Fig 2: A rare heterozygous SUMO4:c.2T>C initiator codon variant detected in 1.8% of ALS patients leads to reduced SUMO4 expression, altered stress granule dynamics, and reduced SUMOylation of VCP. a Electropherograms demonstrating the rare heterozygous SUMO4:c.2T>C p.Met1? variant in leukocyte DNA of four sporadic ALS patients. b SUMO4 mRNA expression in whole blood of SUMO4:c.2T>C variant carrier TALS004-01 and two ALS control patients without the SUMO4:c.2T>C variant was quantified by real-time RT-PCR and normalized to B2M mRNA. SUMO4 mRNA levels were significantly reduced in whole blood of patient TALS004-01 compared to ALS controls (mean ± SD of triplicate samples from three independent experiments). c–e Detection (d) and densitometric quantification (e) of unconjugated SUMO4 protein and all unconjugated SUMO isoforms 1–4 by Western blot analysis of lysates of fibroblasts of patient TALS004-01 harboring the SUMO4:c.2T>C variant (c) and of two sex- and age-matched non-ALS controls without rare SUMO4 variants using anti-SUMO4 and anti-pan-SUMO (detecting SUMO isoforms 1–4) antibodies, showing that SUMO4 protein levels were significantly reduced and levels of all SUMO isoforms were reduced in fibroblasts of patient TALS004-01 compared to controls (mean ± SD from three independent experiments). f, g Detection (f) of stress granules (SGs) in fibroblasts of patient TALS004-01 and two non-ALS controls that were untreated (baseline), treated for 45 min with 0.5 mM sodium arsenite (NaAsO2) or had recovered for 60 min from NaAsO2 treatment (recovery) by immunostaining of the SG marker protein nucleolysin TIAR; nuclei were counterstained with DAPI; scale bar 10 µm. Quantification (g) of SGs from (f), showing significantly higher numbers of SGs in fibroblasts of patient TALS004-01 compared to fibroblasts of non-ALS controls at baseline, upon NaAsO2, and after recovery (mean ± SEM of = 95 cells per individual and condition from three independent experiments with at least 30 cells per individual, condition and experiment). h, i Immunoprecipitation (IP) of VCP, and detection (h) and densitometric quantification (i) of SUMO4-conjugated VCP by Western blot analysis of fibroblast lysates of patient TALS004-01 and non-ALS controls using an anti-SUMO4 antibody, showing that VCP is SUMOylated by SUMO4 and that SUMOylation of VCP by SUMO4 is significantly reduced in fibroblasts of patient TALS004-01 compared to controls (mean ± SD from two independent experiments). j SUMO4 mRNA expression in a human adult multiple tissue cDNA panel was quantified by real-time PCR, normalized to B2M mRNA, and displayed relative to mRNA levels in brain (†). SUMO4 mRNA levels were highest in brain followed by placenta and skeletal (sk.) muscle (mean ± SD of duplicate samples from two independent experiments). *p < 0.05; **p = 0.01; ***p = 0.001; n.s. not significant; according to two-tailed Student’s t test (b, e, i) or one-way ANOVA (g)

Fig 3: Role of SUMO4 expression in JAK2/STAT3 pathway activation in A549 and SK-MES-1 cells. SUMO4 was downregulated in NSCLC cells in combination with the use of a JAK2 inhibitor (AG490). (A) Equal amounts of total cell lysate were analyzed by western blotting. (B) Comparison of protein levels of p-JAK2 and p-STAT3 to GAPDH ratio. *P<0.05; **P<0.01. NSCLC, non-small cell lung cancer; SUMO4, small ubiquitin-like modifier 4; p-, phosphorylated.

Fig 4: SUMO4 expression in NSCLC tissue. (A) SUMO4 staining in adjacent normal lung tissue. (B) Low SUMO4 staining in lung adenocarcinoma tissue. (C) High SUMO4 staining in lung adenocarcinoma tissue. (D) Low SUMO4 staining in lung squamous cell carcinoma tissue. (E) High SUMO4 staining in lung squamous cell carcinoma tissue. All the images were obtained at ×200 magnification. NSCLC, non-small cell lung cancer; SUMO4, small ubiquitin-like modifier 4.

Fig 5: SUMO4-knockdown decreases migration and invasion in NSCLC cells. SUMO4 was downregulated by siRNA in A549, H1650 and SK-MES-1 cells for analysis of its effect on migration and invasion. (A) Western blot analysis shows the expression levels of SUMO4 in the three NSCLC cell lines. (B) Western blot analysis shows the expression levels of SUMO4 and corresponding factors involved in epithelial-mesenchymal transition. (C) Cell migration was detected via the wound healing assay, magnification ×100. (D) Quantitative analysis of (C). (E) Cell invasion was assessed via the Transwell assay. (F) Quantitative analysis of (E) *P<0.05; **P<0.01; ***P<0.001. NSCLC, non-small cell lung cancer; SUMO4, small ubiquitin-like modifier 4; siRNA, small interfering RNA; NC, negative control.