EMBO/soda-nel huggingface dataset:
RAB13 mRNA compartmentalisation spatially orients tissue morphogenesis (2020)
Figure 3

Figure 3. CRISPR-Cas9 excision of the 3'UTR localisation element of RAB13 disrupts mRNA targeting.

A. CRISPR-Cas9 strategy to derive HUVECs with an excision of the LE in the RAB13 3'UTR (∆LE) and parallel generation of wild type (Wt) control cells. The Wt RAB13 exon 8 is represented with its coding sequence in dark and the 3'UTR in clear boxes. The 5' and 3' gRNA-targeted regions are represented with green lines. Arrows: relative positions of the forward (f) and reverse (r) PCR primers used to identify HUVECs with CRISPR-Cas9-mediated excision of the LE.

B. Representative genotyping PCR demonstrates the band size shift in ∆LE HUVECs.

C. Detailed DNA sequence depicting nucleotide positions within the RAB13 3'UTR of Wt and ∆LE HUVECs.

D. Wt and ∆LE HUVEC RNAseq mapped reads depicting RAB13 exon usage.

E. Quantification of RAB13 mRNA smFISH spot number in Wt and ∆LE HUVECs (n=3 experiments; ns: not significant; unpaired t test).

F. Number of RAB13 mRNA smFISH spots plotted against the respective Polarisation Index (PI) (n=29 cells; ns: not significant; linear regression).

G. Left: representative Western blotting (WB) of Wt and ∆LE HUVECs. Right: densitometry analysis of WB data (n=3 samples; ns: not significant; unpaired t test).

H. smFISH co-detection of RAB13 and control GAPDH in Wt and ∆LE motile HUVECs cultured under subconfluent conditions.

I. PI of RAB13 and GAPDH co-detected in Wt and ∆LE HUVECs (n=29 cells; P***<0.001, ns: not significant; Mann-Whitney test).

J. RAB13 PI plotted against respective GAPDH PI. The slope of the coloured lines represents the average RAB13/GAPDH PI ratio; the dashed grey line represents a 1:1 ratio (n=29 cells).

Data information: 3 Wt and 3 ∆LE HUVECs independent clones were used to collect data (E-J). Arrows indicate orientation of RNA localisation; yellow dashed lines outline cell borders; red circles highlight smFISH spots; scale bars = 20 µm (H). Bar charts are presented as means±s.d.
RAB13 mRNA compartmentalisation spatially orients tissue morphogenesis (2020)
Figure 4

Figure 4. RAB13 mRNA polarisation spatially orients filopodia dynamics.

A. Representative time-lapse microscopy of a bEnd.3 cell co-transfected with plasmids expressing Lyn-mCherry, MCP-GFPnls and 24xMS2-RAB13 3'UTR.

B. Frequency of newly formed filopodia formed within 5 µm intervals relative to the nearest MCP-GFPnls particle or a randomised (ctrl) position (n=99 filopodia; **P<0.01, ns: not significant; Pearson r correlation).

C. Distance of newly-formed filopodia to MCP-GFPnls or a ctrl position plotted against filopodia duration (n=99 filopodia; *P<0.05, ns: not significant; Spearman r correlation).

D. Wt and ∆LE HUVECs co-cultured on fibroblast monolayers. Endothelial cells were identified either with an antibody against the endothelial cell marker PECAM-1 (left) or through expression of a nucleofected plasmid encoding the cytoskeletal marker Lifeact-GFP (right).

E. Number of filopodia detected in co-cultured HUVECs (n=30 cells; **P<0.01; unpaired t test)

F. Number of filopodia detected in co-cultured HUVECs within 12 µm intervals relative to cell distal tip (n=30 cells; **P<0.01, P***<0.001, ns: not significant; one-way ANOVA with Bonferroni's correction)

G. Number of filopodia detected in individual clones of co-cultured HUVECs within 12 µm intervals relative to cell distal tip.

H. Illustration of the spatial relationship between RAB13 mRNA localisation and sites of filipodia production.

Data information: 3 Wt and 3 ∆LE HUVECs independent clones were used to collect data (D-G). Arrowheads indicate filopodia (A,D); scale bars = 10 µm (A) and 6 µm (D). Bar charts are presented as means±s.d.
RAB13 mRNA compartmentalisation spatially orients tissue morphogenesis (2020)
Figure 5

Figure 5. mRNA polarisation achieves spatial compartmentalisation of RAB13 translation and protein function.

A Strategy used to detect local protein synthesis in protrusions formed by HUVECs migrating through Transwell membranes.

B. Representative Puro-PLA experiments detecting newly synthesised RAB13 in HUVEC protrusions present in the lower side of Transwell membranes. Puro: puromycin; Aniso: anisomycin; 1ary Abs: primary antibodies.

C. Quantification of RAB13 Puro-PLA punctae normalised to protrusion area (n≥40 protrusions; *P<0.05, ****P<0.0001; Kruskal-Wallis test with Dunn's correction).

D. Representative RAB13 IF assay on migrating HUVECs.

E. Left: representative Western blotting (WB) of siRNA transfected HUVECs. Right: densitometry analysis of WB data (n=3 samples; *P<0.05; paired t test).

F. Control (ctrl) and RAB13 siRNA-treated HUVECs co-cultured on fibroblast monolayers. Endothelial cells were identified with an antibody against the endothelial cell marker PECAM-1.

G. Number of filopodia detected in co-cultured HUVECs (n≥35 cells; **P<0.01; unpaired t test).

H. Number of filopodia detected in co-cultured HUVECs within 12 µm intervals relative to cell distal tip (n≥35 cells; *P<0.05, ns: not significant; Kruskal-Wallis test with Dunn's correction).

I. Illustration of the spatial relationship between the sites of RAB13 mRNA localisation, local translation and RAB13 protein-mediated filopodia distribution.

Data information: white arrowheads indicate Puro-PLA punctae; yellow dashed lines outline protrusion borders (B); black arrowheads indicate filopodia (F); scale bars = 10 µm (B,D) and 6 µm (F). Bar charts are presented as means±s.d.
RAB13 mRNA compartmentalisation spatially orients tissue morphogenesis (2020)
Figure 7

Figure 7. CRISPR-Cas9 editing of the zebrafish rab13 3'UTR perturbs mRNA polarisation.

A. CRISPR-Cas9 strategy to generate the Tg(kdrl:EGFP) rab13+/∆3'UTR zebrafish strain. The wild type (Wt) rab13 exon 8 is represented with its coding sequence in dark and the 3'UTR in clear boxes; the 5' and 3' gRNA-targeted regions are represented with green lines. Arrows: relative positions of the forward (f) and reverse (r) PCR primers used to identify animals with CRISPR-Cas9-mediated deletions (∆) in the rab13 3'UTR.

B. Representative genotyping PCR demonstrates the band size shift in zebrafish harbouring a ∆482 rab13 3'UTR. Asterisk marks a heteroduplex formed between Wt and ∆482 rab13 3'UTR PCR amplicons.

C. Detailed DNA sequence depicting nucleotide positions within the Wt and ∆482 rab13 3'UTR.

D. RNAseq mapped reads depicting rab13 exon usage in Tg(kdrl:EGFP) rab13+/+ and rab13∆3'UTR /∆3'UTR zebrafish embryos. Coloured lines indicate SNPs.

E. qPCR analysis of rab13 mRNA levels in individual 26-28 hpf clutch-matched sibling embryos (n≥9 embryos; ns: not significant; Kruskal-Wallis test with Dunn's correction).

F. smFISH detection of rab13 and kdr mRNA in cultured GFP-expressing endothelial cells extracted from 48 hpf Tg(kdrl:EGFP) rab13+/+ and rab13∆3'UTR /∆3'UTR zebrafish embryos.

G. Polarisation Index (PI) of rab13 and kdr detected by smFISH in individual zebrafish cells (n≥8 cells; **P<0.01, ns: not significant; one-way ANOVA with Bonferroni's correction).

H. rab13 PI plotted against respective kdr PI. The slope of the coloured lines represents the average rab13/kdr PI ratio; the dashed grey line represents a 1:1 ratio (n≥8 cells).

Data information: +/+, +/∆ and ∆/∆ represent Tg(kdrl:EGFP) rab13+/+, rab13+/∆3'UTR and rab13∆3'UTR /∆3'UTR embryos, respectively (E,G,H). Arrows indicate orientation of RNA localisation; yellow dashed lines outline cell borders; red circles highlight smFISH spots; scale bars = 10 µm (F). Bar charts are presented as means±s.d.
RAB13 mRNA compartmentalisation spatially orients tissue morphogenesis (2020)
Figure 8

Figure 8. rab13 mRNA polarisation orients blood vessel morphogenesis.

A. Time-lapse confocal microscopy of representative Tg(kdrl:EGFP) rab13+/+ and rab13∆3'UTR /∆3'UTR embryos. DLAV: dorsal longitudinal anastomotic vessel; HM: horizontal myoseptum.

B. Frequency of ISV ectopic branching occurring at the HM (n=4 experiments; *P<0.05; one-way ANOVA with Bonferroni's correction).

C. Illustration of the role for RAB13 mRNA localisation, local translation and compartmentalisation of RAB13 function in defining the orientation of EC filopodia dynamics, motile EC polarity and blood vessel pathfinding.

Data information: T0= 25 hpf; arrowheads indicate extra branches emerging from the main ISVs at the HM position; scale bars = 50 µm (A). +/+, +/∆ and ∆/∆ represent Tg(kdrl:EGFP) rab13+/+, rab13+/∆3'UTR and rab13∆3'UTR /∆3'UTR embryos, respectively (B). Bar chart is presented as means±s.d.

Extended View Figure Legends:
Human GBP1 is a microbe-specific gatekeeper of macrophage apoptosis and pyroptosis (2019)
Figure 1

Figure 1: GBP1, its GTPase activity and prenylation are required for IFNγ-enhanced cell death upon Toxoplasma infection

(A) IFNγ enhances macrophage host cell death after type I (RH) and type II (Pru) Toxoplasma gondii (Tg) infection. Graphs show cell death as measured by LDH release assay (left) and viability measured by XTT assay (right) in untreated or IFNγ-primed THP-1 cells infected with type I or type II Tg for 24 h.

(B) LDH release assays from THP-1 cells left untreated or primed with IFNγ, transfected with siRNA against indicated GBPs or non-targeting control (CTRL) and infected with indicated strains of Tg for 24 h.

(C) LDH release assay from primary monocyte-derived macrophages (MDM) left untreated or treated with IFNγ, transfected with siRNA against GBP1 or non-targeting control (CTRL) and infected with indicated strain of Tg for 24 hours. Mean ± SEM of n = 4 donors shown.

(D) LDH release assay from indicated wildtype, GBP1 knockout (∆GBP1) or GBP1 reconstituted (∆GBP1+Tet-GBP1) cells infected with type I or type II Tg for 24 hours. Cells were untreated or treated with IFNγ or additionally treated with doxycycline (Dox) as indicated.

(E) Realtime propidium iodide (PI) uptake assay from indicated THP-1 cells infected with type I or type II Tg, and untreated or treated with IFNγ and/or Dox as labelled. AU, arbitrary units.

(F) LDH release assay from wildtype THP-1 or ∆GBP1 cells stably reconstituted with Dox-inducible expression plasmids of the indicated mutants of GBP1. Cells were pre-treated with IFNγ and Dox and infected with either type I or type II Tg for 24 h.

Data information: Graphs in (A-B, D-F) show mean ± SEM from n = 3 independent experiments. * P ≤ 0.05; ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001 for indicated comparisons in (A-D) from two-way ANOVA or comparison between THP-1 WT and other genotypes by one-way ANOVA in (F) following adjustment for multiple comparisons; ns, not significant.
Human GBP1 is a microbe-specific gatekeeper of macrophage apoptosis and pyroptosis (2019)
Figure 2

Figure 2. GBP1 promotes ASC- and CASP8-dependent atypical apoptosis in Toxoplasma-infected human macrophages

(A) Propidium iodide (PI) uptake in indicated wildtype THP-1 or ∆CASP-1, ∆CASP-4, ∆ASC cells, or THP-1 stably expressing GSDMD-targeting miRNA (GSDMDmiR) infected with the indicated strains of Toxoplasma gondii (Tg) for 24 h. Cells were untreated or treated with IFNγ as indicated. Mean area under the curve (AUC) ± SEM from n = 3 experiments are shown. ns, not significant, from two-way ANOVA following adjustment for multiple comparisons.

(B) Real-time apoptosis assay (Annexin V (AnnV)-Glo luminescence) from IFNγ-primed primary human MDMs transfected with non-targeting control (siCTRL; left) or GBP1 siRNA (siGBP1; right) and left uninfected or infected with type I or type II Tg. Depicted is the mean ± SEM of n = 4 donors. AU, arbitrary units.

(C) Immunoblots from primary MDMs infected with the indicated Tg strains for 12 h. Cells were untreated or pre-treated with IFNγ before infection. Treatment with TNFα and cycloheximide (CHX) served as positive control for apoptosis. Images are representative of n = 4 biologically independent experiments.

(D-E) Live time-lapse imaging to visualize apoptotic cell death and measure caspase-3/7 activities using a fluorescent caspase substrate. THP-1 WT were primed with IFNγ or left untreated and infected with either type I or type II Tg. Plot in (D) shows cumulative caspase-3/7 activity from an 18-hour time-course (mean ± SEM from n = 3 independent experiments). Representative phase-contrast images overlaid with fluorescence signal for caspase-3/7 activity (green) images are shown in (E) from IFNγ-primed THP-1 cells infected with type I Tg captured at indicated times post-infection. Scale bar 20 μm.

(F) AnnV-Glo assay from IFNγ-primed THP-1 transfected with siRNA against indicated human caspases and infected with type I or type II Tg. Area under the curve (AUC) from a real-time AnnV-Glo luminescence assays similar to those in (B) are plotted as mean ± SEM from n = 3 independent experiments. * P ≤ 0.05, *** P ≤ 0.001, **** P ≤ 0.0001 from two-way ANOVA for indicated comparisons following adjustment for multiple comparisons; ns, not significant.

(G) AnnV-Glo assay from IFNγ-primed primary human MDMs transfected with siRNA against CASP8, CASP10 and non-targeting control (CTRL) and infected with type I or type II Tg. Area under the curve (AUC) from real-time assays similar to those in (B) are plotted from n = 4 independent experiments. Matched shapes and color of symbols indicate donors. **** P ≤ 0.0001 from two-way ANOVA following adjustment for multiple comparisons; ns, not significant.
Human GBP1 is a microbe-specific gatekeeper of macrophage apoptosis and pyroptosis (2019)
Figure 3

Figure 3: Toxoplasma-infected human macrophages die through AIM2-dependent atypical apoptosis

(A) AnnV-Glo assay from IFNγ-primed THP-1 transfected with siRNA against indicated human NLR/ALR sensors and infected with type I or type II Toxoplasma gondii (Tg). Area under the curve (AUC) from real-time assays are plotted as mean ± SEM from n = 3 independent experiments. **** P ≤ 0.0001 from two-way ANOVA for indicated comparisons following adjustment for multiple comparisons; ns, not significant.

(B) AnnV-Glo assay from IFNγ-primed primary human MDMs transfected with siRNA against AIM2 or non-targeting control (CTRL) and infected with type I or type II Tg. Area under the curve (AUC) from real-time assays are plotted from n = 4 independent experiments. Matched shapes and color of symbols indicate donors. **** P ≤ 0.0001 from two-way ANOVA for indicated comparisons following adjustment for multiple comparisons; ns, not significant.

(C) Representative images (top) from IFNγ-primed THP-1 WT infected with type I Tg for 8 hours and stained for ASC and active caspase-8. Blue, Nuclei; Red, ASC; Green, active caspase-8 (IETD-FITC) and Grey, Tg. Scale bar, 5 μm or 2 μm in the magnified images. Quantification of ASC specks (bottom) in Tg-infected THP-1 WT or ∆CASP1 treated with IFNγ or left untreated at 8 hours post infection, plotted as mean ± SEM from n = 3 independent experiment.

(D) Immunoblots from primary MDMs infected with Tg strains for indicated times. Cells were untreated or pre-treated with IFNγ before infection. Images are representative of n = 4 biologically independent experiments.
Human GBP1 is a microbe-specific gatekeeper of macrophage apoptosis and pyroptosis (2019)
Figure 4

Figure 4: GBP1 targets Toxoplasma vacuoles in human macrophages

(A-B) Representative images (A) and quantification (B) from IFNγ-primed THP-1 ∆GBP1 stably reconstituted with Tet-mCH-GBP1, mCH-GBP1K51A, mCH-GBP1C589A or mCH-GBP1∆589-592 variants or monocyte derived macrophages (MDM) stained for GBP1. Cells were infected with type I or type II Toxoplasma gondii (Tg) for 2 h. Blue, Nuclei; Grey, Tg; Red, mCH-GBP1 or Magenta, GBP1. Scale bar, 5 μm. Graph in (B) shows mean percentage of Tg vacuoles targeted by GBP1 from n = 4 experiments. **** P ≤ 0.0001 for indicated comparisons by two-way ANOVA.
Human GBP1 is a microbe-specific gatekeeper of macrophage apoptosis and pyroptosis (2019)
Figure 5

Figure 5: GBP1 targets Salmonella vacuoles and mediates caspase-4-dependent pyroptosis

(A) LDH release assay from indicated THP-1 cells untreated or treated with IFNγ and infected with Salmonella Typhimurium (STm) WT or a SPI-1 mutant ΔinvA for 4 h at indicated multiplicities of infection (MOI). Mean ± SEM of n = 3 experiments are shown.

(B) Propidium iodide (PI) uptake assay from IFNγ-primed primary MDMs transfected with non-targeting control siRNA (CTRL) or siRNA against GBP1 (left) or indicated lines of THP-1 (right) infected with STm-GFP for 4 h. Area under the curve (AUC) from real-time assay plotted as mean ± SEM from n = 3 independent experiments.

(C) PI uptake assay from IFNγ-primed primary MDMs transfected with indicated siRNA or non-targeting control (CTRL) and infected with STm-GFP for 4 h. Area under the curve (AUC) from real-time assay plotted as mean ± SEM from n = 4 independent experiments.

(D) PI uptake assay from IFNγ-primed or untreated THP-1 ΔCASP-1 cells transfected with indicated siRNA and infected with STm-GFP for 4 h. Area under the curve (AUC) from real-time assay plotted as mean ± SEM from n = 3 independent experiments. Schematic on right shows overview of the pathway leading to caspase-1 and caspase-4 activation.

(E) LDH release assay from indicated THP-1 cells treated with IFNγ and Dox and infected with STm-GFP for 4 h. Shown is the mean ± SEM of n = 3 experiments.

(F-G) Representative images (F) and quantification (G) from IFNγ-primed THP-1 ∆GBP1 cells stably reconstituted with Tet-mCH-GBP1, mCH-GBP1K51A, mCH-GBP1C589A or mCH-GBP1∆589-592 variants or primary MDMs stained for GBP1. Cells were infected with STm-GFP for 2 h. Blue, Nuclei; Grey, STm; Red, mCH-GBP1 or Magenta GBP1. Scale bar, 5 μm. Graph in (G) shows percentage of STm vacuoles targeted by GBP1.

(H) Quantification of GBP1 recruitment to STm-GFP containing vacuoles (SCV) in MDMs pre-treated with IFNγ or left untreated (UT). Mean ± 95% CI of GBP1 fluorescence intensity measured on the indicated number (n) of SCVs for each condition from three independent experiments are shown. **** P<0.0001 by nested t-test.

(I) Representative images from THP-1 ∆GBP1+Tet-mCH-GBP1 cells stably expressing YFP-CASP4C258S. Cells were infected with STm for 2 h. Blue, Nuclei & STm (Hoechst dye); Red, mCH-GBP1; Green, YFP-CASP4C258S. Scale bar, 5 μm.

(J) Quantification of THP-1 ∆GBP1+Tet-mCH-GBP1 cells stably expressing YFP-CASP4C258S and infected with STm for 2 h. Graph shows percentage of STm vacuoles targeted by GBP1, CASP4 or both from n = 3 experiments. n.d. not detected.

Data information: *** P ≤ 0.001, **** P ≤ 0.0001 for indicated comparisons by two-way ANOVA in (B-D, G), comparison between untreated and IFNγ-treated in (A) or THP-1 WT and other genotypes in (E) by one-way ANOVA following adjustment for multiple comparisons; ns, not significant.
Human GBP1 is a microbe-specific gatekeeper of macrophage apoptosis and pyroptosis (2019)
Figure 6

Figure 6: Transfection of pathogen-derived ligands bypasses GBP1 in inducing macrophage death

(A) Cell death measured using Annexin V (AnnV)-Glo assay of THP-1 ∆CASP1 cells transfected with non-targeting control (CTRL) or GBP1 siRNA and treated as indicated. Cells were primed with IFNγ, transfected with soluble Tg antigen (STAg) or both, or left untreated (UT). Area under the curve (AUC) from real-time assay plotted as mean ± SEM from n = 3 independent experiments.

(B) AnnV-Glo assay from naïve or IFNγ-primed THP-1 ∆CASP-1 cells transfected with soluble Tg antigen (STAg) that was untreated (UT) or treated as indicated. Area under the curve (AUC) from real-time assay plotted as mean ± SEM from n = 3 independent experiments.

(C) Immunoblots from IFNγ-primed THP-1∆CASP-1 or THP-1 ∆ASC cells transfected with non-targeting control (CTRL), GBP1, AIM2, CASP-1 or CASP-8 siRNA and then untreated (-) or transfected with STAg for 12 h. Images are representative of n = 2 independent experiments.

(D) AnnV-Glo assay from naïve or IFNγ-primed THP-1 ∆CASP-1 cells transfected with non-targeting control (CTRL), GBP1, AIM2, CASP-1 or CASP-8 siRNA and then untreated (UT) or transfected with STAg as indicated for 18 h. Area under the curve (AUC) from real-time assay plotted as mean ± SEM from n = 3 independent experiments. Schematic on right shows an overview of pathways leading to caspase-8 activation.

(E) Propidium iodide (PI) uptake assay from MDMs transfected with non-targeting control (CTRL) or GBP1 siRNA and then left untreated (UT), treated with LPS or transfection reagent (Lipofectamine) only or transfected with LPS for 4 h. Area under the curve (AUC) from real-time assays plotted as mean ± SEM from n = 4 independent experiments. Schematic on right shows an overview of pathways leading to caspase-4 activation.

Data information: * P ≤ 0.05; **** P ≤ 0.0001 from two-way ANOVA following adjustment for multiple comparisons; ns, not significant.

Expanded View Figure legends
CDK9 and PP2A regulate RNA polymerase II transcription termination and coupled RNA maturation. (2022)
Figure 1

Figure 1. CDK9 inhibition abrogates polyadenylation.

A. Schematic of the nuclear 3'READS experiment.

B. Violin plots of the 3'READS signal on all the TNFα induced genes (n=307) or the set of TNFα induced genes longer than 40 kb (n=111). **** p < 0.0001. Statistical test: Friedman test with Dunn's multiple test correction. Violin plot: minimal to maximal value, dashed horizontal line: median, dotted horizontal lines: interquartile (25% and 75%).

C. Western blot of total pol II, Ser2P, Xrn2, CPSF2, CPSF73, and histone H3 as a loading control, on the chromatin fraction.

D. Western blot of Xrn2, CPSF2, and Nucleolin as a loading control, on the nucleoplasm fraction (the CPSF73 antibody does not provide reliable results on the nucleoplasm fraction).

E. Quantification of the western blots shown in C and D. n=2 biological replicates, mean.

F. Ratio of the different CPA and termination factors to total pol II. n=3 biological replicates, mean ± SEM, p-value: * p < 0.05, ** p < 0.01. Statistical test: two-tailed unpaired t test.
CDK9 and PP2A regulate RNA polymerase II transcription termination and coupled RNA maturation. (2022)
Figure 3

Figure 3. Inhibition of analog-sensitive (as) CDK9 has the same effect on elongation as small molecule CDK9 inhibitors.

A. Western blot of total pol II, Ser2P (Abcam or Cell Signaling), Ser5P (Abcam or Cell Signaling), and histone H3 as a loading control, on the chromatin fraction of CDK9as or wild-type HEK293 cells treated with DMSO or 15 µM 1-NA-PP1 for 15 or 30 minutes.

B. Quantification of the western blots shown in A. n=2 biological replicates, except for Ser2P / pol II (Abcam) CDK9as NA 30 min, Ser5P / pol II (Abcam) CDK9as NA 15 min and NA 30 min, and Ser5P / pol II (Cell Signaling) CDK9as NA 30 min, where n=3 biological replicates.

C. ChIP-qPCR of total pol II and of Ser2P (Abcam or Cell Signaling), and Ser5P (Abcam or Cell Signaling) ratioed to total pol II on KPNB1. n=3 biological replicates, mean ± SEM, p-value: * p < 0.05, ** p < 0.01, *** p < 0.001. Statistical test: two-tailed unpaired t test.