EMBO/soda-nel huggingface dataset:
RIPK1-mediated immunogenic cell death promotes anti-tumour immunity against soft-tissue sarcoma (2020)
Figure 1

Figure 1. The addition of SM to TNF/Mel sensitises cells to RIPK1-dependent cell death.

A. Cell viability analysis using CellTiter-Glo of BN175 cells treated with the indicated agents for 24 hrs and 48 hrs (n = 4 biological replicates). Cell viability values are displayed as a percentage of the relative untreated control. DMSO (Unt), TNF (10 ng/ml), SM (100 ng/ml) and Mel (3.3 µM). SM represents SM-164. Error bars represent SD and statistical analysis was performed with an unpaired t-test, **** P ≤ 0.0001. Exact P values are shown in Appendix Table S1.

B. TNF-induced complex-II immuno-precipitation. BN175 cells were treated with the indicated agents for 8 hrs. FADD immuno-precipitation was performed followed by western blot analysis (n = 3 biological replicates, shown is a representative experiment). DMSO (Unt), zVAD (10 µM), TNF (10 ng/ml), SM (100 ng/ml) and Mel (3.3 µM).

C. Proximity ligation assay between Ripk1 and Casp-8 performed in BN175 cells upon treatment with the indicated agents for 8 hrs (n = 3 biological replicates, shown are representative images). Scale bar represents 10 μm. DMSO (Unt), zVAD (10 µM), TNF (10 ng/ml), SM (100 ng/ml) and Mel (3.3 µM).

D. DEVDase activity assay of BN175 cells treated with the indicated drugs for 6 hrs (n = 3 biological replicates). DMSO (Unt), TNF (10 ng/ml), SM (100 ng/ml) and Mel (3.3 µM). Error bars represent SD and statistical analysis was performed with an unpaired t-test, **** P ≤ 0.0001. Exact P values are shown in Appendix Table S1.

E. Cell viability analysis using CellTiter-Glo of BN175 CRISPR/Cas9 Ripk1 and Casp-8 knockouts (KO) cells treated with the indicated agents for 18 and 48 hrs (n = 3 biological replicates). Cell viability values are displayed as a percentage of the relative untreated control. SM represents SM-164. Error bars represent SD and a one-way Anova was performed to compare the mean value of each treatment to the treated BN175 CRISPR/Cas9 control (Ctrl), **** P ≤ 0.0001. Exact P values are shown in Appendix Table S1
RIPK1-mediated immunogenic cell death promotes anti-tumour immunity against soft-tissue sarcoma (2020)
Figure 3

Figure 3. RIPK1-induced cell death delays tumour growth and prolongs survival

A. Measurements of tumour volume of the respective cohorts (n = 6 animals per cohort). SM represents Birinapant. Error bars represent SD. Statistical analysis was performed with an unpaired t-test, * P ≤ 0.05, *** P ≤ 0.001. Exact P values are shown in Appendix Table S1.

B. The addition of SM to the standard-of-care treatment ILP-TNF/Mel significantly prolonged survival compared to either modality alone (n = 6 rats per cohort). Statistical analysis was performed with a log-rank test, ** P ≤ 0.01. Exact P values are shown in Appendix Table S1.

C. Median TNF/Mel is 0.4472 and TNF/Mel/SM is 1.154. TNF/Mel 25th percentile limit 0.02887 and 75th percentile limit 0.7627. TNF/Mel/SM 25th percentile limit 1.136 and 75th percentile limit 1.67. TNF/Mel Min error bar -0.2389 and Max 0.7687. TNF/SM/Mel Min error bar 1.12 and Max 1.813. Error bars represent SD. Statistical analysis was performed with an unpaired t-test, ** P ≤ 0.01. Exact P values are shown in Appendix Table S1.

D. DEVDase activity assay of parental BN175 cells and drug-tolerant tumour cell lines isolated from ILP-TNF/Mel/SM treated animals (124, 133, 136), treated with the indicated drugs for 6 hrs (n = 4 biological repeats). DMSO (Unt), Mel (3.3 µM), TNF (10 ng/ml) and SM (100 ng/ml).

E. Cell viability analysis using CellTiter-Glo of BN175 parental and 124, 133 and 136 cells treated with the indicated drugs for 24 hrs (n = 3 biological repeats). DMSO (Unt), Mel (3.3 µM), TNF (10 ng/ml) and SM (100 ng/ml). SM represents SM-164. Error bars represent SD. Statistical analysis was performed with a one-way Anova, ns=not significant. Exact P values are shown in Appendix Table S1.

F. Cell viability analysis using CellTiter-Glo of BN175 CRISPR/Cas9 Ripk1 and Casp-8 KO cells treated with the indicated agents for 24 hrs (n = 3 biological replicates). Cell viability values are displayed as a percentage of the relative untreated control. DMSO (Unt), SM (100 ng/ml) and Riboxxol (1 µg/ml). SM represents SM-164. Error bars represent SD and a one-way Anova was performed to compare the mean value of each treatment to the treated BN175 CRISPR/Cas9 control (Ctrl), **** P ≤ 0.0001. Exact P values are shown in Appendix Table S1.
RIPK1-mediated immunogenic cell death promotes anti-tumour immunity against soft-tissue sarcoma (2020)
Figure 4

Figure 4. RIPK1-induced cell death augments tumour immune infiltration

A-I. Flow cytometry analysis of tumours collected on Day 10 following treatment (n = 3 animals per group). (A) Schematic representation of the experimental setup. ILP with TNF/Mel/SM significantly increased infiltration by CD3+ lymphocytes (B) and CD3-CD161+ NK cells (C). While ILP treatment with TNF/Mel significantly reduced Granzyme B positive NK cells (D), the numbers of Granzyme B positive NK cells were restored to normal levels upon co-treatment with SM (TNF/Mel/SM). Importantly, ILP-mediated treatment with TNF/Mel/SM significantly increased the numbers of CD3+CD8+ cytotoxic T cells (E). ILP-TNF/Mel/SM significantly upregulated Granzyme B in CD3+CD8+CD161- cytoxtoxic T cells (F). ILP-TNF/Mel/SM significantly reduced infiltration by CD3+CD4+Foxp3- T cells whilst (G) increasing infiltrations by CD3+CD4+Foxp3+ T regulatory cells (H). A non-significant increase in T regulatory cell ICOS expression was also noted (I). SM represents Birinapant. Error bars represent SD and a one-way Anova was performed * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001; **** P ≤ 0.0001. Abbreviations: positive (+ve), negative (-ve), Granzyme B (GzmB). Exact P values are shown in Appendix Table S1.
RIPK1-mediated immunogenic cell death promotes anti-tumour immunity against soft-tissue sarcoma (2020)
Figure 5

Figure 5. Combination therapy with SM and checkpoint inhibitors further prolongs recurrence-free survival following ILP-mediated TNF/Mel treatment.

A. Measurements of tumour volume of the respective cohorts (n = 6 animals per cohort). SM represents Birinapant. Error bars represent SD.

B. The addition of either anti-CTLA-4 or anti-PD-1 antibodies prolonged survival in comparison to ILP-TNF/Mel/SM alone (n = 6 animals per cohort). SM represents Birinapant. Statistical analysis was performed with a log-rank test, * P ≤ 0.05. Exact P values are shown in Appendix Table S1.

C-E. Tumours were collected 4 days after the ILP procedure and analysed by flow cytometry (n = 3 per cohort). The addition of anti-CTLA-4 antibodies led to a significant increase in CD4+ Foxp3- helper T cells (C) accompanied by a depletion of CD4+ Foxp3+ regulatory T cells (D). The addition of anti-PD-1 antibodies led to an increase in Granzyme B (GzmB) expression on NK cells (E). SM represents Birinapant. Error bars represent SD and a one-way Anova was performed * P ≤ 0.05. Abbreviations: positive (+ve), negative (-ve), Granzyme B (GzmB). Exact P values are shown in Appendix Table S1.
RIPK1-mediated immunogenic cell death promotes anti-tumour immunity against soft-tissue sarcoma (2020)
Figure 6

Figure 6. SM improves anti-tumour immunity of standard-of-care treatment.

A. Schematic representation of the experimental setup.

B. Tumour volumes at the time of surgery following ILP-TNF/Mel or ILP-TNF/Mel/SM (n=6 animals per cohort). SM represents Birinapant. Error bars represent SD. Statistical analysis was performed with an unpaired t-test, ****, P ≤ 0.0001. Exact P values are shown in Appendix Table S1.

C. Individual growth curves following surgery indicated improved control following ILP-TNF/Mel/SM (n=6 animals per cohort).

D. Tumour volume on re-challenge with BN175 sarcoma on contralateral limb of long-term survivors following ILP-TNF/Mel or ILP-TNF/Mel/SM (n=3 animals per TNF/Mel cohort and n=5 per TNF/Mel/SM cohort). Unless otherwise stated, SM represents Birinapant. Error bars represent S.D.

E. ILP-TNF/Mel/SM significantly delayed growth leading to prolonged survival on re-challenge (n=3 animals per TNF/Mel cohort and n=5 per TNF/Mel/SM cohort). SM represents Birinapant. Statistical analysis was performed with a log-rank test, * P ≤ 0.05. Exact P values are shown in Appendix Table S1.

Expanded View Figures
TRIM21-mediated proteasomal degradation of SAMHD1 regulates its antiviral activity (2019)
Figure 1

Figure 1 SAMHD1 suppresses EV71 replication.

(A-D) The expression of SAMHD1 is negatively correlated with EV71 viral replication ability. HEK293T and RD cells were infected with EV71 at a MOI of 0.1, then the cells and supernatants were harvested at the indicated time points. (A) Immunoblotting (IB) analysis of EV71 VP1 in cells, with tubulin as a loading control. EV71-VP1 protein in the supernatants was detected after ultracentrifugation. (B) EV71 viral RNA levels in cell lysates and supernatants were detected by RT-qPCR with GAPDH as a control. (n=3, mean±SD, ns stands for no significance, paired t-test) (C) The mRNA levels of the host restriction factors were detected by RT-qPCR in infected or uninfected HEK293T or RD cells, and the expression levels of the target genes were normalized to GAPDH. (n=3, mean±SD, ns stands for no significance, paired t-test). (D) IB analysis of SAMHD1 and BST-2 protein levels with tubulin as a loading control. The densities of bands were analyzed with ImageJ software to calculate the values relative to that of tubulin.

(E-J) SAMHD1 knockdown enhances EV71 replication. The stable cell lines pLKO.1 and sh-SAMHD1 constructed in HEK293T (E) and RD (H) cells were infected with EV71 at a MOI of 0.1 and 0.05 respectively, and cells and supernatants were harvested at the indicated time points. IB analysis of EV71 VP1 and SAMHD1 in cells was performed with tubulin as a loading control. EV71-VP1 protein in the supernatants was detected after ultracentrifugation. (F and I) EV71 viral RNA in cell lysates was detected by RT-qPCR with GAPDH as a control (n=3, mean±SD, * P < 0.05, ** P < 0.01, paired t-test). (G and J) Viral titers in the supernatants were measured by the cytopathic effect method. The results represent the means ± SD from three independent experiments. Statistical significance was analyzed using Student's t test (*P < 0.05, **P < 0.01).

(K) HEK293T cells transfected with VR1012, which is a eukaryotic expression vector, or Vpx-HA were infected with EV71 at a MOI of 0.1, and then cells and supernatants were harvested at the indicated time points. IB analysis of EV71-VP1, SAMHD1 and Vpx-HA in cell lysates was performed with tubulin as a loading control. EV71-VP1 was detected in the supernatants after ultracentrifugation.

(L) EV71 viral RNA was detected in cell lysates by RT-qPCR with GAPDH as a control (n=3, mean±SD, ** P <0.01, paired t-test).

(M) Viral titres in the supernatants were measured by the cytopathic effect method. The results represent the means ± SD from three independent experiments. Statistical significance was analyzed using Student's t test (**P < 0.01).
TRIM21-mediated proteasomal degradation of SAMHD1 regulates its antiviral activity (2019)
Figure 2

Figure 2 SAMHD1 is degraded by TRIM21 via the proteasomal pathway.

(A) Stable SAMHD1-HA-overexpressing cell lines were constructed in HEK293T and RD cells and detected by IB with pLVX as a negative control.

(B) RD SAMHD1-HA cell lines were treated with dimethyl sulfoxide (DMSO) or 10 μM MG132 for 12 h prior to harvest and subjected to IB with tubulin as a loading control.

(C) RD SAMHD1-HA cell lines were treated as in (B), and then HA immunoprecipitation (IP) and IB were performed. The IP elution was analyzed by mass spectrometry.

(D-E) TRIM21 (D) but not TRIM25 (E) induced the degradation of SAMHD1, and MG132 rescued the TRIM21-mediated degradation of SAMHD1. HEK293T cells were cotransfected with SAMHD1 and VR1012 or TRIM21 or TRIM25 expression plasmids with or without MG132 treatment for 12 h prior to harvest, and then subjected to IB with tubulin as a loading control.

(F) Reverse co-IP confirmed the interaction between SAMHD1 and TRIM21. SAMHD1-flag was transfected with TRIM21-HA into HEK293T cells, and then the cells were treated as in (B) and subjected to HA IP and IB.

(G) Endogenous TRIM21 but not TRIM25 still interacted with SAMHD1. RD-SAMHD1-HA cells treated as in (B) were subjected to TRIM21 or TRIM25 IP and IB.

(H, I) (H) mRNA (n=3, mean±SD, ***P < 0.001, paired t-test) and (I) protein levels of TRIM21 in RD and HEK293T cells.

(J-K) TRIM21 (J) but not TRIM25 (K) colocalized with SAMHD1. Images were taken under a Zeiss LZM710 confocal microscope, Bars, 10μm.
TRIM21-mediated proteasomal degradation of SAMHD1 regulates its antiviral activity (2019)
Figure 3

Figure 3 The degradation of SAMHD1 mediated by TRIM21 affects anti-EV71 restriction of SAMHD1.

(A-D) TRIM21 Knockout in RD cells released SAMHD1 inhibition on EV71 replication. (A) Illustration of gRNA-guided TRIM21 knockout in cells. (B) Negative control or RD-CRISPR-Cas9-TRIM21 RD cells were infected with EV71 at a MOI of 0.05 for the indicated time and harvested for SAMHD1, TRIM21 and EV71-VP1 detection by IB. Tubulin served as a loading control. (C) EV71 RNA levels in (B) were detected by RT-qPCR with GAPDH as a control (n=3, mean±SD, ** P < 0.01, paired t-test). (D) Viral titres in the supernatant were measured by the cytopathic effect method. The results represent the means ± SD from three independent experiments. Statistical significance was analyzed using Student's t test (**P < 0.01).

(E-F) Overexpression of TRIM21 in HEK293T cells increased EV71 replication by degrading SAMHD1. HEK293T cells were transfected with VR1012 or TRIM21-HA for 24 h and then infected with EV71 at a MOI of 0.05. (E) Cells were harvested at the indicated time points and subjected to IB with tubulin as a loading control. (F) EV71 viral RNA in cell lysates was detected by RT-qPCR with GAPDH as a control (n=3, mean±SD, ** P < 0.01, paired t-test)).

(G and H) TRIM21 itself has no effect on EV71 replication. VR1012 or TRIM21 was transfected into pLKO.1 or sh-SAMHD1 HEK293T cells for 24 h and infected with EV71. (G) Cells were harvested and subjected to IB analysis. (H) Viral titers in the supernatants were measured by the cytopathic effect method. The results represent the means ± SD from three independent experiments. Statistical significance was analyzed using Student's t test (*P < 0.05).
TRIM21-mediated proteasomal degradation of SAMHD1 regulates its antiviral activity (2019)
Figure 4

Figure 4 The tissue tropism upon EV71 infection in neonatal mice model is positively correlated with TRIM21 and negatively correlated with SAMHD1.

(A) The mRNA levels of SAMHD1 and TRIM21 in different tissues of neonatal mice were detected by RT-qPCR with GAPDH as a control, and the expression of SAMHD1 and TRIM21 in the heart was set as 100% (n=3, mean±SD, * P < 0.05, paired t-test).

(B) The protein levels of SAMHD1 and TRIM21 in different tissues of mice were examined by Near Infrared Spectrum (NIR) western blot with tubulin as a control.

(C) Immunohistochemical (IHC) staining results of SAMHD1 in various tissues of mice. Bars, 100μm.

(D-F) The mRNA levels of EV71 virus and host factors in mock or EV71 infected mice were detected by RT-qPCR with GAPDH as a control. The expression of EV71 (D), IFNα and IFNβ (E), SAMHD1 and TRIM21 (F) were presented, and the levels of corresponding gene in mock infected mice were set as 100% (n=3, mean±SD, ** P < 0.01, paired t-test).
TRIM21-mediated proteasomal degradation of SAMHD1 regulates its antiviral activity (2019)
Figure 5

Figure 5 The degradation of SAMHD1 by TRIM21 affects the anti-HIV-1 activity of SAMHD1.

(A-I) (A) IB analysis of THP-1 cells treated with scrambled shRNA, TRIM21-specific shRNA or SAMHD1-specific shRNA. Tubulin served as loading control. (B, E, H) Quantification of SAMHD1 and TRIM21 levels in whole cells in which the indicated genes were knocked down by shRNA. Data are normalized to tubulin and expressed as the fold change over cells treated with pLKO.1 without HIV infection (n=3, mean±SD). (C) sh-SAMHD1 and sh-TRIM21 THP-1 cells were treated with PMA for 24 h and infected with the VSV-G pseudotyped reporter HIV-1, then detected 48 h later by flow cytometry analysis (n=3, mean±SD, * P < 0.05, ** P < 0.01, *** P < 0.001, paired t-test). (D) IB analysis of MDM cells from 3 donors transfected with TRIM21 siRNA twice at days 1 and 3, respectively, after MDM differentiation. (F) MDM cells treated with TRIM21 siRNA were infected with VSV-G-pseudotyped HIV-1 reporter and then detected 48 h later by flow cytometry analysis (n=3, mean±SD, ** P < 0.01, paired t-test). (G) IB analysis of Jurkat and KG-1 cells treated with scrambled shRNA or TRIM21-specific shRNA. (I) Jurkat and KG-1 cells treated with TRIM21 shRNA were infected with HIV-1, then detected 48 h later by flow cytometry analysis (n=3, mean±SD, ns stands for no significance, paired t-test).
TRIM21-mediated proteasomal degradation of SAMHD1 regulates its antiviral activity (2019)
Figure 6

Figure 6 The interaction between TRIM21 and SAMHD1.

(A-C) TRIM21 interacts with SAMHD1 via PRY and SPRY domains. (A) Sketch map of TRIM21 WT and mutants. (B) The effect of TRIM21 on the degradation of SAMHD1. SAMHD1-flag was cotransfected with VR1012, TRIM21 WT or the indicated mutant into HEK293T cells for 48 h, and the cells were subjected to IB with tubulin as loading control. (C) SAMHD1-flag was cotransfected with VR1012 or TRIM21 WT or the indicated mutant for 24 h, and the cells were then treated with 10 μM MG132 for 12 h before harvest and subjected to HA IP and IB.

(D) Map of SAMHD1 WT and truncation mutants.

(E) The effect of TRIM21 on SAMHD1 WT or its mutants. SAMHD1-HA or the indicated mutant were cotransfected with VR1012 or TRIM21-flag into HEK293T cells for 48 h, and the cells were subjected to IB with tubulin as a loading control.

(F) SAMHD1-flag 1-547 was cotransfected with VR1012 or TRIM21-HA for 24 h, and the cells were then treated with 10 μM MG132 for 12 h before harvest and subjected to HA IP and IB.

(G) SAMHD1 109-626 followed with a His tag or TRIM21-PRYSPRY followed with a GST tag were expressed in Rosetta (DE3), and pull-down assay were performed with Ni-Sepharose (up) and GST Sepharose (down), respectively.

(H) FRET analysis indicates interaction between YFP-SAMHD1 and CFP-TRIM21. A representative image of SAMHD1-YFP (yellow) and ECFP-TRIM21 (cyan) expressing cells before and after photobleaching the acceptor fluorophore, YFP. The region chosen for photobleaching is marked (white open box), Bars, 10μm. The quantization of fluorescence brightness was analyzed by Image J (n=3, mean±SD, ns stands for no significance, * P < 0.05, paired t-test)

(I) The microscale thermophoresis curve for the interaction between SAMHD1 and TRIM21. Alexa Fluor®647 labelled SAMHD1 at a concentration of 20 nM were incubated with two-fold dilution series of unlabeled TRIM21 (3 μM to 9.16E-05 μM). The curve represents the signal recorded from three measurements. The normalized fluorescence thermophoretic signals were plotted against the concentration of TRIM21 with mean values ± standard deviation as well as a fitting to a 1:1 binding model (NanoTemper® analysis software, F Norm = F Hot / F Cold). The Kd of this interaction was determined to be 270±92nM. The data was a representive of three independent experiments using different dilution.
TRIM21-mediated proteasomal degradation of SAMHD1 regulates its antiviral activity (2019)
Figure 7

Figure 7 Binding sites and ubiquitination sites in SAMHD1 required for TRIM21 recognition.

(A) The effect of TRIM21 on SAMHD1 proteins from various species. HEK293T cells were transfected with VR1012 or TRIM21 and the indicated SAMHD1 expression vector, then subjected to IB analysis.

(B) Identification of amino acids presented only in SAMHD1 of Canis and Gallus but not in other SAMHD1 proteins.

(C) Construction of hSAMHD1 mutants with amino acid alterations.

(D) The effect of TRIM21 on hSAMHD1 mutants. HEK293T cells were transfected with VR1012 or TRIM21 and SAMHD1 mutants for 48 h and then subjected to IB analysis.

(E) SAMHD1-flag WT or the G153S or G183R mutant was cotransfected with VR1012 or TRIM21-HA for 24 h, and the cells were then treated with 10 μM MG132 for 12 h before harvest and subjected to HA IP and IB.

Data information: (A, D-E) Tubulin served as a loading control.

(F) Sketch map of potential ubiquitination sites on SAMHD1.

(G) SAMHD1-HA WT or the indicated mutants were cotransfected with VR1012 or TRIM21-flag into HEK293T cells for 48 h, and the cells were subjected to IB with tubulin as a loading control.

(H) SAMHD1-HA WT and VR1012 or TRIM21 were cotransfected with K48-only and K63-only ubiquitin-flag into HEK293T cells for 24 h, and the cells were then treated with 10 μM MG132 for 12 h before harvest and subjected to HA IP and IB with tubulin as a loading control.

(I) K48-only ubiquitin-flag and SAMHD1-HA WT or mutant K622R were cotransfected with VR1012 or TRIM21 into HEK293T cells as indicated. The cells were then treated with 10 μM MG132 for 12 h before harvest and subjected to HA IP and IB with tubulin as a loading control.

(J) VR1012, SAMHD1-HA WT or the K622R mutant was transfected into HEK293T-shSAMHD1 cells for 24 h. The cells were then infected with EV71 at a MOI of 0.1 and harvested at the indicated time points. SAMHD1-HA, TRIM21 and EV71-VP1 were detected by IB with tubulin as a loading control.
The tRNA methyltransferase Dnmt2 is required for accurate polypeptide synthesis during haematopoiesis. (2015)
Figure 1

Pathological examination of Dnmt2-deficient mice

Giemsa staining of a femoral section from 8-day-old mice showing endochondral ossification defects. Scale bar: 100 μm.

Representative femur sections from 8-day-old wild-type and Dnmt2−/− mice. The staining of perivascular cells (GLS, green) is strongly reduced in Dnmt2−/− bone marrow. Nuclei are counterstained with DAPI. Scale bar: 100 μm.

Quantitative analysis of capillary numbers. GLS-positive capillaries were counted in wild-type (wt) and Dnmt2−/− (D2-) mice, in three independent high-power fields (HPF) per mouse (n = 7).

Quantification of cell numbers in haematopoietic tissues of 8-day-old mice (n = 8).

Representative FACS plots of wild-type and Dnmt2−/− BM stained for Lin− Sca1+ and cKit+ cells.

Quantification of bone marrow LSK cells in wild-type (three independent experiments, each with pooled BM samples from 4 to 6 mice) and Dnmt2−/− bone marrow (three independent experiments, each with pooled BM samples of 5–9 mice).

Restoration of the LSK cell population in adult Dnmt2−/− mice. Frequencies of LSK cells were normalized to the frequencies of LSK cells from the bone marrow of wild-type littermates at the indicated age (2–3 independent experiments, each with pooled bone marrow samples from 3 to 9 mice).

Increased LSK CD34− and reduced LSK CD34+ cell populations in 8-day- and 1-year-old Dnmt2−/− mice compared to wild-type (two independent experiments, each with pooled bone marrow samples from 3–9 mice).

Data information: Data are presented as mean ± SD. Asterisks indicate statistically significant (P < 0.05, t-test) differences. In (G) and (H), asterisks indicate statistically significant (P < 0.05, t-test) differences relative to wild-type (= 1).