,F) RT-qPCR analysis for makers of differentiating progenitor cells from the epidermal lineage on total (whole body derived) RNA from exoc3(RNAi)-treated planarians (E) Expression analysis of differentiation marker prog-2 on day-38 of the injection scheme [n=3 biological replicates; log-transformed data were normally distributed (P>0.05 as per Shapiro-Wilk test and analyzed by one-sided t-test], (F) Expression analysis of marker genes of differentiated cells (prog-1 and Smed-odc-1; Zhu et al, 2015) on day-30, -34 and -38 of the injection scheme [n= 3 biological replicates per group and time point except for prog-1 expression at day 38, which includes 6 biological replicates; log-transformed data were normally distributed (P>0.05 as per Shapiro-Wilk test) and analyzed by multiple t-tests with Holm-Sidak correction for multiple comparisons]. For Smed-odc-1 log-transformed mean centered group data were normally distributed and (P>0.05 as per Shapiro-Wilk test) and analyzed by multiple t-tests with Holm-Sidak correction for multiple comparisons].
Tnfaip2/Exoc3 driven lipid metabolism is essential for stem cell differentiation & organ homeostasis (2020)
Figure 4

Figure 4. Suppression of Smed-exoc3 abrogates organ homeostasis in planarians. (A,B) Planarians were injected with exoc3(RNAi) or with control gfp(RNAi) injections. Organ phenotypes were analyzed on day-38 of the injection protocol. Organ homeostasis was analyzed by staining against specific markers: (A) The structure of the brain/central nervous system (CNS) was analyzed by staining against synapsin (3C11). The cartoon represents the phenotypic change (upper panel). Representative photographs are shown in the lower panel (3 experimental replicates with 2-3 biological replicates per experiment, scale bar: 0.5 mm). Number ratios in the photographs indicate the total number of animals exhibiting the represented phenotype. There was a significant increase in the occurrence of atrophic cephalic ganglia and sensory neuron loss in exoc3(RNAi)-treated planarians vs. controls (cg: cephalic ganglia; sn: sensory neurons). (B) Planarian visual system was analyzed by staining against VC1. Schematic cartoon (upper panel) and representative images (lower panel) are shown (3 experimental replicates with 3 biological replicates per experiment, scale bar: 0.5 mm). Number ratios in the photographs indicate the total number of animals exhibiting the represented phenotype. Arrows point to photosensitive cells (ps); pigmentary cells (pc) and to the optic chiasm (oc).
Tnfaip2/Exoc3 driven lipid metabolism is essential for stem cell differentiation & organ homeostasis (2020)
Figure 7

Figure 7. Tnfaip2 deletion abrogates differentiation potential of ESCs. (A-F) One thousand ESCs of the indicated genotypes were placed into hanging-drop cultures to form embryoid bodies (EBs). After 3 days (referred to as day-0), differentiation was induced by exposure to differentiation medium (days-1 to days-3 after medium change). (A) Representative photographs of EBs of the indicated genotypes at day-0 and day-3 after differentiation induction (experiments were conducted in 3 biological replicates; n=5-42 EBs per biological replicate, scale bar: 0.02 mm). (B-D) RNA-seq analysis was conducted on Tnfaip2-/- EBs vs. WT EBs on day-3 of differentiation induction of EBs (n=3 biological replicates). The heat maps depict the intersection of DEGs with the following gene sets retrieved from the AmiGO 2 data base (Carbon et al., 2009): (B) "stem cell maintenance: positive and negative regulators", (C) "mesoderm development" and (D) "ectoderm development" from the AmiGO 2 data base (Carbon et al., 2009). Tnfaip2 and 5 other stem cell-related genes (Tfcp2l1, Dppa3, Fbxo15, Zfp42 and Klf2) were additionally incorporated based on literature searches. The color scale represents the gene-wise z-score calculated from normalized gene expression levels. The asterisk refers to the gene identified in this study. (E) Representative image of immunofluorescence staining against the ectodermal marker (SOX1) and the mesodermal marker (T alias Brachyury) in WT and Tnfaip2-/- EBs on day-3 of differentiation induction. (3 repeat experiments were conducted on a total number of 9-10 EBs per genotype, scale bar: 20µm). (F) mRNA expression of Tnfaip2 measured by RT-qPCR in ESCs on the indicated days of differentiation induction of WT EBs [n=3 biological replicates; log-transformed data were normally distributed (P>0.05 as per Shapiro-Wilk test), the P value for the upregulation of Tnfaip2 on consecutive time points (day-0, -2 and -3) was calculated starting with the probability of maximum rank-based difference, i.e. having two groups of three data points perfectly separating, p1,b = 0.1 (bidirectional) and P1,u = 0.05 (unidirectional); the probability of finding a triple series of max. difference starting at some timepoint is Ptriple,tp = p1,b · p1,u · p1,u, and finding such a triple somewhere across the 4-step time series is Ptriple = Ptriple,tp + (1- p1,u) · Ptriple,tp = 0.0005.
Tnfaip2/Exoc3 driven lipid metabolism is essential for stem cell differentiation & organ homeostasis (2020)
Figure 9

Figure 9. Restoration of differentiation capacity of Tnfaip2-/- ESCs by TAG supplementation. (A) Heatmap of CPT1A protein expression as measured in proteome analysis of Tnfaip2-/- and WT ESCs and during differentiation induction of EB cultures (n=5 biological replicates). The colour scale represents average log2ratios. (B) Expression of Cpt1a mRNA as determined by qRT-PCR in WT and Tnfaip2-/- EBs at the indicated days of differentiation induction [n=3 biological replicates; log-transformed data were normally distributed (P>0.05 as per Shapiro-Wilk test) and analyzed by multiple t-tests with Holm-Sidak correction for multiple comparisons]. (C-H) Differentiation-induced WT EBs and Tnfaip2-/- EBs were treated with palmitic acid (PA) and palmitoyl-L-carnitine (PC) (8μM each) or a vehicle control (C,D) The diameters of the rosette structure of differentiated WT EBs and Tnfaip2-/- EBs were measured on day-3 after differentiation induction. (n=3 repeat experiments with 7-62 EBs per experiment per group): (C) Representative photographs (scale bar: 0.02 mm) and (D) quantification of the diameter of the rosette structure of the indicated groups [data were not normally distributed (P<0.05 as per Shapiro-Wilk test) and thus analyzed by Mann-Whitney U test with Holm Sidak correction for multiple comparisons]. (E-G) RNA-seq was conducted on day-3 of differentiation induction of Tnfaip2-/- EBs that were either treated with PA/PC or with a vehicle control (n=3 independent pools of 7-62 EBs per group). The heat maps depict the intersection of DEGs with the following gene sets retrieved from the AmiGO 2 data base (Carbon et al., 2009): (E) "stem cell maintenance: positive and negative regulators", (F) "mesoderm development" and (G) "ectoderm development". Five other stem cell-related genes (Tfcp2l1, Dppa3, Fbxo15, Zfp42 and Klf2) were additionally incorporated based on literature searches. The asterisks indicate the reverted changes in gene expression that were seen in Tnfaip2-/- EBs versus WT EBs depicted in Figure 7B-D. The color scale represents the gene-wise z-score calculated from normalized gene expression levels. (H) Representative image of immunofluorescence staining against ectodermal marker (SOX1) and the mesodermal marker (T alias Brachyury) on day-3 of differentiation induction of Tnfaip2-/- EBs +/- co-treatment with PA/PC. Three repeat experiments were conducted with a total number of 9 EBs per group, same experiment as in Fig. 7E (scale bar: 20µm).
Tnfaip2/Exoc3 driven lipid metabolism is essential for stem cell differentiation & organ homeostasis (2020)
Figure 10

Figure 10. Tnfaip2 and Vim act epistatically to impair cellular reprogramming.

(A-G) Oct4-eGFP reporter MEFs or WT MEFs were infected with (A-C) shRNAs or (D,E) combinations of shRNAs. Cells were co-infected with 4 reprogramming factors (OSKM) and double-infected cells were FACS sorted and grown for 14 days, when reprogramming efficiencies were determined by FACS. (A) Representative images of AP staining of iPSC colonies and (B) representative FACS profiles of Oct4-positive iPSCs on day 14, (C) quantification of the percentage of Oct4-GFP+ iPSCs in MEF cultures that were infected with 2 different shRNAs targeting Vim or a scrambled shRNA control [n=3 biological replicates per group; data were normally distributed (P>0.05 as per Shapiro-Wilk test) and analyzed by t-test with Holm-Sidak correction multiple comparisons]. (D) Representative FACS profiles of mouse-specific pluripotency cell surface marker SSEA1+ iPSCs on day 14 of reprogramming and (E) quantification of the percentage of SSEA1+ iPSCs in MEF cultures that were infected with the indicated shRNAs targeting Vim and/or Tnfaip2 [n=5 biological replicates; data were normally distributed (P>0.05 as per Shapiro-Wilk test) but showed unequal variance; data were analyzed by Brown-Forsythe and Welch ANOVA and Dunnett's multiple comparisons test]. (F, G) RT-qPCR was performed to determine (F) Vim mRNA expression in scrambled-shRNA infected MEFs versus shRNA-Tnfaip2-infected MEFs or (G) Tnfaip2 mRNA expression in scrambled-shRNA infected MEFs versus shRNA-Vim-infected MEFs. (F,G) Infected cells were purified by BFP-sorting on day-4 after infection [n=3 biological replicates; log transformed data were normally distributed (P>0.05 as per Shapiro-Wilk test) and analyzed by t-test with Holm-Sidak for multiple comparisons].
Tnfaip2/Exoc3 driven lipid metabolism is essential for stem cell differentiation & organ homeostasis (2020)
Figure 11

Figure 11. Vim and Cpt1a acts downstream of Tnfaip2 in impairing cellular reprogramming. Oct4-eGFP reporter MEFs or WT MEFs were (A-D) infected with a vector expressing the indicated shRNA in combination with the indicated cDNA (OE indicates overexpression), or (E,F) infected with the indicated shRNA with or without combined treatment with Etomoxir - a chemical inhibitor of CPT1A. (A-F) Cells were co-infected with 4 reprogramming factors (4F= Oct4, Sox2, Klf4, c-myc = OSKM) and double-infected cells were FACS sorted for analysis. (A) RT-qPCR analysis on day-4 after infection to determine RNA expression of Tnfaip2 or Vim in Tnfaip2-depleted cells with ectopic expression of Vim (left) or in Vim-depleted cells with ectopic expression of Tnfaip2 (right). Scramble shRNA (shScr)-infected MEFs served as a control [n=3 biological replicates; log-transformed data were normally distributed (P>0.05 as per Shapiro-Wilk test) and analyzed by t-test]. (B) RT-qPCR analysis on day-4 after infection to determine RNA expression of Vim or Tnfaip2 in Tnfaip2-depleted cells with ectopic expression of Vim (left) or in Vim-depleted cells with ectopic expression of Tnfaip2 (right). Scramble shRNA (shScr)-infected MEFs served as a control [n=3 biological replicates; log-transformed data were normally distributed (P>0.05 as per Shapiro-Wilk test) and analyzed by t-test]. (C) Representative images of FACS profiles and (D) quantification of SSEA1+ iPSCs on day 14 after infection of MEF cultures with the indicated combination of shRNAs plus cDNAs (OE) or scrambled shRNA control [n=3 independent cultures per group; data were normally distributed (P>0.05 as per Shapiro-Wilk test) and analyzed by one-way ANOVA and Dunnett's multiple comparison test ] (E) Representative FACS profiles and (F) quantification of the percentage of SSEA1+ iPSCs in MEF cultures on day-18 after infection with shRNAs targeting Tnfaip2 or a scrambled shRNA control with or without continuous treatment with Etomoxir [n=5 biological replicates; data were not normally distributed (P<0.05 as per Shapiro-Wilk test) and analyzed by Mann-Whitney U test with Holm Sidak correction for multiple comparisons].
Caspase-8 in endothelial cells maintains gut homeostasis and prevents small bowel inflammation in mice (2022)
Figure 1

Fig. 1: Acute loss of Casp8 in adult ECs is lethal and impairs intestine homeostasis.

A. Schematic representation of Casp8 deletion in adult ECs indicating the time points for tamoxifen treatment, analysis of the phenotype (early (day 15) or late (day 18)), and the timeframe of disease progression.

B-C. Graphs showing the relative weight (B; n= 15 WT, 12 ECko; curve comparison) and the survival (C; n= 15 WT, 11 ECko; Log-Rank test) of mice upon tamoxifen treatment.

D. Representative images of the intestine of Casp8WT and Casp8ECko mice, with mild and severe hemorrhages at a late disease stage.

E-F. Representative images of H&E staining (E) and quantification of intestinal pathology (F) of the small intestine at an early disease stage in Casp8WT and Casp8ECko mice (enteropathy score; n= 6 WT, 6 ECko; two tailed unpaired Student's t-test). Scale bars: 100µm; insets 50µm.

G-H. Quantification of intestinal pathology (enteropathy score, G; n= 8 WT, 7 ECko; two tailed unpaired Student's t-test) and hemorrhages into the mucosa (H; n= 4 WT, 5 ECko; two tailed unpaired Student's t-test) at a late disease stage in Casp8WT and Casp8ECko mice.

I. Overview of H&E stained swiss roll preparations of Casp8WT and Casp8ECko mice. Scale bars: 2mm.

J. Representative images of H&E staining of the small intestine of Casp8WT (j') and Casp8ECko mice with mild (j'') and more severely (j''') affected areas at a late disease stage (white arrows point to hemorrhages, asterisk points to focal erosion of the epithelium). Scale bars: 100µm.

Data information: Data shown as mean ± SEM. **P <0.01, *** P <0.001, **** P <0.0001, ns: not significant.
Caspase-8 in endothelial cells maintains gut homeostasis and prevents small bowel inflammation in mice (2022)
Figure 2

Fig. 2: Loss of Casp8 in ECs leads to small bowel inflammation.

A-B. QPCR analysis of genes associated to immune and bacterial response (A), vessel permeability and EC activation (B) in small intestine lysates at a late disease timepoint in Casp8WT and Casp8ECko mice (n= 4 WT, 3-4 ECko, biological replicates, multiple t-test with Holm-Sidak method).

C-D. Graphs revealing that significantly upregulated cytokines in the proteome profiler in Casp8ECko mice are associated to the complement cascade (C) and mucosal healing (D) (n= 4 WT, 4 ECko, multiple t-test with Holm- Sidak method).

E-F. Representative images (E; area between dotted lines shows crypts) and quantification (F) of Ki67 staining in small intestine at an early disease stage in Casp8WT and Casp8ECko mice (n= 5 WT, 6 ECko two tailed unpaired Student's t-test). Scale bars: 100µm.

G. QPCR analysis of the proliferation markers Ki67 and c-myc in ileal lysates from late disease stage Casp8WT and Casp8ECko mice (n= 4 WT, 3-4ECko, two-way ANOVA with Sidak method).

H. Representative images of E-Cadherin staining, showing intact epithelial barrier integrity in Casp8ECko mice in healthy areas (h'), and disrupted barrier integrity in intestinal lesions (h'') at late disease stages compared to Casp8WT mice (n= 4 WT, 4 ECko). Scale bars: 100µm.

I. Representative pictures of CD45 and NIMPR14 staining at late disease stages (n= 5 WT, 5 ECko), showing accumulation of CD45+ and NIMPR14+ cells (black arrowheads) in Casp8ECko intestine at sites of impaired epithelial barrier integrity and tissue necrosis (red asterisks). Scale bars: 100µm.

J. Representative images of CD4, F4/80 and MPO stainings in small intestines at a late disease stage showing accumulation of these cells at the crypt base in Casp8ECko mice (arrowheads) (n= 4 WT, 4 ECko). Scale bars: 50µm.

Data information: Data shown as mean ± SEM. * P <0.05, ** P <0.01, *** P <0.001, **** P <0.0001.
Caspase-8 in endothelial cells maintains gut homeostasis and prevents small bowel inflammation in mice (2022)
Figure 3

Fig. 3: Dysfunction of the lymphatic vasculature and GVB breakdown precede disease manifestation in Casp8ECko mice.

A. Representative pictures showing Peyer´s Patches in Casp8WT mice, and different severities of hemorrhage formation in Casp8ECko mice at an early disease stage. Scale bars: 1mm.

B-C. Quantification of lymphatic vessel density (B) and representative pictures of CD31 (pan EC) and Lyve1 (lymphatic) staining (C) in Peyer's Patches in Casp8WT and Casp8ECko mice at an early disease stage (n = 9 WT, 7-10 ECko Peyer's Patches are pooled from 4-6 mice per genotype; one-way ANOVA with tukey's multiple comparison). Scale bars: 200µm.

D-F. Representative images (D) and quantifications (E, F) of stainings for CD31, Lyve1 and DAPI (nuclei) in small intestinal wholemounts at an early disease stage (blue arrowheads point to defective lacteals, white arrowheads indicate edema) in Casp8ECko mice (E; n= 7 WT, 6 ECko. F; n= 7 WT, 10 ECko; two tailed unpaired Student's t-test). Scale bars: 100µm.

G. Representative images of small intestine sections of mice injected with a 70kDa fluorescently labeled dextran and stained for CD31 at an early disease stage. White arrowheads point to extravasated dextran in Casp8ECko mice (n= 5 WT, 5 ECko). Scale bars: 50µm.

H. Quantification of extravasated dextran in small intestine and brain lysates in Casp8WT and Casp8ECko mice at an early disease stage (n= 4 WT, 4-5 ECko; two-way ANOVA with tukey's multiple comparison). RFU: relative fluorescent units.

I. Representative images of CD31 and VE-Cadherin staining in intestinal wholemounts at an early disease stage (n= 5 WT, 5 ECko) in Casp8WT and Casp8ECko mice (red arrowheads point to VE-Cadherin 'empty' vessel patches). Scale bars: 25µm.

Data information: Data shown as mean ± SEM. * P <0.05, ** P <0.01, *** P <0.001, **** P <0.0001, ns: not significant.
Caspase-8 in endothelial cells maintains gut homeostasis and prevents small bowel inflammation in mice (2022)
Figure 4

Fig. 4: Vascular dysfunction is rescued, and intestine homeostasis restored in Casp8ECko/ MLKLko mice.

A-B. Representative images (A) and quantification (B) of CD31, TUNEL and DAPI staining in small intestine sections of Casp8WT and Casp8ECko mice at an early disease stage (red dotted lines: epithelium; white dotted lines: endothelium) in (n= 5 WT, 5 ECko, multiple t-tests with Holm-Sidak method). Scale bars: 100µm.

C. Schematic representation of the strategy of tamoxifen treatment and timepoint of analysis in Casp8WT/MLKLko or Casp8ECko/MLKLko mice.

D-E. Graphs showing survival and relative weight of Casp8WT/MLKLko and Casp8ECko/MLKLko mice upon tamoxifen treatment. (D; n= 5-8 WT/ MLKLko, 5-8 ECko/MLKLko; Log-Rank test, E; n= 7 WT, 8 ECko; two tailed unpaired Student's t-test).

F. Representative pictures of intestines of Casp8WT/MLKLko and Casp8ECko/MLKLko mice at 30 days after tamoxifen treatment (n= 8 WT, 8 ECko).

G. Representative images of stainings for CD31, Lyve1 and DAPI in small intestinal wholemounts from Casp8WT/MLKLko and Casp8ECko/MLKLko mice at 30 days after tamoxifen treatment. Scale bars: 100µm.

H-K. Quantification of vessel area (H, I) and lacteal length (J, K) at two different timepoints after tamoxifen treatment in Casp8WT/MLKLko and Casp8ECko/MLKLko mice (n= 4-6 WT/MLKLko, 5 ECko/MLKLko; two tailed unpaired Student's t-test).

L. Graph showing quantification of extravasated 70kDa Dextran from small intestine tissue of Casp8WT/MLKLko and Casp8ECko/MLKLko mice at 30 days after tamoxifen treatment (n= 7 WT/MLKLko, 6 ECko/MLKLko; two tailed unpaired Student's t-test).

M-N. Representative pictures (M) and quantification (N) of lymphatic vessel density in Peyer's Patches of Casp8WT/MLKLko and Casp8ECko/MLKLko mice at 140 days after tamoxifen treatment (n = 8 WT, 7 ECko Peyer's Patches are pooled from 4 mice per genotype; two-tailed unpaired students t-test). Scale bars: 200µm.

O. Representative pictures of H&E stainings of small intestine tissue of Casp8WT/MLKLko and Casp8ECko/MLKLko mice (n= 4-6 WT, 4-5 ECko). Scale bars: 100µm.

P-Q. Quantification of intestinal pathology in Casp8WT/MLKLko and Casp8ECko/MLKLko mice at two different timepoints after tamoxifen treatment (enteropathy score; n=4-6 WT/MLKLko, 5 ECko/MLKLko; two tailed unpaired Student's t-test).

Data information: All data is shown as mean ± SEM. ns: not significant.