EMBO/soda-nel huggingface dataset:
VEGF-B Signaling Impairs Endothelial Glucose Transcytosis by Decreasing Membrane Cholesterol Content (2020)
Figure 1

Figure 1. VEGF-B regulates endothelial glucose uptake and transcytosis

A) 2-NBD-glucose uptake in murine pancreatic endothelial cells (MS1) and primary human umbilical vein endothelial cells (HUVEC) treated for 2 h with VEGF-B167 (B167) or VEGF-B186 (B186) compared to control treated cells. Data presented as mean ± SEM from representative experiments performed in triplicates. Statistical evaluation using one-way ANOVA and Dunnett´s multiple comparisons test, p-value: * <0.05, ** <0.01, *** <0.001 (compared to untreated control). Scale bar, 20 μm.

B) Trans-cellular 2-NBD-glucose transport kinetics in MS1 cells treated for 2 h with VEGF-B186. Data presented as mean ± SEM from three individual experiments. Statistical evaluation using t-test, p-value: ** <0.01 (compared to untreated control).

C) Cardiac glycogen content in 17 weeks old male Vegfb+/+ (n=6), Vegfb+/- (n=6) and Vegfb-/- mice (n=5) presented as mean ± SEM. Statistical evaluation using one-way ANOVA and Dunnett´s multiple comparisons test, p-value: * <0.05 (compared to Vegfb+/+).

D) Glucose uptake and accumulation (18F-DG PET) in hearts of HFD fed wild-type (WT) mice pre-treated for 1 week with 2H10 antibody (n=4) compared to control antibody treated mice (n=4). Data presented as mean ± SEM. Statistical evaluation using two-way ANOVA, p-value: **** <0.0001.

E) Linear regression analysis of the 18F-DG PET data in D assessing differences in initial (T=0.9-6.5 min) glucose uptake rate. Data presented as mean curve slope (m) ± SEM. Statistical evaluation comparing slope differences using analysis of covariance (ANCOVA), p-value: * <0.05.

Data information: See also Figure EV1.
VEGF-B Signaling Impairs Endothelial Glucose Transcytosis by Decreasing Membrane Cholesterol Content (2020)
Figure 2

Figure 2. VEGF-B regulated glucose uptake is mediated by GLUT1, NRP1 and VEGFR1.

A) Relative mRNA expression of the Slc2a glucose transporter (GLUT) family members (1-13) in cardiac ECs derived from Vegfb+/+ (n=4) and Vegfb-/- (n=4) mice. Data represent normalized PLIER values (mean ± SEM) from microarray analysis performed on n=4+4 RNA samples (one RNA sample/mouse).

B) mRNA expression levels of the SLC2A transporter (GLUT) family members (1-14) in HUVECs. Data presented as mean ± StDev relative to RPL19 expression from 3 independent experiments.

C) mRNA expression levels of VEGFB, SLC2A1, NRP1 and FLT1 (VEGFR1) in HUVECs. Data presented as mean ± StDev relative to RPL19 expression from 3 independent experiments.

.

D) 2-NBD-glucose uptake in response to 2 h treatment with VEGF-B186 (B186) in HUVECs exhibiting siRNA-mediated knock-down of FLT1, NRP1, SCL2A1, KDR (VEGFR2) or control siRNA. Data presented as mean ± SEM from three individual experiments. Statistical evaluation using one-way ANOVA and Sidak´s multiple comparisons test, p-value: * <0.05 (comparisons made to respective untreated control).

E) Representative images of GLUT1 (left panels) and CD31 (right panels) immunohistochemistry performed on consecutive sections from paraffin embedded mouse hearts. Scale bar, 100 μm.

F) mRNA expression analysis from enriched cardiac endothelial cells (open columns, n=4 mice) or whole heart (closed columns, n= 3 mice). Data presented as mean ± StDev relative to Rpl19 expression. Enrichment factor is calculated as mean cardiac EC/mean whole heart and presented in the box.

Data information: See also Fig EV2 and Appendix Fig S1.
VEGF-B Signaling Impairs Endothelial Glucose Transcytosis by Decreasing Membrane Cholesterol Content (2020)
Figure 3

Figure 3. VEGF-B signaling changes microdomain localization of GLUT1 in the plasma membrane

A) Immunofluorescence labeling of GLUT1 (white), early endosomes (EEA1, green) and nuclei (blue) in HUVECs treated with VEGF-B186 for 10 min, 30 min or 2 h. Scale bar, 10 µm.

B) GLUT1 protein expression in HUVECs treated with VEGF-B186 for 15 min or 2 h. Data presented as mean ± StDev of 3-4 independent experiments relative to β-Actin expression. Statistical evaluation using t-test revealed no significant changes. Western blots shown in right panel.

C) Sucrose gradient (15-50% sucrose) fractions of HUVEC cellular lysates after 2 h VEGF-B186 stimulation were analyzed with Western blots shown in Appendix Fig S2. Different cellular sub-compartments were identified by accumulation of specific protein markers: EEA1 for early endosomes, LIMPII for late endosomes/lysosomes, flotillin for plasma membrane/lipid rafts and calnexin for endoplasmic reticulum (ER) (left panels). Amount and sub-cellular distribution of VEGFR1, NRP1 and GLUT1 protein in control or VEGF-B186 stimulated HUVECs shown in panels to the right. Data represent % protein relative to total protein of interest measured from a representative experiment.

D) Quantification of GLUT1 protein in % relative to total measured protein in HUVECs after 2 h VEGF-B186 stimulation in different sucrose fractions (upper panel). Data from two independent experiments are shown. Green arrows mark fractions with increased GLUT1 content and red arrows mark fractions with decreased GLUT1 content after VEGF-B186 stimulation. Changes in GLUT1 content in response to VEGF-B treatment are shown in the table (bottom panel).

Data information: See also Appendix Fig S2.
VEGF-B Signaling Impairs Endothelial Glucose Transcytosis by Decreasing Membrane Cholesterol Content (2020)
Figure 4

Figure 4. VEGF-B reduces membrane cholesterol prior to reduction of glucose uptake and increased FA uptake

A) Representative confocal (grey) and epifluorescence (blue) images of filipin staining in HUVECs in response to 2 h VEGF-B186 stimulation (left panels). Cellular cholesterol content measured by quantification of fluorescent filipin staining. Data presented as mean ± SEM from a representative experiment performed in triplicates. Statistical evaluation using t-test, p-value: * <0.05. Scale bars, 10 µm.

B-D) Measurement of cholesterol content (B), glucose uptake (C) and FA uptake (D) in HUVECs after VEGF-B186 stimulation for different time-points. Data presented as mean ± SEM from representative experiments performed in triplicates. Statistical evaluation using t-test, p-value: * <0.05, ** <0.01, *** <0.001 (compared to respective control).

E) Colorimetric quantification of non-esterified cholesterol extracted from HUVECs stimulated with VEGF-B186 for 20 min. Data are presented as mean ± SEM of three individual experiments. Statistical evaluation using t-test, p-value: * <0.05.

F) Uptake of a fluorescent cholesterol tracer (TopFluor) in HUVECs after VEGF-B186 stimulation for 20 min or 2 h. Data presented as mean ± SEM from a representative experiment performed in triplicates. Statistical evaluation using one-way ANOVA and Dunnett´s multiple comparisons test, p-value: ** <0.01, *** <0.001 (compared to respective untreated control).

G) Quantification of the major cholesteryl ester (CE) species in HUVECs after VEGF-B186 stimulation for 20 min, analyzed by lipid mass spectrometry. Data presented as mean ± StDev of an experiment performed in triplicates.

Data information: See also Fig EV5.
VEGF-B Signaling Impairs Endothelial Glucose Transcytosis by Decreasing Membrane Cholesterol Content (2020)
Figure 5

Figure 5. Cholesterol content and glucose uptake are tightly linked in ECs

A) Measurement of cellular cholesterol content (upper panel) and glucose uptake (lower panel) in HUVECs treated with VEGF-B186 for 1 h or after 15 min treatment with cholesterol-extracting methyl-beta-cyclodextrin (MbCD). Data presented as mean ± SEM from a representative experiment performed in triplicates. Statistical evaluation using one-way ANOVA and Dunnett´s multiple comparisons test, p-value: *** <0.001 (compared to untreated control).

B) Measurement of cellular cholesterol content (upper panel) and glucose uptake (lower panel) in HUVECs treated with VEGF-B186 for 15 min alone or supplemented with soluble methyl-beta-cyclodextrin complexed with cholesterol (5, 10 or 20 μg/ml cholesterol). Data presented as mean ± SEM from a representative experiment performed in triplicates. Statistical evaluation using one-way ANOVA and Dunnett´s multiple comparisons test, p-value: * <0.05, ** <0.01, *** <0.001 (compared to control w/o cholesterol).

Data information: See also Fig EV5.
VEGF-B Signaling Impairs Endothelial Glucose Transcytosis by Decreasing Membrane Cholesterol Content (2020)
Figure 6

Figure 6. VEGF-B regulates endothelial cholesterol handling in an LDL receptor-dependent manner

A) mRNA expression analysis of lipoprotein receptors in HUVECs. Data presented as mean ± StDev relative to RPL19 expression from 3 independent experiments.

.

B-C) Cellular cholesterol content (B) and glucose uptake (C) in HUVECs treated with control siRNA or siRNA targeting LDLR or Scavenger receptor B1 (SCARB1, SRBI) followed by 15 min stimulation with VEGF-B186. Data presented as mean ± SEM from representative experiments performed in triplicates. Statistical evaluation using one-way ANOVA and Dunnett´s multiple comparisons test, p-value: * <0.05, ** <0.01, *** <0.001 (compared to respective control).

D) Cellular cholesterol content of ECs freshly isolated from Ldlr+/+ and Ldlr-/- mice stimulated for 15 min with VEGF-B186. Data presented as mean ± SEM from a representative experiment performed in triplicates. Statistical evaluation using one-way ANOVA and Tukey´s multiple comparisons test, p-value: *** <0.001 (compared to Ldlr+/+ control).

E) Cellular cholesterol content (left panel) and glucose uptake (right panel) of HUVECs treated with de novo cholesterol synthesis inhibitor simvastatin. Data presented as mean ± SEM from a representative experiment performed in triplicates. Statistical evaluation using one-way ANOVA and Tukey´s multiple comparisons test, p-value: * <0.05, ** <0.01, *** <0.001 (compared to untreated control). p-value: # <0.05, ### <0.001 (compared to simvastatin control).

F) Cell surface biotinylated LDLR protein levels in HUVECs after 20 min of VEGF-B167 or VEGF-B186 stimulation. Data presented as mean ± SEM from three independent experiments. Western blot shown below. Statistical evaluation using t-test, p-value: * <0.05 (compared to control).

G) Representative images of cell surface distribution of the LDLR by immunofluorescence staining of non-permeabilized cells (white, left panels), Dil-LDL cell surface binding (evaluated at 4 °C, middle panels) and Dil-LDL uptake (evaluated at 37 °C, right panels) in HUVECs in response to VEGF-B186 stimulation for 10 min. Scale bar, 10 µm. Overview pictures are found in Appendix Fig S3. See also Appendix Fig S3-5.
MitoTev-TALE: a monomeric DNA editing enzyme to reduce mutant mitochondrial DNA levels (2018)
Figure 1

Figure 1. Development of a mitoTev-TALE targeting the m.8344A>G mitochondrial DNA mutation.

A) The structure of a mitoTev-TALE. It consists in a mitochondrial localization sequence (MLS) and a TALE-DNA binding domain which is fused to a monomeric endonuclease (I-TevI), through a flexible linker (linker).

B) Diagram of mtDNA illustrating the binding site for two monomeric mitoTev-TALEs (8.5 and 11.5 RVDs) designed to target the m.8344 A>G in the tRNALys gene. The two mitoTev-TALEs differ in the number of RVDs. The A:T base pair at position T0 is shown in green and the G:C discriminatory base pair in red. The I-TevI endonuclease domain targets a 5'CNNNG3' sequence, in this case 5'CACTG3' shown in green which is spaced by 12 bp from the TALE-DNA binding site.

C) HEK293T cells were transfected for 48 hours with each mitoTev-TALE (11.5 and 8.5 RVDs) and total cell extracts were used for western-blots with anti-T2A antibody. Molecular weights of the two different mitoTev-TALEs are ~85KD and ~70KDa, respectively. Immunocytochemistry using the same antibody along with Mitotracker red was performed in COS7 cells, 24 hours after transfection. Nuclei were counterstained with DAPI (blue).

D) 11.5RVD and 8.5RVD mitoTev-TALEs both co-localized with mitochondria, as seen in the merge image. Scale bar: 10 µm.
MitoTev-TALE: a monomeric DNA editing enzyme to reduce mutant mitochondrial DNA levels (2018)
Figure 2

Figure 2. Monomeric mitoTev-TALEs reduce mutant mtDNA loads in low mutant cybrids harboring the m.8344A>G mtDNA point mutation.

A) MtDNA heteroplasmy analyzed by PCR/RFLP, 24 hours post mitoTev-TALE and mitoTALEN transfection. The RFLP analysis shows increased %WT mtDNA in sorted cells when compared to the the untransfected. mitoTALEN monomers positive for both eGFP and mCherry were isolated as "Yellow". The "Black" cells represent the mitoTALEN sorted population of cells negative for eGFP and mCherry and the "GFP-+" population represents mitoTev-TALE sorted cells with low levels of eGFP fluorescence. GFP++ were cells positive for GFP after mitoTev-TALEs transfections.

B) Quantification of the % of WT load in cybrid sorted populations after transfection.

C) Representative RFLP analysis showing the change in heteroplasmy at 1 day and 15 days, after sorting.

D) Quantification of the heteroplasmic shift over time.

E) Determination of the levels of mtDNA (ND1/ACTB) by qPCR of mutant cybrids transfected with MERRF mitoTev-TALE.

F) mtDNA levels in wild-type sorted cells transfected with the MERRF mitoTev-TALE. Data is expressed as percentage of the Untransfected cells (%UNT).

Data information: Data are expressed as mean±SEM of n=5-12 independent experiments (panel B), n=3 (panel D) independent experiments, n=3-6 independent experiments (panel E) and n=6 independent experiments (panel F), Statistical analyses were performed by the use of unpaired two-tailed Student's t-test, *p<0.05, **p<0.005, ***p<0.001, ****p<0.0001. For exact p values and n number see Appendix Table 1.
MitoTev-TALE: a monomeric DNA editing enzyme to reduce mutant mitochondrial DNA levels (2018)
Figure 3

Figure 3. The MERRF mitoTev-TALE effectively shifts heteroplasmy in MERRF high mutant cybrids.

A) RFLP analyses of high mutant cybrids transfected with the MERRF mitoTev-TALE. The sorted cells were analyzed at different times of growth by PCR/RFLP as previously described.

B) Quantification of WT loads of 6-8 independent experiments/sortings, analyzed at different time-points. C) Total mtDNA levels determined by qPCR in 6-7 separate sortings/experiments.

D) Flow cytometry diagram of gated sorted cells. The GFP-+ represent the cells with low levels of fluorescence, GFP++ showed intermediate levels of fluorescence while GFP+++ had the highest levels. E) Quantification of mtDNA levels by PrimeTime qPCR probes. The mtDNA levels were analyzed in the different populations of sorted cells. Data are expressed as mean±SEM of 3-4 separate experiments.

F) mtDNA levels were also determined in the WT cybrids as described above. Data are expressed as mean±SEM of 4-7 separate experiments.

Data information: Statistical analyses were performed by a two-tailed Student's t-test.; UNT vs GFP-+/GFP++ *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. For more details see Appendix Table1.
MitoTev-TALE: a monomeric DNA editing enzyme to reduce mutant mitochondrial DNA levels (2018)
Figure 4

Figure 4. The MERRF mitoTev-TALE significantly improves mitochondrial oxidative phosphorylation function of high mutant cybrids.

A) Oxygen consumption rate (OCR) upon sequential injection of oligomycin (Oligo), FCCP and rotenone (Rot) + antimycin (AA), in untransfected, sorted "GFP-+" and "GFP++". The error bars represent the mean±SEM of 5-7 separate experiments.

B) Quantification of 4-6 independent experiments comparing the basal respiration, maximal respiration, spare respiratory capacity and ATP-linked respiration of the UNT versus "GFP-+", "GFP++" and WT cells. The cells were analyzed between 25-28 days after sorting.

C) Coupling efficiency, measured as the ratio of ATP-linked/Basal respiration x 100. Error bars represent the mean±SEM of 4-6 separate experiments.

D) Representative membrane of a mitochondrial protein synthesis experiment. The results were normalized to the gel loading. Graph represents 5 separate experiments (with exception of wild-type samples, n=2).

E) Western-blot showing the levels of NDUFB8 and COXI in untransfected and sorted cells. The SDHA and tubulin levels were also analyzed. The panel also shows the quantification of these markers normalized by the stain-free loading of 6 independent sortings/experiments.

Data information: Statistical analysis was performed using two-tailed Student's t-test between two groups (UNT vs GFP-+; UNT vs GFP++ and UNT vs WT), *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. For exact p values and n numbers see Appendix Table1.
Interferon-mediated repression of miR-324-5p potentiates necroptosis to facilitate antiviral defense (2022)
Figure 2

Fig 2 MiR-324-5p negatively regulates MLKL expression

A HT-29 cells were transfected with NC, siRIPK1 or miR-324-5p. After 48 h, cells were treated with TNF-α (T) or T+S+Z for an additional 6 h. Western blotting analysis of p-RIPK1, RIPK1, p-RIPK3, RIPK3, p-MLKL, MLKL and β-actin.

B Western blotting analysis of RIPK1, RIPK3, MLKL and β-actin levels in HT-29 cells that were transfected with NC, siRIPK1, siRIPK3, MLKL siRNA oliogs ( siMLKL), and miR-324-5p.

C HT-29 cells were harvested 48 h after transfection with NC, siMLKL, and miR-324-5p. MLKL expression was analyzed by qPCR (upper) or RT-PCR (lower).

D, E MKN45 (D) and 174T (E) cells were harvested 48 h after transfection with NC, siMLKL, and miR-324-5p. qPCR analysis for the expression of MLKL (upper) and western blotting analysis of MLKL and β-actin (lower).

F HT-29 cells were transfected with NC, siMLKL, miR-324-5p, or anti-miR-324-5p. After 48 h, cells were treated with DMSO or T+S+Z for an additional 24 h. Cell survival was determined by measuring ATP levels.

G Western blotting analysis of MLKL and β-actin in HT-29 cells that were harvested 48 h after transfection with NC, siMLKL, miR-324-5p, or an anti-miR-324-5p.

Data information: Data are represented as the means ± SD of three biological replicates. Statistical analyses were performed using unpaired Student's t-test. All experiments were performed at least three times, and representative data are shown.
Interferon-mediated repression of miR-324-5p potentiates necroptosis to facilitate antiviral defense (2022)
Figure 3

Fig 3 MiRNA-324-5p targets the 3'UTR of MLKL mRNA in a seed region-independent manner

A Schematic representation of the miR-324-5p target binding site in the 3'UTR of MLKL. Two mutated forms (mutant#1 and mutant#2) of MLKL-3'UTR were generated.

B HEK293T cells were transfected with NC, MLKL siRNA oligos targeting the 3'UTR of MLKL (siMLKL-3'UTR), or miR-324-5p, together with pmirGLO-MLKL-3'UTR or the mutated form of MLKL-3'UTR shown in A. Relative luciferase activity analysis of MLKL-3'UTR. Data are presented as firefly luciferase activity/renilla luciferase activity ± SD.

C HEK293T cells were transfected with NC, miR-324-5p, or the mutated form of miR-324-5p together with pmirGLO-MLKL-3'UTR. Relative luciferase activity analysis of MLKL-3'UTR. Data are presented as firefly luciferase activity/renilla luciferase activity ± SD (n=5, biological replicates).

D Western blotting analysis of MLKL and β-actin in HeLa-endogenous MLKL cells (HeLa cells stably expressing human RIPK3) and HeLa-exogenous MLKL cells (Mlkl-/- HeLa cells stably expressing human RIPK3 and the coding sequence (CDS) of MLKL) that were harvested 48 h after transfection with NC, miR-324-5p, or MLKL siRNA oligos targeting the CDS region of MLKL (siMLKL-CDS).

E qPCR analysis for the expression of MLKL in HeLa-exogenous MLKL cells that were harvested 48 h after transfection with NC, miR-324-5p, siMLKL-3'UTR, or siMLKL-CDS.

F, G HeLa-endogenous MLKL cells (F) and HeLa-exogenous MLKL (G) were transfected with NC, miR-324-5p, siMLKL-3'UTR, or siMLKL-CDS. After 48 h, cells were treated with T+S+Z for 24 h. Cell survival was determined by measuring ATP levels.

H HeLa cells expressing MLKL(1-190 aa) fused to DmrB (HeLa-MLKL(1-190)-Dmir cells) were transfected with NC, miR-324-5p, siMLKL-3'UTR, or siMLKL-CDS. After 48 h, cells were treated with DMSO or the dimerization agent AP20187 (60 nM) for 24 h. Cell survival was determined by measuring ATP levels.

Data information: The number of surviving cells was normalized to the number of surviving control cells, which were treated with DMSO. In B, and E-H, data are represented as the means ± SD of three biological replicates. Statistical analyses were performed using unpaired Student's t-test. All experiments were performed at least three times, and representative data are shown.
Interferon-mediated repression of miR-324-5p potentiates necroptosis to facilitate antiviral defense (2022)
Figure 4

Fig 4 MiR-324-5p-mediated regulation of MLKL is species specific

A, B Evolutionary conservation of the CCT(CCU) region in MLKL-3'UTR. The DNA sequences of MLKL flanking the CCT(CCU) region from various species were aligned by DNAman.

C African green monkey kidney epithelial (Vero) cells were transfected with NC, siMLKL, or miR-324-5p for 48 h. qPCR analysis for the expression of MLKL.

D MEFs were transfected with NC, siMLKL, or miR-324-5p for 48 h. qPCR analysis for the expression of MLKL and western blotting analysis of MLKL and β-actin.

E MEFs were transfected with NC, siMLKL, or miR-324-5p. After 48 h, cells were treated with DMSO or T+S+Z for 24 h. Cell viability was determined by measuring ATP levels.

F HEK293T cells were transfected with NC or miR-324-5p, together with pmirGLO-mouse MLKL-3'UTR or the mutated form of mouse MLKL-3'UTR. Relative luciferase activity analysis of MLKL-3'UTR. Data are presented as firefly luciferase activity/renilla luciferase activity ± SD (n = 5, biological replicates).

Data information: In (C-E), data are represented as the means ± SD of three biological replicates. Statistical analyses were performed using unpaired Student's t-test. All experiments were performed at least three times, and representative data are shown. β-actin serves as an internal control (D).
Interferon-mediated repression of miR-324-5p potentiates necroptosis to facilitate antiviral defense (2022)
Figure 5

Fig 5 The IFN-JAK-STAT1 signaling pathway negatively regulates miR-324-5p

A U937 cells were treated with 100 ng/ml IFN-α, 100 ng/ml IFN-β, 100 ng/ml IFN-γ, 100 ng/ml LPS, or 25 μg/ml poly(I:C) for 24 h. Identical concentrations were used in later experiments unless otherwise stated. qPCR analysis for the expression of MLKL.

B U937 cells were treated with IFN-α, IFN-β, IFN-γ, LPS, or poly(I:C) for 24 h. and western blotting analysis of MLKL and β-actin (left). Quantification of MLKL normalized to β-actin levels (right).

C U937 cells were treated with PBS, IFN-α, IFN-β, IFN-γ, LPS, or poly(I:C) for 24 h. qPCR analysis for the expression of miR-324-5p.

D, E U937 cells were transfected with NC, siMLKL, or miR-324-5p. After 48 h, cells were treated with IFN-β (D) or IFN-γ (E) for 24 h. Western blotting analysis of MLKL and β-actin.

F, G U937 cells were incubated with 300 nM ruxolitinib for 2 h prior to IFN-β (F) or IFN-γ (G) treatment. After 24 h, qPCR analysis for the expression of miR-324-5p. After 48 h, western blotting analysis of p-STAT1, STAT1, MLKL and β-actin.

H, I U937 cells were transfected with NC, or STAT1 siRNA oliogs (siSTAT1). After 48 h, cells were treated with IFN-β (Η) or IFN-γ (Ι). qPCR analysis for the expression of miR-324-5p. Western blotting analysis of STAT1, MLKL and β-actin.

J HT-29 cells were treated with IFN-γ for 24 h. The cell lysate was collected for ChIP analysis. STAT1 binding to miR-324-5p promoter DNA region was determined by Chip-qPCR. The amount of precipitated DNA was calculated as percent input.

Data information: Data are represented as the means ± SD of three biological replicates. Statistical analyses were performed using unpaired Student's t-test. All experiments were performed at least three times, and representative data are shown.