EMBO/soda-nel huggingface dataset:
Gata6 promotes hair follicle progenitor cell renewal by genome maintenance during proliferation (2016)
Figure 6

Figure 6. Genome-wide transcriptional changes following Gata6 depletion.

(A) Heatmap of differentially expressed genes (adjusted P-value < 0.05 and fold change > 1.5) from RNA-seq (n=3 different WT and iKO cell lines). (B) qRT-PCR validation of candidate genes selected from RNA-seq analysis using RNA from cultured cells (average SD, n=4). *Unpaired t-test P-values: Edaradd=0.03, Apcdd1=0.005, Gls2=7x10-5, Ppargc1b=3x10-4, Rab17=2x10-5, Foxn1=0.03. Mann-Whitney U test P-values: Edaradd=0.06, Apcdd1=0.03, Gls2=0.03, Ppargc1b=0.03, Rab17=0.03, Foxn1=0.03. (C) Table summarizing selection of candidate genes from (B). Genes were selected based on known function or skin phenotype related to Gata6 iKO phenotype. (D) Staining of ROS by DHE. LY83583 treated cells served as positive control with elevated ROS. (E) Quantification of fold change in ROS at 6 and 24 hours after TM compared to WT, based on fluorescence intensity of DHE (average SD, n=2). (F) Analysis of transcription factor binding site overrepresentation (oPOSSUM) in differentially expressed genes from RNA-seq. Points represent transcription factors plotted as Z.score of the frequency of transcription factor binding site occurrences in the promoters of downregulated genes compared to the background rate of occurrences in all gene promoters and Fisher P-value of the proportion of downregulated gene promoters containing each transcription factor binding site compared to the frequency in all gene promoters. (G) Comparison of Gata6 iKO downregulated genes with genes upregulated in Eda transgenic (Tg) skin. Genes related to DNA replication and repair are found in the set of genes shared between the two datasets. (H) qRT-PCR of selected genes found to be overlapping between Gata6 iKO and Eda transgenic analyzed in Gata6 WT and iKO keratinocytes (average SD, n=4). *Unpaired t-test P-values: Mcm3=0.001, Mcm6=0.02, Mcm10=0.04, Exo1=0.02, Pold1=0.02. Mann-Whitney U test P-values: Mcm3=0.03, Mcm6=0.03, Mcm10=0.03, Exo1=0.03, Pold1=0.06.
Gata6 promotes hair follicle progenitor cell renewal by genome maintenance during proliferation (2016)
Figure 7

Figure 7. Edaradd-NF-B signaling are downstream of Gata6.

(A) Model of potential Gata6 role upstream of Edaradd/NF-B. (B) qRT-PCR confirmation of the change in Edaradd mRNA expression in-vivo from mouse whole skin (average SD, 1d n=3, 2d n=4). *Unpaired t-test P-values: 1d = 0.02, 2d = 0.05. Mann-Whitney U test P-values: 1d=0.06, 2d=0.03. (C) ChIP-qPCR for Gata6 at three conserved binding sites within the Edaradd promoter. GAPDH served as a negative control. Binding site 1, 2, and 3 are located 50 bp, 1 kb, and 1.5 kb upstream of the transcription start site, respectively (average SD, n=4). *Unpaired t-test P-values GAPDH=0.4, 1=0.002, 2=0.8, 3=0.2. Mann-Whitney U test P-values: GAPDH=0.3, 1=0.06, 2=0.9, 3=0.2. (D) Western blot from whole skin lysate. Note decrease of Edaradd and activated pNF-B in the iKO. (E-F) Staining for Edaradd and quantification show decrease in Edaradd protein levels in the matrix of Gata6 iKO. *Unpaired t-test P-value = 3.5x10-14. n=100 follicles from 3 mice per genotype. (G-H) Staining for pNF-B and quantification show decrease of active NF-B in the Gata6 iKO matrix. *Unpaired t-test P-value = 2.2x10-16. n=50 follicles from 3 mice. (I) Rescue of Gata6 iKO keratinocyte growth by transfection with pGata6 or pEdaradd. Cell plates of keratinocytes are stained with Rhodanile Blue (n=3 cell lines each). (J) Quantification of H2A.X in cells stably transfected with pMock, pGata6, or pEdaradd plasmids, followed by induction with TM for 6 hours (average SD, n=4 with > 500 cells counted per condition). *Unpaired t-test P-values = 2x10-4 (iKO pGata6 vs pMock), 2x10-4 (iKO pEdaradd vs pMock). (K) qRT-PCR analysis of Mcm10 expression in pMock, pGata6, and pEdaradd WT and iKO expressing cells (average SD, n=4). *Unpaired t-test P-values: 8x10-4 (iKO pGata6 vs pMock), 0.03 (iKO pEdaradd vs pMock). Mann-Whitney U test P-values: 0.03 (iKO pGata6 vs pMock), 0.03 (iKO pEdaradd vs pMock).
Presynaptic inhibition upon CB1 and mGluR2/3 receptor activation requires ERK/MAPK phosphorylation of Munc18-1 (2016)
Figure 1

Figure 1. Munc18-1 is an extracellular signal-regulated kinase (ERK) substrate

(A) Yeast two-hybrid assays to test for interaction between Munc18-1 and ERK1/2. pGAL4-Munc18-1 (M18) and pGAL4-Empty (control) were used as baits and ERK1 and ERK2 as prey. Both ERK1 and ERK2 bind Munc18-1 and allow growth on selection plates (minus Histidine (-HIS) and minus Histidine and Adenosine (-ADE)).

(B) In vitro kinase assays showing Munc18-1 phosphorylation by ERK. Radioactive phosphoblot (32P) and immunoblot (IB) of in vitro ERK kinase assay using immunoprecipitated Munc18WT, Munc184A (M18T78A,S158A,S241A,T574A), Munc183A (M18S158A,S241A,T574A), and Munc18AA (M18S241A,T574A) from HEK293T cells incubated with recombinant ERK2. The phosphoblot shows phosphorylated Munc18-1 (pMunc18-1) and auto-phosphorylated ERK2 (pERK2).

(C) Quantification of phospho-Munc18-1 intensity in wild-type and Munc18AA mutant expressed as the amount of 32P incorporation in Munc18-1 normalized to total Munc18-1 levels. Phosphorylation of Munc18WT was set to 100%. (Munc18WT: 100±3%; Munc18AA: 24±2%, n=3, * p = 0.01).

(D) ERK1 in vitro kinase assay showing ERK-dependent 32P-incorporation in Munc18WT immuno-precipitated from HEK293T cells. Western Blot stained with Munc18-1 antibody shows total precipitated Munc18-1 levels were identical in all conditions.

(E) Alignment of amino acid sequence of Munc18-1 in different species. ERK consensus site (P-X-S/T-P) (yellow) around phosphorylation site S241 (red) is highly conserved.

(F) Three-dimensional structure of Munc18-1 (blue, green and yellow ribbons) and syntaxin 1 (purple and red ribbons). Zoom (right) of grey box in full structure (left) illustrates position of the ERK phosphorylation sites S241 (red) and T574 (blue). The T574 site has been reported as phosphorylation site for Cdk5 (Shuang et al, 1998).
Presynaptic inhibition upon CB1 and mGluR2/3 receptor activation requires ERK/MAPK phosphorylation of Munc18-1 (2016)
Figure 2

Figure 2. ERK phosphorylates Munc18-1 at S241 in vivo

(A) Experimental setup of fear conditioning paradigm. Mice placed in a fear- conditioning box received no shock (NS), an immediate shock (IS) or delayed shock (DS) and were decapitated 30 min after returning to the home cage. Dorsal and ventral hippocampi were subjected to cellular fractionation (P2). DS and IS significantly increased the levels of phosphorylated ERK1/2, a hallmark of ERK activation.

(B) Activity values across three different training paradigms to ascertain that foot shock was received in the IS and DS group.

(C) Simplified schematic representation of dorsal and ventral hippocampal connections and their main functions (see, Fanselow & Dong, 2010).

(D) Western Blot analysis of P2 synaptosomal fractions of ventral hippocampi from the fear conditioning paradigm stained for pM18S241, total Munc18-1, pSynapsin, total synapsin, pERK and GAPDH as loading control.

(E) Quantification Munc18-1 phosphorylation in dorsal hippocampus expressed as ratio pM18S241 over total Munc18-1 levels. No shock was set to 1 (NS: 1.0±0.19, n=5; DS: 4.01±0.39, n=5; IS: 2.88±0.37, n=5; **p<0.01; ***p<0.001).

(F) Quantification of Munc18-1 phosphorylation in ventral hippocampus expressed as ratio pM18S241 divided by total Munc18-1 levels. (NS: 1.0±0.32, n=5; DS: 4.39±0.55, n=5; IS: 3.62±0.50, n=5; **p<0.01; ***p<0.001).

(G) Quantification of synapsin phosphorylation in dorsal hippocampus expressed as ratio pSynapsin divided by total synapsin levels. (NS: 1.0±0.31, n=5; DS: 1.88±0.43, n=5; IS: 1.18±0.34, n=5; n.s.). Inset shows quantification of ERK2 phosphorylation in dorsal hippocampus expressed as ratio pERK2 divided by total ERK2 levels. (NS: 1.0±0.11, n=5; DS: 3.51±0.41, n=5; IS: 3.98±0.24, n=5; **p<0.01).

(H) Quantification of synapsin phosphorylation in ventral hippocampus expressed as ratio pSynapsin divided by total synapsin levels. (NS: 1.0±0.20, n=5; DS: 3.32±0.46, n=5; IS: 3.30±0.60, n=5; **p<0.01).
Presynaptic inhibition upon CB1 and mGluR2/3 receptor activation requires ERK/MAPK phosphorylation of Munc18-1 (2016)
Figure 3

Figure 3. ERK phosphorylates Munc18-1 during increased network activity

(A) Western Blot of whole cell lysate of adult mouse brain slices treated with the proteasome inhibitor MG132 (10 M) and DMSO, the GABAA receptor blocker GABAzine (GBZ, 10 M) or the voltage-gated sodium channel blocker TTX (2 M) for 4 h. Blots were stained for pMunc18S241, Munc18-1 and tubulin as loading control.

(B) Quantification of (A). ERK phosphorylation of Munc18-1 in adult mouse brain slices expressed as ratio pM18S241 divided by total Munc18-1 levels. Vehicle (DMSO) was set to 1 (DMSO: 100 ± 0.4%; GBZ: 1.73 ± 0.12; TTX: 0.052 ± 0.002, n = 4, * p<0.05, ** p<0.01).

(C) Western Blot of whole cell lysate of adult mouse brain slices treated with proteasome inhibitor MG132 (10 M) and MEK inhibitor PD98059 (10 M) for 4 h. Proteasome block increases the levels of phosphorylated Munc18-1 and ERK1/2, which can be prevented by MEK inhibition. Blots were stained for pMunc18S241, Munc18, pERK1/2 and ERK1/2.

(D) Quantification of (C). ERK phosphorylation of Munc18-1 in adult mouse brain slices expressed as ratio pM18S241 divided by total Munc18-1 levels. Vehicle (DMSO) was set to 1 (DMSO: 100 ± 0.8%; MG132: 375 ± 14.2%; MG132 + PD98059: 24 ± 3%, n = 3, ** p<0.01).

(E) DIV14 rat cortical cultures treated with type 2 mGluR agonist LY379268 (1 M) for 5 min or BDNF (100ng/ml) for 48 h in the presence of 2 M TTX and stained for pMunc18S241, Munc18, pERK and tubulin as loading control.
Presynaptic inhibition upon CB1 and mGluR2/3 receptor activation requires ERK/MAPK phosphorylation of Munc18-1 (2016)
Figure 4

Figure 4. ERK-dependent phosphorylation of Munc18-1 controls synaptic transmission

Hippocampal autaptic neurons of munc18-1 null mutant mice were rescued with lentiviruses expressing M18WT, M18S241A or M18S241D. Electrophysiological recordings were performed between DIV14-17.

(A) Amplitude of excitatory synaptic response (EPSC) (M18WT: 1.37±0.21 nA, n=35; M18S241A: 2.53±0.31 nA, n=45; M18S241D: 0.24±0.05 nA, n=17, ***p<0.001, **p<0.01, *p<0.05).

(B) Percentage of cells that do not respond to electrical stimulation (M18WT: 15.5%; M18S241A: 0%; M18S241D: 56.9%). These cells are not included in (A). Insert: representative recording of single EPSCs. Scale bar represents 10 ms and 0.5 nA.

(C) Typical examples of spontaneous vesicle release. Scale bar represents 0.1 s and 25 pA.

(D) Spontaneous release frequency (M18WT: 7.05 ± 1.28 Hz, n=32; M18S241A: 14.26 ± 2.04 Hz, n=37; M18S241D: 0.64 ± 0.29 Hz, n=11; ***p<0.001, **p<0.01).

(E) Amplitude of spontaneous release events (M18WT: 15.40 ± 1.06 pA, n=17; M18S241A: 15.03 ± 0.61 pA, n=20; M18S241D: 10.98 ± 0.72 pA, n=10; **p<0.01).

(F) Size of the readily releasable pool (RRP) assessed with single hyperosmotic sucrose application (500 mM, 3.5 s, M18WT: 0.74 ± 0.15 nC, n=7; M18S241A: 1.6 ± 0.33 nC, n=10; M18S241D: could not be determined; * p < 0.05).

(G) Neurons were stimulated with two consecutive pulses with varying inter-stimulus interval to assess release probability. Paired pulse ratio calculated as ratio of the second to the first synaptic response is plotted as function of inter-stimulus interval (M18WT, n=25; M18S241A, n=19; M18S241D, n=17); * p < 0.05, ** p < 0.01). All data are expressed as mean ± SEM. See also Figure EV2 and EV3.
Presynaptic inhibition upon CB1 and mGluR2/3 receptor activation requires ERK/MAPK phosphorylation of Munc18-1 (2016)
Figure 5

Figure 5. Munc18S241D expression levels are reduced due to proteasomal degradation

(A-F) Munc18-1 protein levels were measured in glia free cultures of munc18-1 null mutant neurons expressing M18WT, M18S241A or M18S241D.

(A) Typical examples showing Munc18-1 protein localization and intensity in neurites of M18WT, M18S241A or M18S241D neurons. MAP2 in blue, VAMP2 in red and Munc18-1 in green. Scale bar represents 2 m.

(B) Mean somatic Munc18-1 intensity (M18WT: 1682 ± 206 a.u., n = 46; M18S241A: 1719 ± 238 a.u., n = 36; M18S241D: 582 ± 98 a.u., n = 33; *** p < 0.001).

(C) Mean synaptic Munc18-1 intensity (M18WT: 1281 ± 161 a.u.; M18S241A: 1390 ± 180 a.u.; M18S241D: 210 ± 30 a.u.; *** p < 0.001).

(D) Mean synaptic Munc18-1 intensity as a function of radial distance from the soma.

(E) Mean synaptic Munc18-1 intensity in cultures with 3 times the standard number of neurons to increase network activity (M18WT: 1072 ± 123 a.u., n = 36; M18S241A: 1480 ± 170 a.u., n = 32; M18S241D: 251 ± 70 a.u., n = 34; * p < 0.05, *** p < 0.001).

(F) Mean synaptic Munc18-1 intensity in cultures treated with GABAA receptor antagonist Bicuculline (40 μM, 48 h). (M18WT: 761 ± 121 a.u., n = 26; M18S241A: 1482 ± 150 a.u., n = 31, ** p < 0.01).

(G) Mean synaptic syntaxin intensity in autaptic cultures (M18WT: 875 ± 35, n = 51; M18S241A: 1058 ± 42, n = 46; M18S241D: 458 ± 19, n=40, ** p < 0.01, ***p<0.001).

(H) Western Blot of lysates from high-density neuronal cultures from munc18-1 null mutant mice rescued with M18WT, M18S241A or M18S241D. Prior to lysis, cells were treated for 6 h with MG132 (10 M) or DMSO as control. Western Blots were stained for Munc18-1 and tubulin as loading control.

(I) Quantification of (H). Munc18-1 levels were normalized to tubulin levels. Effect of proteasome inhibition was quantified as ratio between MG132 and DMSO treatment of each mutant (M18WT: 1.62 ± 0.41; M18S241A: 1.26 ± 0.16; M18S241D: 7.53 ± 1.17, n = 3; **p<0.01).

(J) Ubiquitination of different Munc18-1 mutants in HEK293T cells. Western Blot of denatured Munc18-1 immuno-precipitations from cells expressing Munc18-1 mutants and HA-tagged ubiquitin. Prior to lysis, cells were treated for 8 h with MG132 (10 M). Ubiquitin covalently attached to Munc18-1 can be observed as a smear above the characteristic 68 kD Munc18-1 band on Western blots stained for HA-tag.

(K) Quantification of immuno-precipitation in (J). Level of Munc18-1 ubiquitination was quantified as ratio of immunoprecipitated ubiquitin and Munc18-1 levels. M18WT was set to 1 (M18WT: 1 ± 0.06; M18S241A: 0.84 ± 0.57; M18S241D: 2.70 ± 0.07, n = 3; **p<0.01). All data are expressed as mean ± SEM.
Presynaptic inhibition upon CB1 and mGluR2/3 receptor activation requires ERK/MAPK phosphorylation of Munc18-1 (2016)
Figure 6

Figure 6. Munc18-1 functions in endocannabinoid-induced depression of glutamate release

(A) Western blot from whole cell lysate of munc18-1 null mutant neurons rescued with M18WT and incubated with WIN55,212-2 (5 M) for 30 s. Cells were harvested after the indicated time. CB1R activation leads to a rapid and transient increase in pERK activity.

(B-D) Hippocampal autaptic neurons of munc18-1 null mutant mice were rescued with lentiviruses expressing M18WT or M18S241A. CB1R agonist WIN55,212-2 (5 M) was applied at t = 60 s for 30 s.

(B) Amplitude of excitatory synapse response normalized to t=0 s. Neurons were stimulated by a single action potential every 30 s for 4 min (M18WT n = 21, M18S241A n = 24, *p<0.05). Note the broken y-axis.

(C) Ratio of the frequency of spontaneous release events after (t = 180 s) and before (t = 0 s) WIN55,212-2 application (M18WT: 58.0 ± 2.3 %, n = 21; M18S241A: 84.2 ± 5.6 %, n = 24, * p < 0.05).

(D) Ratio of amplitude of spontaneous release events after (t=240 s) and before (t=0 s) WIN55,212-2 application (M18WT: 85.0 ± 2.7 %, n = 21; M18S241A: 83.1 ± 2.6 %, n = 24).

(E-G) Autaptic neurons of munc18-1 null mutant mice rescued with lentiviruses expressing M18WT were superfused with WIN55,212-2 (1 M), WIN55,212-2 (1 M) plus AM251 (1 M) or vehicle only (DMSO).

(E) Amplitude of excitatory synapse response normalized to t=0 s. CB1R agonist WIN55,212-2 (1 M) or vehicle only (DMSO) were applied at t=60 s for 30 s. (WT + WIN n = 13; WT + DMSO n = 16, * p < 0.05). Note the broke Y-axis.

(F) Paired pulse ratio (20 ms interval) at t = 0 and t = 240 s in M18WT neurons superfused with CB1R agonist WIN55,212-2 (1 M) at t = 60 s for 30 s. WIN55,212-2 application increases PP ratios indicative of a reduction of synaptic release probability (t = 0 s: 0.94 ± 0.1, n = 13; t = 240 s: 1.36 ± 0.2, n = 16, * p < 0.05).

(G) Amplitude of excitatory synapse response normalized to t = 0 s. CB1R agonist WIN55,212-2 (1 M) was applied together with the CB1R antagonist AM251 (1 M) at t = 60 s for 30 s. (WT + WIN n = 13, same trace as 6F; WT + WIN + AM251 n = 15, * p < 0.05). Note the broke Y-axis.

(H-J) Hippocampal autaptic neurons of munc18-1 null mutant mice were rescued with M18WT or M18S241A. mGluR2/3 agonist LY379268 (1 M) was applied by bath-perfusion for 4 min.

(H) Ratio of amplitude of evoked release events after (t = 240 s) and before (t = 0 s) LY379268 application (M18WT: 55.0 ± 4.7 %, n = 14; M18S241A: 81.1 ± 3.6 %, n = 16, * p < 0.05).

(I) Ratio of frequency of spontaneous release events after (t = 240 s) and before (t = 0 s) LY379268 application (M18WT: 63.0 ± 5.7 %, n = 14; M18S241A: 85.1 ± 3.4 %, n = 16, * p < 0.05).

(J) Ratio of the amplitude of spontaneous release events after (t = 240 s) and before (t = 0 s) WIN55,212-2 application (M18WT: 81.0 ± 1.3 %, n = 14; M18S241A: 82.1 ± 3.3 %, n = 16).
Using a bistable animal opsin for switchable and scalable optogenetic inhibition of neurons (2021)
Figure 1

Figure 1 - Lamplight can be used for switchable and tuneable control Go activity in Hek293T cells

A) Hek293T cells expressing Lamplight-1D4 (anti-1D4 = green, DAPI = blue, Top) and schematic of BRET assay, measuring interaction of liberated Gβγ-Venus with GRK3 fragment-Nanoluciferase (Bottom). Scale bar = 20µm.

B) Lamplight-driven response to increasing intensity 1s 405nm light

C) 405nm irradiance response curve

D) Response to 1s 405nm (blue line) and 525nm light (green line)

E) Response to 405nm (blue line) followed by 525nm light of different intensities (green line)

F) Schematic of Lamplight photoequilibrium. In dark, Lamplight is mostly in inactive (R) state. 405nm light shifts equilibrium (blue arrow) so opsin is mostly in active (M) state, while 525nm light shifts (green arrow) Lamplight into R state. Shifts caused by concurrent 525nm and 405nm partially cancel out leading to a small increase of opsin in M state.

G) Response to 405nm alone or with 525nm light

H-I) Representative time courses of Lamplight-driven responses to 8s mixed stimuli (grey bar) with different 405nm: 525nm ratios from 1 (100% 405nm) to 0 (100% 525nm) from (H) dark or (I) after 8s 405nm light (blue bar)

J) Sustained Lamplight-driven activity (mean BRET ratio 20-80s after stimulus) after exposure to mixed stimuli from dark (ON) or 405nm light-adapted state (OFF). Lamplight data fit with linear trendline. Best fitting parameters for ON, slope = 0.311, Y-intercept = 0.04, R2 = 0.85. For OFF, slope = 0.30, Y-intercept = 0.07, R2 = 0.69).

Data information: For (B-E, G), error bars = SEM, N = 3-4 biological replicates. All intensities are for total Lamplight effective log photons/cm2/s. Timing of light stimuli shown by vertical line/bar
TLR4-dependent shaping of the wound site by MSCs accelerates wound healing (2020)
Figure 1

Figure 1. LPS primed MSCs activate neutrophils with enhanced NET.

(a) MSCs were exposed to increasing LPS concentrations, thereafter washed and co-cultured with PMA activated neutrophils. Incubation of neutrophils with PMA alone served as positive control and DMSO treated neutrophils as negative control. Scale bars: 50μm.

(b) Quantification of NET bound elastase indicative of NET formation. Statistical analysis was performed using one way ANOVA, values are represented as mean ± SEM, n=3 biological replicates.

(c) Representative microphotographs of murine wound sections immunostained for Ly6G (neutrophils, green) and DNA-histone (displayed as expulsed streaks in red indicative of NETs). Wounds injected with LPS primed MSCs (upper row, outer right panel) depict enhanced NET formation (magnified inset) and increased expression of activation markers like neutrophil elastase (NE, lower panel, outer left panel) compared to wounds injected with non-primed MSC (middle panels) or PBS injected wounds (outer left panels). Murine skin wounds treated with PBS injections served as control. Scale bar: 10μm (upper row) and 50μm (lower row). To facilitate comparison, areas inside the rectangles are shown at 5x magnification in the insets.
TLR4-dependent shaping of the wound site by MSCs accelerates wound healing (2020)
Figure 2

Figure 2. LPS priming leads to profound transcriptomic reprogramming in MSCs.

(a) Hierarchical clustering analysis of RNAseq expression profile from MSCs stimulated with LPS for 6 and 24 h. The heat-map shows the gene expression profile of non-primed MSCs and MSCs which were LPS primed for 6 and 24h. Red color represents upregulation, while blue color depicts down regulation in gene expression, each data point represents FPKM in log2 value.

(b-c) Pathway analysis depicts that cytokine-cytokine receptor interactions, genes encoding secreted soluble factors, an ensemble of genes encoding extracellular matrix, chemokine receptors which bind distinct chemokines, GPCR ligand binding, cytokine and inflammatory response, chemokine signaling pathway and IL-5 signaling are the most dominant pathways as assessed by transcriptional changes when non-primed and LPS primed MSCs were compared at 6 and 24 h after LPS priming.

(d) Venn diagram analysis displays up-regulated and down-regulated gene numbers indicated in blue and pink color, respectively. Purple color indicates the number of shared genes of the up-regulated and down-regulated genes at 6h and 24h after LPS stimulation. This implies rapid changes of expression of the same genes.

(e-g) Validation of the selected genes uncovered from global RNAseq analyses by qRT-PCR analyses displays up-regulation of CXCL-6, IL-8 and IL-1β after LPS treatment. Statistical analysis was performed using one way ANOVA, values are represented as mean ± SEM, n=3 biological replicates.

(h-i) ELISA results depict an up-regulated expression of CXCL-6 after 24h of LPS treatment, while a significantly increased IL-8 expression after 6 and 24h of LPS treatment. Statistical analysis was performed using one way ANOVA, values are represented as mean ± SEM, n=3 biological replicates.

(j) Western blot analysis showed up-regulation of IL-1β expression upon LPS priming of LPS primed MSCs. Actin served as control. Densitometry graph shows significant increase after 6h LPS stimulation compared to non-primed MSCs. Statistical analysis was performed using one way ANOVA, values are represented as mean ± SEM, n=3 biological replicates.