EMBO/soda-nel huggingface dataset:
S-acylated Golga7b stabilises DHHC5 at the plasma membrane to regulate cell adhesion (2019)
Figure 4

Figure 4: Proteomic analysis of context-dependent DHHC5-associated protein complexes.

(A) FLAG-DHHC5 immunoprecipitations were performed using lysates from Hela cells co-expressing either wild-type (WT) or palmitoylation deficient mutant (m) Golga7b. The interaction of DHHC5 with its substrate Flot2 was measured by immunoblotting and is significantly reduced in the presence of palmitoylation deficient mutant Golga7b, quantification normalised to input, *=p<0.05, ratio paired t-test, n=3.

(B) Venn diagram comparing the overlap of numbers of proteins immunoprecipitated when FLAG-DHHC5 is expressed alone or co-expressed with either WT or mutant Golga7b. IF images are taken from figures EV2 and EV3 to illustrate DHHC5 localisation.

(C) Volcano plot of DHHC5 interactors from AP-MS analysis from HeLa cells co-expressing FLAG-DHHC5 and WT Golga7b highlighting interacting proteins involved in clathrin-mediated endocytosis, cell:cell adhesion and known substrates of DHHC5.

(D) Validation of novel DHHC5 interactors by co-IP: immunoblots of Dsg2from Hela cells co-expressing DHHC5 and either Wild-type (WT) or mutant (m) Golga7b and quantification normalised to input, *=p<0.05, ratio paired t-test, n=3.

(E) Co-IP immunoblots of clathrin heavy chain from Hela cells co-expressing DHHC5 and either wild-type (WT) or mutant (m) Golga7b and quantification normalised to input, *=p<0.05, ratio paired t-test, n=3.

(F) Co-IP immunoblots of ZO-1 protein from Hela cells co-expressing DHHC5 and either wild-type (WT) or mutant (m) Golga7b and quantification normalised to input, **=p<0.01 ratio paired t-test, n=3.

(G) Bar graph comparing the number of the cell:cell adhesion proteins identified in each protein interaction dataset. All scale bars 10μm. All error bars represent s.e.m.
S-acylated Golga7b stabilises DHHC5 at the plasma membrane to regulate cell adhesion (2019)
Figure 5

Figure 5: Inhibition of endocytosis restores DHHC5 to the plasma membrane in the presence of mutant Golga7b.

(A) 30 minute DMSO treatment of HeLa cells co-expressing WT DHHC5 (FLAG tagged) and mutant Golga7b (HA tagged).

(B) 30 minute Dynasore treatment (25µM) of HeLa cells co-expressing WT DHHC5 (FLAG tagged) and mutant Golga7b (HA tagged).

(C) Non-targeting control siRNA treatment (10nM) of HeLa cells expressing mutant Golga7b (HA tagged). Images are detecting endogenous DHHC5 and the HA tagged Golga7b

(D) AP2µ siRNA treatment (10nM) of HeLa cells that were subsequently transfected with mutant Golga7b (HA tagged). Images are detecting endogenous DHHC5 and the HA tagged Golga7b.

(E) Quantification of plasma membrane signal of DHHC5 in (A and B). ****=p<0.001, unpaired t-test. N=37, +Dynasore. N=28, +DMSO.

(F) Quantification of plasma membrane signal of DHHC5 in (C and D). ****=p<0.001, unpaired t-test. All scale bars 10μm.

(G) Biotin endocytosis assays of HeLa cells with endogenous DHHC5, over-expression of DHHC5 alone, co-expression of DHHC5 and WT Golga7b, co-expression of DHHC5 and mutant Golga7b or expression of the DHHC5 C-terminal mutant. Biotinylated cell surface proteins endocytosed within 15 minutes were purified using streptavidin resin and blots probed with an antibody to DHHC5. The lack of signal in the pull-down of the DHHC5 C-terminal mutant or when DHHC5 and WT Golga7b are co-expressed indicates that DHHC5 is stabilised at the plasma membrane in these conditions. The robust signal in the pull-down of endocytosed proteins from cells co-expressing DHHC5 and mutant Golga7b shows a high rate of endocytosis of DHHC5 in this condition. N=3, *=p<0.05, paired t test"

Data information: All N numbers refer to the number of cells imaged and quantified, all experiments repeated 3 times. Error bars represent s.e.m.
S-acylated Golga7b stabilises DHHC5 at the plasma membrane to regulate cell adhesion (2019)
Figure 6

Figure 6: Desmogelin-2 palmitoylation by DHHC5/Golga7b regulates its plasma membrane localisation and incorporation into desmosomes.

(A) ABE purifications from HeLa cells treated with 10nM non-targeting siRNA (nt siRNA) or DHHC5 siRNA were immunoblotted for Dsg2. Loss of the band in the +HA pull-down in the DHHC5 siRNA condition shows that Dsg2 palmitoylation is dependent on DHHC5.

(B) Confocal images of DHHC5 and Dsg2 in nt siRNA treated A431 cells three hours after calcium was reintroduced to the media in calcium switch experiments.

(C) Confocal images of DHHC5 and Dsg2 in DHHC5 depleted A431 cells three hours after calcium was reintroduced to the media in calcium switch experiments.

(D) Confocal images of DHHC5, Dsg2 and Golga7b in Golga7b depleted A431 cells three hours after calcium was reintroduced to the media in calcium switch experiments. See Appendix Figure S6 for quantification of data in panels B-D.

(E) Immunoblots and quantification of Dsg2 pulled-down in cell surface biotinylation assays in nt siRNA, DHHC5 siRNA or Golga7b siRNA treated conditions. Quantification of the intensity of the DSG2 signal in the pulldown is normalised to input. A significant reduction of cell surface Dsg2 occurs when DHHC5 or Golga7b are depleted by siRNA. *=p<0.05, paired t-test. N=4, Error bars represent s.e.m.

(F) Immunoblot of an ABE purification for Dsg2 in cells with either normal media or low calcium (5µM calcium) conditions, with quantification of the intensity of the DSG2 signal in the pulldown normalised to input. There is a significant reduction in the Dsg2 signal in the +HA pull-down from low calcium cells showing that Dsg2 is significantly less palmitoylated in this condition. *=p<0.05, paired t-test, N=4, error bars represent s.e.m. All scale bars 10μm.
S-acylated Golga7b stabilises DHHC5 at the plasma membrane to regulate cell adhesion (2019)
Figure 7

Figure 7: Cell adhesion is regulated by DHHC5/Golga7b.

(A) 10x brightfield images of A431 cells treated with non-targeting siRNA at time 0 (left) and after 48 hours (right) of 5 ng/ml EGF treatment.

(B) 10x brightfield images of A431 cells treated with DHHC5 siRNA at time 0 (left) and after 48 hours (right) of 5ng/ml EGF treatment.

(C) Quantification of cell scattering of A431 cells, showing a significant increase in cell scatter after 48 hours of DHHC5 knockdown and therefore a decrease in cell adhesion was observed. N=3 individual repeats, n=31 fields of view. **= p<0.005, unpaired t-test.

(D) 10x brightfield images of A431 cells treated with non-targeting siRNA at time 0 (left) and after 48 hours (right) of 5ng/ml EGF treatment.

(E) 10x brightfield images of A431 cells treated with Golga7b siRNA at time 0 (left) and after 48 hours (right) of 5ng/ml EGF treatment.

(F) Quantification of cell scattering of A431 cells, showing a significant increase in cell scatter after 48 hours of Golga7b knockdown and therefore a decrease in cell adhesion, was observed. N=3 individual repeats, n=31 fields of view. **= p<0.005, unpaired t-test.

(G) Plot showing the resistance of A431 cells from a trans-epithelial electrical resistance assay treated with either DHHC5 siRNA, Golga7b siRNA or non-targeting siRNA. A significant reduction in TEER demonstrates a decrease in cell adhesion after both DHHC5 and Golga7b depletion. N=12, ****=p<0.001, unpaired t-test, ***=p<0.01, unpaired t-test.

Data information: Error bars represent s.e.m.
Transnuclear mice reveal Peyer's patch iNKT cells that regulate B cell class switching to IgG1 (2019)
Figure 1

Figure 1. iNKT TN mice have increased numbers of tissue resident iNKT cells.

(A) Diagram representing the placement of various tissues analyzed for iNKT cells. mLN = mesenteric lymph node; sdLN = skin-draining lymph node.

(B) Relative iNKT cell yield in various tissues from TN mouse lines compared to C57BL/6 mouse lines. Tissues were isolated from indicated C57BL/6 or iNKT TN mouse lines and stained with anti-CD3 and CD1d (PBS57) tetramer.

(C) Spleen, mLN, sdLN, liver, adipose, and lung lymphocytes from Jα18-/- or Vα14 mice were stimulated in vitro with RAW-CD1d cells and 1μg α-GalCer. An additional sample of Vα14 lymphocytes from each organ was plated with RAW-CD1d cells but no α-GalCer. Supernatants were collected after 24 hours and cytokine concentration determined by cytokine bead array. Error bars are SD of mean values from three different mice per group. Results shown are representative of two independent experiments where n=3 biological replicates.
Transnuclear mice reveal Peyer's patch iNKT cells that regulate B cell class switching to IgG1 (2019)
Figure 2

Figure 2. iNKT cells from TN and C57BL/6 mice show similar influence of tissue microenvironment on NKT1, NKT2, and NKT17 subsets.

(A-F) Lymphocytes from the indicated tissues of C57BL/6 and Vα14 mice were stained with anti-CD3 and CD1d (PBS57) tetramer, before they were fixed, permeabilized, and stained with antibodies to T-bet, RORγt, and PLZF. Results shown are gated on CD3+CD1d-tetramer+ cells. (G) The percentage of CD3+ CD1d-tetramer+ iNKT cells in each organ that stained positively for PLZF, T-bet, and RORγt are shown. **p<0.01, Mann-Whitney test. Error bars are SD.

Data information: Results shown are representative of three independent experiments where n=3 biological replicates.
Transnuclear mice reveal Peyer's patch iNKT cells that regulate B cell class switching to IgG1 (2019)
Figure 3

Figure 3. iNKT TN mice show increased IgG1 production and IgG1+ B cells in the mLN and Peyer's patches.

(A) Lymphocytes from the Peyer's patches of C57BL/6 and Vα14 mice were stained with anti-CD3 and CD1d (PBS57) tetramer.

(B) Percentage of lymphocytes that were CD3+CD1d-tetramer+ iNKT cells among the Peyer's patches of C57BL/6, Vα14, and Jα18-/- mice are shown. Mann-Whitney test. Error bars are SD. C57BL/6 n=22; Vα14 n=16; Jα18-/- n=8.

(C-E)Mice were analyzed for total fecal IgA (C) total fecal IgG1 (D) or total serum IgG1 (E) by ELISA. Mann-Whitney test. Error bars are SD. C57BL/6 n=14; Vα14 n=11; Jα18-/- n=13.

(F) Percentages of total B cells that were IgG1+ in the spleen, mLN, and Peyer's patches of C57BL/6, Vα14, and Jα18-/- mice are shown. Mann-Whitney test. Error bars are SD. C57BL/6 n=15; Vα14 n=15; Jα18-/- n=5
Transnuclear mice reveal Peyer's patch iNKT cells that regulate B cell class switching to IgG1 (2019)
Figure 4

Figure 4. PP-NKT cells produce IL-4 in vitro and in vivo.

(A) CD1d (PBS57) tetramer+ CD3+ cells were sorted from spleens or PP of 3 different Vα14 TN mice along with CD4+CD3+CD1d tetramer- cells from PP (PP CD4). RNAseq was performed.

(B) Heatmap of FPKM values for the indicated Tfh genes across each RNAseq sample.

(C-D) Spleen, mLN, and PP lymphocytes from Vα14 and C57BL/6 mice were stimulated with PMA and ionomycin. Lymphocytes were stained with anti-CD3, CD1d (PBS57) tetramer, anti-IL-4, and anti-IFNγ. Percentages of iNKT cells and non-iNKT T cells within the population of CD3+IL-4+ cells and CD3+IFNγ+ cells, n = 5 mice for C57BL/6 group and n = 4 mice for Vα14 group. Error bars are SD. (E) Vα14 TN mice were administered α-GalCer either 2μg intravenously or 5μg by oral gavage. Mice were given Brefeldin A intraperitoneally after 30 min, and tissues were harvested 3 hours later. Cells from spleen, mesenteric lymph node, and Peyer's patches of Vα14 TN mice were permeabilized, fixed, and stained with antibodies to IL-4, IFNγ, and IL-17. Plots shown are gated on CD1d-(PBS57)-tetramer+ CD3+ iNKT cells.

(F) Quantification of data from C, n=2 mice per group. Mann-Whitney test. Error bars are SEM.
Transnuclear mice reveal Peyer's patch iNKT cells that regulate B cell class switching to IgG1 (2019)
Figure 5

Figure 5. iNKT cells provide indirect help for B cell class switching to IgG1 in vitro.

(A) Pooled spleen and LN cells from a Vα14 TN mouse were cocultured with wild type B cells with or without 1μg α-GalCer and with or without agnostic anti-CD40. IgM was measured by ELISA of culture supernatants 4 days later.

(B) Pooled spleen and LN cells from a Vα14 TN mouse were cocultured with anti-CD40 activated wild type B cells with or without 1μg α-GalCer and with blocking antibody to CD1d (1μg/mL, clone 1B1) or isotype control as indicated. IgM, IgG1, and IgA were measured by ELISA of 4 day culture supernatants.

(C-G) Spleen, mLN or PP cells from Vα14 TN iNKT mice and PP cells from an IL-4-/- mouse were cocultured with anti-CD40 activated wild type or CD1d-/- B cells. 1μg α-GalCer was added to the cultures to specifically activate iNKT cells. Blocking antibodies to IL-4 or isotype were added as indicated. IL-4 (C) and IgG1 (D-E) were measured by ELISA of culture supernatants. IgG1+ and IgA+ class switched B cells were enumerated by flow cytometry after 4 days of coculture (F-G). Representative of 2 independent experiments. np= not performed.

Data information: Mann-Whitney test. Error bars are SD of triplicate samples. *p<0.05; **p<0.01; ***p<0.001.
Transnuclear mice reveal Peyer's patch iNKT cells that regulate B cell class switching to IgG1 (2019)
Figure 6

Figure 6. Oral vaccination with a-GalCer leads to specific antibody production in NKT cell proficient mice.

(A) Mice were administered ovalbumin by oral gavage, with or without α-GalCer at the indicated time points, at the indicated amounts. Mice used were Jα18-/- (n=10 total), iNKT transnuclear (n=7 total), or littermate controls of iNKT TN mice that did not inherit the Vα14 allele (B6, n=12 total).

(B-C) OVA-specific antibody titers from feces and blood were determined by ELISA from the mice in (A). Shown are optical density values for samples collected on day 52 post-vaccination (7 days after the final boost). Error bars are SD.

(D) Cells from spleen, mesenteric lymph node, and Peyer's patches of Vα14 TN mice were permeabilized, fixed, and stained with the indicated antibodies. Plots shown are gated on CD1d-(PBS57)-tetramer+ CD3+ iNKT cells.

(E) Quantification of data from E, n=3 mice per group. Mann-Whitney test. Error bars are SEM. *p<0.05.
Transnuclear mice reveal Peyer's patch iNKT cells that regulate B cell class switching to IgG1 (2019)
Figure 7

Figure 7. PP iNKT cells exhibit a unique gene expression profile compared to spleen iNKT and PP CD4+ cells.

(A) Principle component analysis of RNAseq samples from biological replicates of PP-iNKT, spleen iNKT, and PP CD4+ cells

(B) Differential expression analysis comparing transcriptomes of spleen iNKT cells versus PP-iNKT cells.

(C) Heatmap of FPKM values from genes uniquely upregulated in PP-iNKT cells as compared to both spleen iNKT and PP CD4 cells. Red indicates higher expression and blue indicates lower expression.

(D) Spleen and PP cells from Vα14 TN mice were stained with the indicated antibodies, analyzed by flow cytometry and gated on CD1d (PBS57) tetramer+CD3+ cells.

(E) Quantification of data from D, n=11 mice per group. Mann-Whitney test. Error bars are SEM.
YTHDF1-Mediated Translation Amplifies Wnt-Driven Intestinal Stemness (2020)
Figure 1

Figure 1. Wnt signaling promotes YTHDF1 expression at the translational level

(A) Immunoblot analysis of mouse intestinal crypts treated with Wnt3a (60 ng/mL) for indicated time. The relative protein level was quantified and shown under each band.

(B) Polysome profiles of mouse intestinal crypt treated with or without Wnt3a for 30 min. The right panels show the distributions of Ythdf1 and Actb in polysome fractions. Data are represented as mean ± SEM. *p<0. 05, **p<0. 01 (3 biological replicates, t-test).

(C) Immunoblot analysis of SW620 cells with or without APC overexpression. The relative protein level was quantified and shown under each band.

(D) Polysome profiles of SW620 cells with or without APC overexpression. The right panels show the distributions of YTHDF1 and ACTB in polysome fractions. Data are represented as mean ± SEM. *p<0. 05, **p<0. 01 (3 biological replicates, t-test).

(E) Dual-Luciferase assay wih a construct bearing the 5'UTR of YTHDF1 in SW620 cells with or without APC overexpression. Data are represented as mean ± SEM. *p<0. 05, **p<0. 01 (3 biological replicates, t-test).

(F) YTHDF1 protein expression in intestinal tissue from 4 pairs of Apc+/+ and Apcmin/+ mice. N, normal tissue. T, tumor tissue. A, tumor adjacent tissue.

(G) YTHDF1 staining in matched samples of human CRC tissue and adjacent non-tumor tissue. Scale bar, 450 μm. The right panel shows the statistics of IHC scores. Chi-square test; n = 75 for each group.
YTHDF1-Mediated Translation Amplifies Wnt-Driven Intestinal Stemness (2020)
Figure 2

Figure 2. YTHDF1 is required for efficient intestinal regeneration

(A) The representative jejunum from wildtype (Ythdf1CTL) and Ythdf1cKO mice. Left: hematoxylin and eosin staining (H&E); middle: BrdU staining; right: quantification of crypt height and BrdU+ cells. Scale bar, 50 μm. Data are represented as mean ± SEM. (9 biological replicates, t-test).

(B) Immunoblot analysis of small intestines from Ythdf1CTL and Ythdf1cKO mice after 12 Gy IR.

(C) YTHDF1 (left) and Ki67 (right) staining of small intestine from wildtype mice 72 hr after 12 Gy IR. Scale bar, 50 μm. Red arrows indicate the proliferating cells.

(D) Small intestines from Ythdf1CTL and Ythdf1cKO mice 72 hr after 12 Gy IR. Left: H&E staining; middle: Ki67 staining; right: quantification of crypt height and Ki67+ cells. Scale bar, 50 μm. Vertical bars indicate the length of the crypts. Data are represented as mean ± SEM. ****p<0. 0001 (9 biological replicates, t-test).

(E) RT-qPCR analysis of small intestines from Ythdf1CTL and Ythdf1cKO mice 72 hr after 12 Gy IR. Data are represented as mean ± SEM. *p<0. 05, **p<0. 01 (5 biological replicates, t-test).
YTHDF1-Mediated Translation Amplifies Wnt-Driven Intestinal Stemness (2020)
Figure 3

Figure 3. Ythdf1 deletion reduces Wnt-driven tumorigenesis in vivo

(A) Small intestines from 24-week-old Apcmin/+; Ythdf1CTL and Apcmin/+; Ythdf1cKO mice.

(B) H&E staining of small intestines from 24-week-old Apcmin/+; Ythdf1CTL and Apcmin/+; Ythdf1cKO mice. Scale bar, 1 mm. The black arrows indicate the tumors.

(C and D) Tumor number (C) and size distribution (D) from the small intestines of 24-week-old Apcmin/+; Ythdf1CTL and Apcmin/+; Ythdf1cKO mice. Data are shown as mean ± SEM (6 mice for each group). **p<0. 01, ***p<0. 001 (t-test).

(E) Workflow of the AOM/DSS-induced colitis associated cancer model.

(F) Colons from Ythdf1CTL and Ythdf1cKO mice on day 84 of AOM/DSS induction.

(G) H&E staining of colons from Apcmin/+; Ythdf1CTL and Apcmin/+; Ythdf1cKO mice after AOM/DSS induction. Scale bar, 200 μm.

(H and I) Colon tumor number (H) and size distribution (I) from Ythdf1CTL and Ythdf1cKO mice on day 84 of induction. Data are shown as mean ± SEM (6 mice for each group). **p<0. 01 (t-test).
YTHDF1-Mediated Translation Amplifies Wnt-Driven Intestinal Stemness (2020)
Figure 4

Figure 4. YTHDF1 is required for maintenance of intestinal stem cells

(A) Fluorescent in situ hybridization for Ythdf1 mRNA in Ythdf1CTL and Ythdf1cKO small intestines. Scale bar, 50 μm.

(B) Immunoblot analysis of fractionated murine intestine enriched for villus cells and crypt cells.

(C) Flowcytometry analysis of YTHDF1 protein expression in GFPhigh/low/neg populations from Lgr5-GFP-IRES-creERT2 mice.

(D) Morphology of organoids from Lgr5-creERT2:Ythdf1fl/fl mice in Wnt3a-conditioned medium without or with 4-OHT induction and infected with lentivirus expressing YTHDF1 or YTHDF1 mutant. Scale bar, 250 μm.

(E) Quantification of differentiated versus undifferentiated organoids from (D). Data are represented as mean ± SEM. **p<0. 01 (4 biological replicates, t-test).

(F) Relative mRNA levels of intestinal stem cell and differentiation marker genes in organoids described in (D). Data are represented as mean ± SEM. 4 biological replicates. *p<0. 05, **p<0. 01, ***p<0. 001 (t-test).
YTHDF1-Mediated Translation Amplifies Wnt-Driven Intestinal Stemness (2020)
Figure 5

Figure 5. Transcriptome-wide identification of YTHDF1-regulated mRNAs

(A) Scatterplot of mRNA expression and ribosome profiling data from control and YTHDF1 knockdown cells. The upregulated (red) and downregulated (blue) genes in translational efficiency (TE) are highlighted.

(B) The relative m6A peak coverage in control cells for transcripts grouped according to TE changes after YTHDF1 knockdown. POI: peak over input. Down, genes significantly downregulated in YTHDF1 knockdown cells (<0.5 fold). Unchanged, genes not significantly changed. Up, genes significantly upregulated (>2 fold). The upper and lower quartiles and the median are shown for each group. Mann-Whitney test. **p<0. 01. ***p<0. 001.

(C) Violin plots showing TE change between control and YTHDF1 knockdown cells for not-methylated (non-m6A) and methylated (m6A) transcripts. The upper and lower quartiles and the median are indicated for each group. Mann-Whitney test.

(D) Cumulative distributions of TE change for non-m6A and m6A transcripts as in (C). Kolmogorov-Smirnov test.

(E) Heatmap showing the TE of Wnt signaling components in control and YTHDF1 knockdown cells. The values show the fold change of the indicated color of the heatmap.
YTHDF1-Mediated Translation Amplifies Wnt-Driven Intestinal Stemness (2020)
Figure 6

Figure 6. TCF7L2 is a key functional target of YTHDF1

(A) Immunoblot analysis of HCT116 cells with YTHDF1 knockdown. Two major bands were detected for human TCF7L2 due to alternative splicing.

(B) Polysome profiles of HCT116 cells with or without YTHDF1 knockdown. The right panels show the distributions of TCF7L2, and ACTB in polysome fractions. Data are represented as mean ± SEM. *p<0. 05, **p<0. 01 (3 biological replicates, t-test).

(C) Immunoblot analysis of crypts from Ythdf1CTL and Ythdf1cKO mice. Multiple bands were detected for mouse TCF7L2 representing different isoforms.

(D) Immunoblot analysis of mouse intestinal crypts treated with Wnt3a for 60 min.

(E) MeRIP-qPCR analysis of m6A levels of TCF7L2 in crypts treated with or without Wnt3a. Data are represented as mean ± SEM. *p<0. 05 (3 biological replicates, t-test).

(F) RIP analysis of the interaction of YTHDF1 with TCF7L2 mRNA. The enrichment was measured by qPCR and normalized to input. Data are represented as mean ± SEM. *p<0. 05 (3 biological replicates, t-test).

(G) β-catenin/TCF4 reporter activity in YTHDF1 knockdown cells infected with lentivirus expressing TCF7L2. Data are represented as mean ± SEM. *p<0. 05, **p<0. 01, ***p<0.001 (3 biological replicates, t-test).

(H) Morphology of organoids from Lgr5-creERT2:Ythdf1fl/fl mice in Wnt3a-conditioned medium without or with 4-OHT induction and infected with lentivirus expressing TCF7L2. Scale bar, 250 μm.

(I) Quantification of differentiated versus undifferentiated organoids from (H). Data are represented as mean ± SEM. **p<0. 01, ***p<0. 001 (3 biological replicates, t-test).