EMBO/soda-nel huggingface dataset:
SPINK6 inhibits human airway serine proteases and restricts influenza virus activation (2021)
Figure 4

Figure 4. Exogenous and endogenous SPINK6 affect virus maturation and viral replication in human airway organoids.

(A) Confocal imaging (left) shows intrinsic SPINK6 (green) expression in the epithelial cells in 2D human airway organoids. Nuclei and actin filaments are counterstained and shown as blue and purple respectively. Scale, 10 µm. Flow cytometry analysis demonstrates HAT and SPINK6 expression in human airway organoids. The plots (middle) show the representative result of one experiment performed 3 times. The percentages of HAT and SPINK6 positive cells (right) are shown as mean and SD of 3 independent experiments. (B) At 24h after inoculation of H1N1/pdm or mock inoculation in triplicate, 2D human airway organoids were harvested to detect mRNA expression levels of the indicated genes normalized with GAPDH. Data represent the mean and SD of a representative experiment performed three times. (C) 2D human airway organoids were incubated with the fluorogenic substrate in triplicate for 1 hour in the presence of wtSPINK6 or mutSPINK6, and applied to fluorescence assay. The fluorescence intensity of mutSPINK6-treated organoids is arbitrarily set as 1. Data represent the mean and SD of a representative experiment performed three times. (D) After inoculation of H1N1/pdm, 2D human airway organoids were incubated with wtSPINK6 or PBS in triplicate for 24 hours (left). Cell-free culture media were harvested for viral titration (right) and examination of HA cleavage by WB after 10-fold concentration (middle). Viral titer data present the mean and SD of triplicated samples. (E) 2D airway organoids were applied to fluorescence assay after incubation with α-SPINK6 or isotypic IgG in triplicate for 4 hours (left). Data present mean and SD of triplicated samples (right). Data present the mean and SD of triplicated samples. (F) After inoculation of H1N1/pdm, 2D human airway organoids were incubated with an α-SPINK6 or isotype IgG in triplicate for 24 hours (left). Cell-free culture media were harvested for viral load detection (right) and examination of HA cleavage by WB after 10-fold concentration (middle). Viral load data present the mean and SD of triplicated samples.

Data information: *, P<0.05; **, P<0.01; ***, P<0.001. Student's t test.
SPINK6 inhibits human airway serine proteases and restricts influenza virus activation (2021)
Figure 5

Figure 5. SPINK6 treatment suppresses viral growth and improves survival in mouse influenza infection.

(A) Regime of the mouse experiment. Balb/c mice were a mouse-adapted strain of pandemic H1N1 viru. At 24 hours before inoculation, 8, 24 and 36 hours post-inoculation, two groups of mice were intranasally administered with wtSPINK6 protein or mutSPINK6 protein. Ten mice treated with wtSPINK6 or mutSPINK6 were monitored daily for disease signs, body weight and survival for 14 days. Five mice treated with wtSPINK6 or mutSPINK6 were sacrificed at 3 and 4 days after the viral challenge. Lung tissues were collected for the quantification of viral growth and immunofluorescence staining. (B) Survival rates of mice treated with wtSPINK6 and mutSPINK6 was analyzed with Mantel-Cox test. (C) Viral load and viral titer in the lung homogenates of mice treated with wtSPINK6 and mutSPINK6. Data represent mean ± SD. **, P < 0.01; ***, P< 0.001. Student's t test. (D) Mouse lung tissues are applied to immunofluorescence staining to identify the viral NP positive cells (green). Nuclei are counterstained with DAPI (blue). Representative confocal images of virus-infected cells in the indicated mice. Scale bar, 10 µm.
SPINK6 inhibits human airway serine proteases and restricts influenza virus activation (2021)
Figure 6

Figure 6. The association SNP genotypes are correlated to differential SPINK6 mRNA expression in human tissues. (A) The association SNP rs1432689 genotypes are correlated to differential SPINK6 mRNA expression in over 1000 human lung tissues from three centers. The box denotes interquartile range; the thick line and diamond within the box are the median and mean respectively; whiskers are the minimum and maximum and open dots are outliers. Linear regression was used for eQTL data analysis. Data of three cohorts were applied to meta-analysis and resulted in the P-value of the association SNP. (B) rs1432689 genotypes are correlated to SPINK6 expression in human lung tissues and esophagus mucosa from GTEx database. Sample sizes for each genotype are shown in the x-axis. The y-axis shows the normalized expression of SPINK6 (Norm. expression) in the indicated trusses. Median and interquartile range are represented by a white line and dark box respectively. Data distribution is colored with light blue; a wider section of the violin plot indicates the data on the section have a higher frequency. Linear regression was used for data analysis.

Table 1. Genetic variation of SPINK6 SNP rs1432689 is associated with the susceptibility to human H7N9 infection.

Variant Distribution

& Analysis

1.44

0.00852

1.78

0.00834

OR, odds ratio
Inducing mitophagy in diabetic platelets protects against severe oxidative stress (2016)
Figure 1

Figure 1. DM oxidative stress is associated with p53 phosphorylation, mitochondrial dysfunction, and apoptosis

A ROS levels were measured by flow cytometry after staining with specific oxidative stress detection dyes (CellROX green, Molecular Probes, USA) in HC (n=4) and DM (n=4) platelets. (**p=0.032 vs. HC).

B Western blot analysis of ROS downstream signaling molecules p53 and phosphorylated p53 in HC (#1-3) and DM (#1-8) patient platelets. Quantification analysis on HC (n=5) and DM (n=18) individuals. (**p=0.0025 vs. HC). GAPDH served as the loading control.

C Fluorescent immunostaining for phosphorylated p53 in HC and DM patient platelets. Graph indicates HC and DM platelets positive for phosphorylated p53 signal. The y-axis indicates percentage of phosphorylated p53 positive cell from total cells (**p=0.0064 vs. HC). Representative figure from n=5.

D Mitochondrial membrane potential (ΔΨm) was measured by MitoTracker staining (50nM for 15 min) in HC and DM patient platelets. Graph indicates signal intensity of MitoTracker fluorescence in HC and DM platelets. The y-axis indicates fold of signal intensity in HC and DM platelet (**p=1.1608E-05 vs HC). Signal intensity of each group was converted to fold change compared with HC values. Representative figure from HC (n=6) and DM (n=4).

E ΔΨm and platelet apoptosis were measured by flow cytometry analysis. ΔΨm was detected using TMRM and apoptosis was assessed with Annexin-V (PS externalization). Representative figure from n=3.

F Western blot analysis of apoptosis-related pp53, p53, cytochrome c, and active caspase 3 in HC and DM platelets. Tubulin was used as the loading control. Representative figure from n=3.

G Western blot analysis showing release of cytochrome c (Cyto c) into the cytosol in DM. Representative figure from n=3.

H EM of platelets mitochondria from HC and DM patients demonstrating examples of mitochondria damage in DM platelets (DM) compared to a typical healthy mitochondria (HC). Arrows indicate disrupted outer membranes.

I Quantification of mitochondria in HC (n=33) and DM (n=17) patient platelets. The y-axis indicates average number of mitochondria in HC and DM single platelet EM view (**p=5.50811E-05 vs. HC).

J Quantification of healthy and damaged mitochondria in HC and DM platelets.
Inducing mitophagy in diabetic platelets protects against severe oxidative stress (2016)
Figure 2

Figure 2. Autophagy is activated in DM platelets.

A Western blot analysis of autophagy markers, Beclin1, ATG3, ATG7, ATG12-5, and LC3I/II, and the mitophagy-related proteins Parkin and PINK1 in HC (#1-3) and DM platelets (#1-8).

B Quantification of autophagy markers and mitophagy-related proteins from HC (#1-5) and DM (#1-18) individuals. (Beclin1; **p=0.0003, ATG12; *p=0.0388, ATG3; *p=0.0213, ATG7; p=0.4051, LC3; *p=0.0298, PINK; *p=0.0155, Parkin; *p=0.0103 vs. HC). GAPDH served as a loading control.

C The three panels represent autophagy stages found in DM platelets. Autophagosome structures containing/enclosing organelles are indicated by the black box. Shown for each panel is an enlargement of the autophagosome (DM1 and 2) or the autolysosome (DM3). Dashed structures are an outline of the autophagosome membranes.

D LC3 immuno-EM analysis of HC and DM platelets. Arrows indicate immunogold-labeled LC3 clusters. No clusters were found in HC platelets. Representative areas of clusters of gold labeling in DM patients (DM1-3) are presented. Shown in the insets are enlargements of the gold clusters adjacent to mitochondria-like structures.

E Double staining for MitoTracker (mitochondria membrane potential) (50nM for 15 min) and LC3 (autophagy) in HC and DM platelets. Boxed platelets are enlarged in Panel F.

F A typical HC platelet has high membrane potential and low LC3 in contrast to a representative DM platelet which has low membrane potential and high LC3.

G Quantification of autophagosome positive platelets using 6 HC and 4 DM samples after staining using CoxIV (mitochondria) and LC3 (autophagy marker). The y-axis indicates percentage of LC3 positive cells in total cells (**p=0.006 vs. HC).
Inducing mitophagy in diabetic platelets protects against severe oxidative stress (2016)
Figure 3

Figure 3. Autophagy is activated in platelets through an oxidative stress-induced JNK pathway

A Representative western blot analysis of recognized autophagy signaling molecules (pJNK, JNK, pAKT, and AKT, pmTOR, and mTOR) in HC (#1-3) and DM (#1-8) patient platelets. Quantification analysis of autophagy upstream signaling molecules on HC (#1-5) and DM (#1-18) individuals. (pAKT/AKT; p=0.0747, pJNK/JNK; *p=0.001, pmTOR/mTOR; p=0.3356 vs. HC). GAPDH was used as the loading control.

B Representative western blot analysis using HC and HG platelets with or without H2O2. And assessing for LC3I/II. GAPDH was used as the loading control.

C Platelet suspensions were incubated in HG for 2 hours and with H2O2 (1mM). ROS levels were evaluated using a detection kit (Enzo Life Science) for 60 mins at 37 ºC.

D Western blot analysis of p53, JNK and LC3I/II in HC platelets treated with H2O2 (1mM for 1 hour) alone or with NAC (100 μM for 30 min). GAPDH was used as the loading control.

E Confocal microscopy was used to corroborate the Western analysis using triple staining for Mitotracker, CoxIV, and LC3 in HC or H2O2. The insets highlight the association of low membrane potential with high levels of LC3 and increased colocalization of Cox IV with LC3 in H2O2-treated platelets.

F HC platelets were pretreated with H2O2 to induce autophagy and then treated with or without SP600125 (JNK inhibitor) at 3 concentrations (1, 5 and 10 µM) to assess for inhibition of autophagy. This Western blot is representative of three independent experiments. Quantification of the three independent experiments is provided at right. (pJNK/JNK in H2O2/SP600125; **p=0.0286 vs. H2O2 group, LC3II in H2O2; *p=0.0241 vs. HC group, LC3II in H2O2/SP600125; **p=0.0044 vs. H2O2 group, n=3 for each group).

G Triple staining of CoxIV, LC3, and LAMP1 in HC and H2O2 with/without SP600125-treated platelets using confocal microscopy. Arrow indicates localization of LC3 and LAMP1. Graph indicates colocalization between LC3, LAMP1 and CoxIV signal. The y-axis indicates fold change of colocalization between LC3 and LAMP1 in the mitochondria area. Signal intensity of each group was converted to fold and compared with HC values (H2O2;*p=0.023 vs. HC, H2O2/SP600125;*p=0.04 vs. H2O2 group).
Inducing mitophagy in diabetic platelets protects against severe oxidative stress (2016)
Figure 4

Figure 4. Human platelet mitophagy protects platelets from increased oxidative stress

A Western blot analysis of LC3 and the apoptosis markers phosphorylated p53, p53, and LAMP1 in HC platelets. Platelets were treated with high glucose (25mM), with or without CCCP (10µM). This Western blot is representative of three independent experiments.

B Quantification analysis of phosphorylated p53 and LAMP1 (HG; *p=0.018 vs DMSO group, HG/CCCP; **p=0.001 vs HG group, n=4 for each group).

C The recognized autophagy inhibitor (2mM 3-Methyadenine; 3MA for 1h) was used to treat DM platelets compared to HC assessing for phosphorylated p53, p53, and LC3I/II

D Quantification analysis of phosphorylated p53/p53 was performed. Each value indicated the average band density from a total of three independent samples (DM/3MA; *p=0.026 vs. DM group).

E Western blot analysis of PLPs transfected with control or ATG3 siRNA, with or without treatment of 1 mM H2O2. Each lane displayed a different transfected group.

F Quantification analysis of ATG3, phosphorylated p53 (ser15), p53, and GAPDH in each group. (ATG3 in ATG3 siRNA; **p=0.0003 vs control group, pp53 in ATG3 siRNA/ H2O2; *p=0.0145 vs. H2O2 group).
Inducing mitophagy in diabetic platelets protects against severe oxidative stress (2016)
Figure 5

Figure 5. Murine platelet mitophagy protects against enhanced oxidative stress.

A Western blot analysis of phosphorylated p53 (ser15), p53, LC3I/II, and GAPDH in WT mouse platelets. Mouse platelets were treated high glucose (25mM) with or without CCCP (10µM).

B Quantification analysis of phosphorylated p53 (ser15) and LC3 II (pp53 in HG; **p=0.009 vs WT group, pp53 in HG/CCCP ; *p=0.022 vs HG group, LC3 II in HG/CCCP ; *p=0.028 vs HG group, n=3 for each group).

C ΔΨm and platelet apoptosis were measured by flow cytometry analysis in WT or WT_H2O2 or WT_ H2O2/3MA in mouse platelets. ΔΨm was detected using TMRM and apoptosis levels were assessed with Annexin-V (PS externalization). Representative of n=3.

D Graph indicates the percentage of TMRM positive cells in total cell population (H2O2; **p=0.002 vs. not treated group, H2O2/3MA; **p=0.008 vs. not treated group, NS means no significance, n=3).

E Graph indicates the percentage of Annexin V positive cell in total cell population (H2O2; NS, p=0.058 vs. not treated group, H2O2/3MA; **p=0.004 vs. not treated group, **p=0.008 vs. H2O2 group, NS means no significance, n=3).

F Representative western blot analysis of LC3 I/II in analysis in WT or WT/H2O2 or WT/H2O2/3MA in mouse platelets (n=3).

G Representative western blot analysis of phosphorylated p53, and Parkin in Parkin -/- mice compared to WT. Quantification analysis of phosphorylated p53 and Parkin (Parkin; **p=0.0001 vs. WT, pp53; *p=0.019 vs. WT, n=4).

H ΔΨm and platelet apoptosis were measured by flow cytometry analysis in WT or WT_H2O2 and Parkin -/- or Parkin -/-_H2O2 mouse platelets. ΔΨm was detected using TMRM (WT_ H2O2; **p= 0.002 vs. WT group, Parkin-/-_ H2O2; **p= 1.61525E-06 vs. Parkin-/- group, n=3)

I Apoptosis level was assessed with Annexin-V (PS externalization). Graph indicates the percentage of Annexin V positive cell in total cell population (Parkin-/- : *p=0.010 vs. WT group, Parkin-/-_ H2O2 ;**p= 0.008 vs. WT_ H2O2; group, Parkin-/-_ H2O2; **p=0.001 vs. Parkin-/- group, NS means no significance n=3)

J Western analysis in WT and Parkin -/- demonstrating the inability to induce LC3 despite the addition of H2O2. Shown graphically are the LC3II to LC3I ratios (WT_ H2O2;*p=0.028 vs. WT group, n=3)
Inducing mitophagy in diabetic platelets protects against severe oxidative stress (2016)
Figure 6

Figure 6. Activation of platelet is regulated by induced mitophagy.

A Representative western analysis of pp53 in WT (n=6), DM (n=6) and PINK1-/-DM mice (n=3). GAPDH served as the loading control (DM ; **p=0.006 vs. WT group, PINK1-/-DM; **p=0.004 vs. DM group).

B Measurement of p-selectin translocation to the cell membrane by flow cytometry analysis in WT (n=3), DM (n=3) and PINK -/- DM (n=3) mice platelets. Graphs indicate mean fluorescence intensity (MFI) in each group. (*p<0.05 vs. WT or DM).

C Quantification of vessel occlusion time after carotid arterial flow measurement (WT and DM; and PINK -/- : **p<0.01 vs. WT or DM).

D Plot of carotid arterial flow by Doppler flow probe after 2 min FeCl3 injury, indicative of occlusive thrombosis. Flow was plotted as percentage of baseline flow before injury (DM: n=3, PINK-/- DM; n=3).
KLF10 promotes nonalcoholic steatohepatitis progression through transcriptional activation of zDHHC7 (2022)
Figure 1

Figure 1. Hepatic KLF10 is induced in NASH mice and patients.

(A) Heatmap of genes in the livers of HFD-feeding induced NAFL mice and WD/CCl4-treatment induced NASH mice. n=5. The color gradient represents log2(Fold change).

(B) Relative mRNA levels of KLF10 in the livers from NAFL and NASH mice. n=5. (C) Protein levels of KLF10 examined by western blots (left) and its expression was normalized to the β-actin (right). n=3. Results represent three independent experiments.

(D) Representative immunohistochemistry of KLF10 protein expression in the liver sections from NAFL and NASH mice. The bar chart showed the fold change of KLF10 positive cells per field (NASH vs. NAFL). n=5.

(E) Correlations between KLF10 and NAS, inflammation score, fibrosis scores, and hepatic triglyceride contents (TG) from NAFL mice (n=7) and NASH mice (n=8).

(F) H&E staining, Sirius red staining, and KLF10 immunofluorescence staining of liver sections from NAFL and NASH patients. n=3.

(G) Gene expression analysis in liver tissues from NAFL and NASH patients using GEO datasets (GSE48452 and GSE61260) from NCBI database. In the dataset GSE48452, n=17 for NAFL patients and n=15 for NASH patients. In the dataset GSE61260, n=21 for NAFL patients and n=24 for NASH patients.

(H) Spearman correlations between KLF10 expression and NAS in liver tissues from NAFL and NASH patients using GEO datasets (GSE48452). Left panel, n=32 (including 17 NAFL patients and 15 NASH patients). Right panel, n=21 (including 6 NAFL and 15 NASH patients).

Data information: * P <0.05, ** P <0.01-. Results are shown as mean ± SD. Student's t test (B-D, G). Spearman's correlation (E, H).
KLF10 promotes nonalcoholic steatohepatitis progression through transcriptional activation of zDHHC7 (2022)
Figure 2

Figure 2. Hepatic KLF10 overexpression promotes NASH pathogenesis in mice.

(A-H) 8-week-old male C57BL/6J mice were administered with AAV-GFP or AAV -Klf10 through tail vein injection, and then kept on WD/CCl4 for 12 weeks. n=5 per group. (A) Scheme for the experimental strategy. (B) Protein levels of KLF10 in the livers (left) and its expression was normalized to the β-actin (right). (C) H&E staining of liver sections. (D) CD68 immunofluorescence staining of liver sections (left) and its quantitation (right). (E) Body weight (BW), liver/body weight ratio, serum AST and ALT levels, and hepatic TG contents of two groups of mice. (F) Sirius red staining of liver sections (left) and its quantitation (right). (G) Relative mRNA levels of genes involved in the hepatic inflammation and fibrosis. (H) Protein levels of TNF-α in the livers (left) and its expression was normalized to the β-actin (right).

(I-P) 8-week-old male C57BL/6J mice were administered with AAV-GFP or AAV -Klf10 through tail vein injection, and then kept on HFD-feeding for 12 weeks. n=5 per group. (I) Scheme for the experimental strategy. (J) Protein levels of KLF10 in the livers (left) and its expression was normalized to β-actin (right). (K) H&E staining of liver sections. (L) CD68 immunofluorescence staining of liver sections (left) and its quantitation (right). (M) Body weight (BW), liver/body weight ratio, serum AST and ALT levels, and hepatic TG contents. (N) Sirius red staining of liver sections (left) and its quantitation (right). (O) Relative mRNA levels of genes involved in the hepatic inflammation and fibrosis. (P) Protein levels of TNF-α in the livers (left) and its expression was normalized to the β-actin (right).

Data information: * P < 0.05, ** P < 0.01, *** P < 0.001. Results are shown as mean ± SD. Student's t test (B, D-G, H, J, L-P).
KLF10 promotes nonalcoholic steatohepatitis progression through transcriptional activation of zDHHC7 (2022)
Figure 3

Figure 3. KLF10 transcriptionally activates zDHHC7 expression.

(A) Heatmap of genes in mouse primary hepatocytes transfected with lentivirus vectors expressing KLF10 (KLF10OE) or empty vector (Ctrl), P＜0.001. n=5 per group. The color gradient represents log2(Fold change).

(B) Relative mRNA (n=5) and protein (n=3) levels of zDHHC7 in MPHs expressing KLF10 or empty vector (Ctrl). The protein level of KLF10 or zDHHC7 was normalized to β-actin. Results represent three independent experiments.

(C-D) Spearman correlations between KLF10 and zDHHC7 in liver tissues from NASH and NAFL patients using GEO datasets (GSE48452 and GSE61260). In the dataset GSE61260 (C), n=83 (including 38 healthy controls, 21 NAFL patients and 24 NASH patients). In the dataset GSE48452 (D), n=73 (including 41 healthy controls, 17 NAFL patients and 15 NASH patients).

(E) Relative mRNA levels of DHHCs family in HepG2 cells overexpressing KLF10 or empty vector (ctrl). n=5. Results represent three independent experiments.

(F) Protein levels of zDHHC7 in the livers of HFD mice or WD/CCl4 mice as described in the Figure 2. The protein level of zDHHC7 was normalized to the β-actin. n=5.

(G) Luciferase reporter assays showing the activity of different truncation of zDHHC7 promoter fragments in HepG2 cells. n=3. Results represent three independent experiments.

(H) The transcriptional activity of wild-type or mutant zDHHC7 promoter in HepG2 cells. n=3. Results represent three independent experiments.

(I) Chromatin immunoprecipitation analysis of KLF10 binding activity at the zDHHC7 promoter. Soluble chromatin was prepared from the livers of WD/CCl4 treatment-induced NASH mice and immunoprecipitated with antibodies against KLF10 or against IgG. The final DNA extractions were amplified using pairs of primers that cover the regions of zDHHC7 and GAPDH gene promoters.

(J) Quantitative real-time RCR results of ChIP assays. IgG, immunoglobulin G. n=3. Results represent three independent experiments.

Data information: * P <0.05, ** P <0.01, *** P <0.001. Results are shown as mean ± SD. Student's t test (B, E, F, H, J). Spearman's correlation (C, D).
KLF10 promotes nonalcoholic steatohepatitis progression through transcriptional activation of zDHHC7 (2022)
Figure 4

Figure 4. KLF10 overexpression promotes CD36 palmitoylation in hepatocytes. (A-E) The liver tissues were collected from WD/CCl4-treated mice expressing KLF10 or control as described in the Figure 2A. (A) Representative immunofluorescence staining of CD36 and β-catenin. The arrows represent the colocalization of CD36 and β-catenin. (B) Quantitative results of CD36 colocalization rate. n=5. Results represent three independent experiments. (C) Expression of CD36 in whole livers and plasma membrane fractions. Protein levels were normalized to the β-actin or ATP1a1, respectively. n=5. Results represent three independent experiments. (D) Protein levels of palmitoylated CD36 in mouse livers. (E) Quantitative results of CD36 colocalization rate. n=3. Results represent three independent experiments.

(F-J) The liver tissues were collected from HFD-fed mice expressing KLF10 or control as described in the Figure 2I. (F) Representative immunofluorescence staining of CD36 and β-catenin. The arrows represent the colocalization of CD36 and β-catenin. (G) Quantitative results of CD36 colocalization rate. Results represent three independent experiments. (H) Expression of CD36 in whole livers and plasma membrane fractions. Protein levels were normalized to the β-actin or ATP1a1, respectively. n=5. Results represent three independent experiments. (I) Protein levels of palmitoylated CD36 in mouse livers. (J) Quantitative results of CD36 colocalization rate. n=3. Results represent three independent experiments.

Data information: * P < 0.05, ** P < 0.01, *** P < 0.001. Results are shown as mean ± SD. Student's t test (B, C, E, G, H, J).