EMBO/soda-nel huggingface dataset:
MiR-342 controls Mycobacterium tuberculosis susceptibility by modulating inflammation and cell death (2021)
Figure 2

Figure 2 MiR-342-3p enhances the production of inflammatory cytokines

(A, B) Survival of Mir342+/+C3H mice and their wild-type littermate controls (C3H) (n=10) (A), or Mir342-/-B6 mice and their wild-type littermate controls (B6) (n=10) (B) after aerosol infection with around 400 CFU Mtb. Data are shown as the Kaplan-Meier curves.

(C, D) Mtb bacterial loads in lungs, spleens and livers of Mir342+/+C3H mice and their littermates (C3H) (C), or Mir342-/-B6 mice and their littermates (B6) at 21 days post-infection (dpi). Data are shown as the medians ± interquartile ranges of n = 3 biological replicates.

(E, F) Tuberculosis lung lesions of Mir342-/-B6 mice and their littermates (B6) (E), or Mir342+/+C3H mice and their littermates (C3H) (F) were detected by haematoxylin/eosin staining at 21 dpi. Representative images from n = 3 biological replicates are shown. Scale bar = 100 μm.

(G, H) Secretion of cytokines TNF-α, IL-1, IL-6 and CXCL15 in BMDMs obtained from Mir342+/+C3H and their littermates (C3H) (G), or from Mir342-/-B6 and their littermates (B6) (H) after Mtb stimulation, was detected by ELISA. Data are shown as the mean ± s.e.m. of n = 3 biological replicates.

Data information: Log-rank (Mantel-Cox) test (A, B), Mann-Whitney U test (C, D) and ANOVA followed by Bonferroni post hoc test (G, H) were used for data analysis. *, p < 0.05, **, p < 0.01.
MiR-342 controls Mycobacterium tuberculosis susceptibility by modulating inflammation and cell death (2021)
Figure 3

Figure 3 MicroRNA-342-3p directly targets SOCS6

(A) Predicted binding sites of miR-342-3p and Socs6 3′-UTR.

(B) HEK-293T fibroblasts were co-transfected with miR-342-3p mimic and luciferase reporter construct containing wild-type or mutated UTR. After 24 hours, cells were collected for luciferase assays. The Y-axis showed relative luciferase activity. Data are shown as the mean ± s.e.m. of n = 3 biological replicates.

(C, D) MiR-342-3p mimic or inhibitor was transfected to NIH-3T3 fibroblasts. After 48 hours, cells were collected for qRT-PCR (C, data are shown as the mean ± s.e.m. of n = 3 biological replicates) and western blotting (D, representative blots from n = 3 biological replicates are shown) to detect SOCS6 expression.

(E) RAW264.7 cells were transfected with Myc-tagged Ago2 in the presence of miR-342-3p mimic or scramble for 24 hours. Immunoprecipitation was performed with Myc-antibody or IgG, and Socs6 mRNA was detected by qRT-PCR. Data are shown as the mean ± s.e.m. of n = 3 biological replicates.

(F-H) C3H BMDMs were transfected with miR-342-3p mimic or SOCS6-overexpressing lentivirus, followed by stimulation with Mtb or z-VAD (20 μM) for 24 hours. Cell death mechanisms (F), cell viabilities (G) and Mtb growth rates (H) were analyzed respectively. Data are shown as the mean ± s.e.m. of n = 3 biological replicates.

(I-K) B6 BMDMs were transfected with miR-342-3p inhibitor or Socs6 siRNA, followed by stimulation with Mtb or z-VAD (20 μM) for 24 hours. Cell death mechanisms (I), cell viabilities (J), Mtb growth rates (K) were analyzed respectively. Data are shown as the mean ± s.e.m. of n = 3 biological replicates.

(L) Relative expressions of miR-342-3p and Socs6 in PBMCs from healthy individuals (n=27) and TB patients (n=34) were detected by qRT-PCR. The correlation between miR-342-3p and Socs6 was analyzed by Spreaman test. Spearman rank correlation: r = -0.532, p < 0.001, n = 61.

Data information: ANOVA followed by Bonferroni post hoc test (B, C, E-K) was used for data analysis. *, p < 0.05, **, p < 0.01. Abbreviations: n.s., not significant. NC, negative control.
MiR-342 controls Mycobacterium tuberculosis susceptibility by modulating inflammation and cell death (2021)
Figure 4

Figure 4 SOCS6 participates in anti-Mtb immunity

(A, B) Survival of Mir342-/-B6 and Mir342-/-B6 mice supplemented with Socs6 siRNA (n=10) (A), or Mir342+/+C3H and Mir342+/+C3H mice supplemented with SOCS6-overexpressing vector (n=10) (B), after aerosol infection with around 400 CFU Mtb. Data are shown as the Kaplan-Meier curves.

(C, D) Mtb bacterial loads in lungs, spleens and livers of Mir342-/-B6 and Mir342-/-B6 mice supplemented with Socs6 siRNA (C), or Mir342+/+C3H and Mir342+/+C3H mice supplemented with SOCS6-overexpressing vector (D) at 21 dpi. Data are shown as the medians ± interquartile ranges of n = 3 biological replicates.

(E, F) Tuberculosis lung lesions of Mir342-/-B6 and Mir342-/-B6 mice supplemented with Socs6 siRNA (E), or Mir342+/+C3H and Mir342+/+C3H mice supplemented with SOCS6-overexpressing vector (F) at 21 dpi were detected by haematoxylin/eosin staining. Representative images from n = 3 biological replicates are shown.Scale bar = 100 μm.

Data information: Log-rank (Mantel-Cox) test (A, B) and Mann-Whitney U test (C, D) were used for data analysis. **, p < 0.01.
MiR-342 controls Mycobacterium tuberculosis susceptibility by modulating inflammation and cell death (2021)
Figure 5

Figure 5 SOCS6 regulates the expression of chemokines via modulating JAK/STAT signaling

(A) ELISA was performed to detect the secretion of cytokines TNF-α, IL-1, IL-6 and CXCL15 in Socs6+/+ and Socs6-/- BMDMs during Mtb stimulation. Data are shown as the mean ± s.e.m. of n = 3 biological replicates.

(B) Phosphorylation states of STAT1 in response to Mtb stimulation in Socs6-/- BMDMs were examined by western blotting. Fludarabine treatment concentration was 10 μM and the treatment time was 24 hours. Representative blots from n = 3 biological replicates are shown.

(C-F) Relative expressions of chemokines CCL5, CXCL10, ICAM1 (C), and Caspase 3, Caspase 7, Caspase 8 (F) in Mtb-stimulated Socs6+/+ or Socs6-/- BMDMs were detected by qRT-PCR. Data are shown as the mean ± s.e.m. of n = 3 biological replicates. Cluster analysis of clinical samples. Heatmaps showed relatively high (red) or low (green) expression levels of Socs6 and inflammatory factors in PBMCs from healthy individuals (n=27) (D) and TB patients (n=34) (E).

Data information: ANOVA followed by Bonferroni post hoc test (A, C, F) was used for data analysis. **, p < 0.01. Abbreviation: n.s., not significant.
MiR-342 controls Mycobacterium tuberculosis susceptibility by modulating inflammation and cell death (2021)
Figure 6

Figure 6 SOCS6 determines cell death mechanisms via modulating recruitment of RIPK3

(A) Relative expressions of RIPK1 and RIPK3 were analyzed by western blotting in Socs6-/- BMDMs stimulated with Mtb for 0-24 hours. Representative blots from n = 3 biological replicates are shown.

(B) Socs6-/- BMDMs were stimulated with Mtb for 0-24 hours, cell lysates were collected and immunoprecipitated using an anti-RIPK1 antibody. The recruitment of Caspase 8, RIPK3, FADD and MLKL was analyzed by immunoblots. The lower panel represents the immunoblot analysis of whole cell lysates. Representative blots from n = 3 biological replicates are shown.

(C) Socs6-/- BMDMs were transfected with plasmids expressing Myc-RIPK3 or Myc-RIPK3 K51A mutant for 24 hours. Afterwards, transfected cells were stimulated with Mtb for 12 hours, cell lysates were collected and immunoprecipitated using an anti-RIPK1 antibody. The recruitment of Caspase 8, RIPK3, FADD and MLKL was analyzed by immunoblot. The lower panel represents the immunoblot analysis of whole cell lysates. Representative blots from n = 3 biological replicates are shown.

(D-F) Cell viabilities (D), cell death mechanisms (E), or Mtb growth rates (F) of Mtb-infected Socs6-/- BMDMs that were transfected with plasmids expressing Myc-RIPK3 or Myc-RIPK3 K51A mutant as indicated. Z-VAD treatment concentration was 20 μM and the treatment time was 24 hours. Data are shown as the mean ± s.e.m. of n = 3 biological replicates.

Data information: ANOVA followed by Bonferroni post hoc test (D-F) was used for data analysis. *, p < 0.05, **, p < 0.01. Abbreviation: n.s., not significant.
MiR-342 controls Mycobacterium tuberculosis susceptibility by modulating inflammation and cell death (2021)
Figure 7

Figure 7 A20 controls degradation of RIPK3 through ubiquitin chains

(A) RAW264.7 cells were pretreated with or without E64d/PepA or MG132 for 4 hours, and then stimulated with or without Mtb for 12 hours as indicated. Cell lysates were collected and immunoprecipitated using an anti-RIPK3 antibody, and the ubiquitination of endogenous RIPK3 was detected using an anti-Ub antibody. Representative blots from n = 3 biological replicates are shown.

(B, C) Socs6+/+ BMDMs were transfected with miR-342-3p mimic or Socs6 siRNA (B), and Socs6-/- BMDMs were transfected with plasmid expressing Myc-SOCS6 (C) for 24 hours as indicated. Transfected cells were then stimulated with Mtb for another 12 hours, cell lysates were collected for immunoprecipitation, and ubiquitination of endogenous RIPK3 was detected by immunoblot. Representative blots from n = 3 biological replicates are shown.

(D) RAW264.7 cells were transfected with A20 siRNA, cell lysates were collected for immunoprecipitation and immunoblot. Representative blots from n = 3 biological replicates are shown.

(E-G) RAW264.7 cells were transfected with plasmids expressing A20 and RIPK3 (E), or A20 and RIPK3 mutants (G) as indicated, cell lysates were collected for immunoprecipitation and immunoblot. Myc-A20 or Flag-RIPK3 purified from transfected HEK-293T cells was incubated with ATP, E1, E2 and ubiquitin as indicated. The in vitro ubiquitination of RIPK3 was analyzed by immunoblot using an anti-Ub antibody (F). Representative blots from n = 3 biological replicates are shown.
MiR-342 controls Mycobacterium tuberculosis susceptibility by modulating inflammation and cell death (2021)
Figure 8

Figure 8 A20-mediated cell death mechanism is independent of inflammatory responses

(A, B) Production of cytokines TNF-α, IL-1, IL-6, CXCL15 (A), or chemokines Ccl5, Cxcl10, Icam1 (B) in the A20-/-Socs6+/+, A20+/+Socs6+/+, A20-/-Socs6-/- and A20+/+Socs6-/- BMDMs after Mtb stimulation. Data are shown as the mean ± s.e.m. of n = 3 biological replicates.

(C-E) Cell death mechanisms (C), cell viabilities (D), or Mtb growth rates (E) of A20-/-Socs6+/+, A20+/+Socs6+/+, A20-/-Socs6-/- and A20+/+Socs6-/- BMDMs stimulated with Mtb. Z-VAD treatment concentration was 20 μM and the treatment time was 24 hours. Data are shown as the mean ± s.e.m. of n = 3 biological replicates.

(F) Schematic representation of miR-342 regulated anti-Mtb immunity.

Data information: ANOVA followed by Bonferroni post hoc test (A-E) was used for data analysis. *, p < 0.05, **, p < 0.01. Abbreviation: n.s., not significant.

Expanded View Figure Legends
Autophagy induction enhances TDP43 turnover and survival in neuronal ALS models (2014)
figf1

(a,b) Histograms of TDP43(WT)-EGFP (a) and TDP43A315T-EGFP (b) levels in individual neurons. Increasing amounts of DNA significantly shifted the distribution of log-transformed expression levels toward higher values (P 0.01 for all comparisons, two-sided Kolmogorov-Smirnov test). TDP43(WT)-EGFP, n = 1,287 neurons, with 290–342 cells per group; TDP43A315T-EGFP, n = 1,290, with 315–332 neurons per group. Values were pooled from 12 wells per condition, with experiments performed in duplicate. (c) Automated fluorescence microscopy. Primary rodent cortical neurons were transfected with mApple and TDP43-EGFP, and survival was determined by repeated imaging at regular intervals. The last time at which the cell was noted to be alive (red arrows) was used as the time of death. Cells that survive the entire length of the experiment (cyan arrow) were censored. Scale bar, 25 μm. (d,e) Cumulative risk of death over time for neurons transfected with EGFP, TDP43(WT)-EGFP (d) or TDP43A315T-EGFP (e). Supplementary Table 1 lists the number of neurons, hazard ratios, 95% CI and P values for each condition, as determined by Cox proportional hazards analysis. Results were pooled from 12 wells per condition, with experiments performed in duplicate. (f,g) Linear regression of calculated hazard ratio (± s.e.m.) as a function of total TDP43(WT)-EGFP (R2 = 0.9353) (f) or TDP43A315T-EGFP (R2 = 0.9574) (g) level. The total TDP43 level was determined by quantitative immunocytochemistry using TDP43-specific antibodies (Supplementary Fig. 3) and normalized to the amount of TDP43 in nontransfected neurons. Dashed lines, reference (HR = 1).
Autophagy induction enhances TDP43 turnover and survival in neuronal ALS models (2014)
figf2

(a) Primary neurons were labeled with 35S-labeled methionine, and TDP43 was immunoprecipitated using anti-TDP43 antibodies (IP-TDP43) or nonspecific IgG antibodies (IP-IgG). (b) Log-normal plot of normalized TDP43 levels. TDP43 half-life was determined by fitting a first-order exponential curve to the data (R2 = 0.8984). Values were pooled from five independent experiments. (c) Potential confounders in half-life analyses. Top, total protein ideally decreases over time in a predictable manner (top right panel). Middle, toxicity reduces the amount of measured protein, potentially shortening the calculated half-life (middle right panel). Bottom, protein aggregation might also shorten half-life estimations if the protein cannot be immunoprecipitated (black arrows, bottom right panel) or lengthen it if the protein is more stable and successfully immunoprecipitated (gray arrows, bottom right panel). (d) OPL. Primary cortical neurons were transfected with EGFP and TDP43(WT)-Dendra2, photoconverted then imaged by AFM. Dead cells (arrows) were excluded at the time of death. Pre, before photoconversion. (e) The half-life of TDP43(WT)-Dendra2 was determined by fitting a first-order exponential curve to the data (R2 = 0.9558). (f) Including cells with aggregates prolonged TDP43(WT)-Dendra2 half-life (pink line, R2 = 0.9003; *P 0.0001, F 15.34, extra sum-of-squares F-test). Excluding cells that died over the 36-h experiment also prolonged half-life (cyan line, R2 = 0.7171; **P 0.0001, extra sum-of-squares F-test). Error bars represent s.e.m. Values were pooled from eight wells per condition, with experiments performed in quadruplicate.
Autophagy induction enhances TDP43 turnover and survival in neuronal ALS models (2014)
figf3

(a) Scatter plot depicting half-life measurements for individual cells expressing TDP43(WT)-Dendra2 (cyan, n = 206) and TDP43A315T-Dendra2 (red, n = 251). *P = 0.0029, U = 11,797, Mann-Whitney test. (b) Probability density plot for single-cell half-life measurements, demonstrating a reduced half-life of TDP43A315T-Dendra2 (red, n = 251), compared to TDP43(WT)-Dendra2 (cyan, n = 206; **P = 0.0007, two-sided Kolmogorov-Smirnov test). Values were pooled from eight wells per condition, with experiments performed in quadruplicate. (c,d) Probability density plots of single-cell half-life measurements for TDP43(WT)-Dendra2 (c) and TDP43A315T-Dendra2 (d). At 0.5 μM, all three of the compounds significantly reduced TDP43(WT)-Dendra2 half-life compared to vehicle control (DMSO, n = 110; FPZ, n = 96, f* P = 1 × 10−4; MTM, n = 129, m* P = 2 × 10−7; NCP, n = 111, n* P = 2 × 10−5, two-sided Kolmogorov-Smirnov test). The compounds also reduced TDP43A315T-Dendra2 half-life, in comparison to vehicle control (DMSO, n = 120; FPZ, n = 155, f* P = 2 × 10−9; MTM, n = 152, m* P = 0.004; NCP, n = 111, n* P = 0.005, two-sided Kolmogorov-Smirnov test). Colored hash marks represent values from individual neurons in b–d. Values were obtained from eight wells per condition, with experiments performed in triplicate.
Autophagy induction enhances TDP43 turnover and survival in neuronal ALS models (2014)
figf5

(a) FPZ (n = 490) and MTM (n = 580) but not NCP (n = 523) lowered total TDP43(WT)-EGFP levels in comparison to vehicle (DMSO, n = 1,007). (b) Histogram of normalized, log-transformed TDP43(WT)-EGFP levels. (c) Each compound also reduced TDP43A315T-EGFP levels (DMSO, n = 983; FPZ, n = 550; MTM, n = 542; NCP, n = 493). (d) Histogram of single-cell TDP43(A315T)-EGFP levels. Asterisks in b and d denote median values for cells exposed to vehicle (gray), FPZ (green), MTM (blue) or NCP (purple). For FPZ, MTM and NCP versus DMSO in a and d, P 1 × 10−3 by two-sided Kolmogorov-Smirnov test. (e) No compound reduced TDP43(WT)-EGFP inclusions 48 h after transfection (DMSO, n = 360; FPZ, n = 194; MTM, n = 234; NCP, n = 203). (f) FPZ and MTM but not NCP reduced TDP43A315T-EGFP inclusions at 48 h (DMSO, n = 327; FPZ, n = 226; MTM, n = 262; NCP, n = 253). (g) No compound affected TDP43(WT)-EGFP localization 24 h after transfection (DMSO, n = 256; FPZ, n = 212; MTM, n = 250; NCP, n = 241). (h,i) Each compound (h) prevented TDP43A315T-EGFP cytoplasmic mislocalization (DMSO, n = 233; FPZ, n = 268; MTM, n = 263; NCP, n = 270) and reduced cytoplasmic TDP43(A315T)-EGFP levels (DMSO, n = 125; FPZ, n = 102; MTM, n = 121; NCP, n = 144. *P 0.05; NS, P > 0.05) (i). For a, c and e–i, *P 0.05; nonsignificant (NS), P > 0.05, one-way ANOVA with Dunnett's test. Error bars represent s.e.m. Values in a–d were pooled from 8–12 wells per condition from six experiments, values in e–h were pooled from eight wells per condition from three experiments, and values in i were pooled from five wells per condition from two experiments.
Autophagy induction enhances TDP43 turnover and survival in neuronal ALS models (2014)
figf6

(a,b) Primary neurons transfected with TDP43(WT)-EGFP (a) or TDP43A315T-EGFP (b) were treated with autophagy inducers and survival determined by AFM. Data were obtained from eight wells per condition, with experiments performed in triplicate. Supplementary Table 2 lists the number of neurons per condition, hazard ratios, 95% CI and P values, as determined by Cox hazards analysis. (c) Autophagic induction improved survival in TDP43M337V HB9-positive human iPSC-derived MNs. (d) FPZ and MTM also reduced the risk of death in MAP2-positive human iPSC-derived MNs Values in c and d were pooled from 18 wells per condition, with experiments performed in triplicate. (e) Astrocytes differentiated from human iPSCs exhibit mutant TDP43-related toxicity that is mitigated by FPZ and MTM. Values were pooled from 18 wells per condition, performed in triplicate. Supplementary Table 3 lists the number of human iPSC-derived neurons and astrocytes per condition, hazard ratios, 95% CI and P values, as determined by Cox hazards analysis.
High salt activates p97 to reduce host antiviral immunity by restricting Viperin induction (2021)
Figure 3

Figure 3. Identification of USP33 as a key deubiquitinase controlling Viperin protein stability.

A RT-qPCR analysis of Viperin mRNA in RAW264.7 treated with mIFNβ (300 IU/ml, 12 hrs) immediately after addition of NaCl (+17, 34 and 51 mM).

B Western blot analysis of FH-Viperin protein in stable FH-Viperin-expressing HeLa cells pretreated with additional NaCl (+34 mM) and then treated with CHX (50 μM) for 6 and 12 hrs.

C RAW264.7 were treated with MG132 (10 μM) and additional NaCl (+34 mM), followed by mIFNβ (300 IU/ml) treatment for 12 hrs. Viperin protein levels were analyzed by immunoblotting.

D Immunoprecipitation analysis of Viperin ubiquitination in RAW264.7 treated with mIFNβ (300 IU/ml) for 12 hrs immediately after addition of NaCl (+34 mM).

E Western blot analysis of FH-Viperin protein in stable FH-Viperin-expressing HeLa cells treated with PR-619 (10 μM) together with additional NaCl (+34 mM) for 12 hrs.

F Mass spectrometry analysis of potential Viperin-binding deubiquitinases in HEK293T cells transfected with Flag-Viperin.

G Immunoprecipitation analysis of the interaction between USP33 and Flag-Viperin in HEK293T cells transfected with Flag-Viperin.

H Immunoprecipitation analysis of the interaction between Viperin and FH-USP33 in 2fTGH cells transfected with FH-USP33 and then treated with IFNα (1,000 IU/ml) for 12 hrs.

I Western blot analysis of Flag-Viperin levels in HEK293T cells co-transfected with Flag-Viperin and increasing amount of Flag-HA (FH)-USP33.

J Western blot analysis of Viperin in 2fTGH cells transfected with control shRNAs (shCtrl) or shRNAs against USP33 (shUSP33) and then treated with IFNα (1,000 IU/ml) for different times.

K, L Western blot analysis of Viperin protein in Usp33+/+ or Usp33-/- MEF cells treated with IFNβ (500 IU/ml) (K) or infected with VSV (MOI=1.0) (L) as indicated.

M Western blot analysis of Viperin protein in Usp33-/- MEF cells transfected with FH-USP33 (wild type, WT; C194S and H673Q, DM) and then stimulated with mIFNβ (500 IU/ml) for 12 hrs.

N Immunoprecipitation analysis of Viperin ubiquitination in HEK293T cells co-transfected with Viperin and FH-USP33 (WT or DM).

O Western blot analysis of Flag-Viperin in stable Flag-Viperin-expressing HEK293T cells transfected with shCtrl or shUSP33 and then treated with CHX (50 μM) as indicated.

Data information: Data (A) represent mean and SD of three biological replicates; Data (B-E, G-O) are representative of at least two biological replicates. For all statistical testing, p values were calculated using two-tailed unpaired Student's t-test. NS, not signiﬁcant (p > 0.05).
High salt activates p97 to reduce host antiviral immunity by restricting Viperin induction (2021)
Figure 4

Figure 4. High salt reduces cellular USP33 and therefore decreases Viperin protein stability.

A, B Western blot analysis of USP33 proteins in HT1080 (A) or THP1 (B) cells treated with additional NaCl (+17, 34 and 51 mM) for 12 hrs.

C Western blot analysis of USP33 levels in the spleen tissues of mice fed an NSD or HSD for 7 days or 30 days as indicated.

D Immunoprecipitation analysis of Viperin ubiquitination in HEK293T cells co-transfected with Flag-Viperin, HA-Ub and shUSP33, then treated with additional NaCl (+34 mM) for 12 hrs.

E Western blot analysis of Viperin levels in 2fTGH cells transfected with shCtrl or shUSP33 and then infected with VSV (MOI=1.0, 12 hrs) immediately after addition of NaCl (+34 mM).

F Western blot analysis of Viperin in the splenocytes of Usp33+/+ or Usp33-/- mice treated with mIFNβ (500 IU/ml, 12 hrs) immediately after addition of NaCl (+34 mM).

G RT-qPCR analysis of VSV RNA levels in 2fTGH cells transfected with shCtrl or shUSP33 and then infected with VSV (MOI=1.0, 24 hrs) immediately after addition of NaCl (+34 mM).

H Mouse splenocytes of Usp33+/+ or Usp33-/- mice were infected with VSV (MOI=1.0, 24 hrs) immediately after addition of NaCl (+34 mM). VSV-G protein levels were analyzed by Western blot.

I Survival curves of 8-week-old Usp33-/- mice fed an NSD (0.5% NaCl) or HSD (4% NaCl) for 7 days and then infected with VSV (1x108 PFU per gram body mouse, n=10).

Data information: Data (A-F, H) are representative of at least two biological replicates; Data (G) represent mean and SD of three biological replicates, and p values were calculated using two-tailed unpaired Student's t-test; Data (I) show mean and SEM of ten biological replicates, and p value was calculated using logrank (Mantel-Cox) tests. For all statistical testing: NS, not signiﬁcant (p > 0.05). ***p < 0.001.