Fig 2: Dermal White Adipose Tissue (DWAT) atrophy and increased adipocyte SIX1 expression in SSca) Hematoxylin and eosin staining of human skin biopsies from GENISOS cohort participants. top Control skin. middle early SSc-representative image bottom established representative SSc-skin sample. Clinical and demographic features of biopsies individuals are provided in Supplementary Table 4. Boxes contain dermal white adipose tissue. Dotted boxes denote DWAT areas. b) Representative images of dual in situ hybridization for SIX1 (teal) and FABP4 (pink) in 15 SSc and 5 demographically-matched control biopsies. Hematoxylin (purple) co-stain labels nuclei. “A” marks a dermal adnexal structure. Black arrows point to SIX1 transcript signal. Dash dot outlines denote the interior periphery of adipocytes.

Fig 3: Six1 silence can trigger apoptosis especially mitochondrial pathway apoptosis. (A) Six1 knock-down promoted apoptosis in SGC-7901 cells. Apoptotic cells were stained with Annexin V and 7-AAD. (B) Histogram showed the percent of apoptotic cells in NC and Six1 knock-down groups. (C) Six1 silence decreased the mitochondrial membrane potential (Δψm) and triggered mitochondrial apoptosis detected by JC-1 assay. JC-1 displays red fluorescence in cells with high Δψm, whereas JC-1 exhibits green fluorescence in cells with low Δψm. (D) Flow cytometry showed the distribution ratio of JC-1 red and JC-1 green cell population. (E) Histogram calculated the relative ratio of red against green fluorescence. *, p < 0.05.

Fig 4: Combined knockdown of SIX1, SIX2, and SIX4 does not inhibit myogenic differentiation. FSHD1 (54–2) myoblasts were transfected with siRNAs targeting SIX1, SIX2, and SIX4, and differentiated to generate multinucleated myotubes. A Multinucleated myotubes with clusters of nuclei for the individual and combined knockdown of SIX1/2/4. B Immunofluorescent staining for nuclei (blue) and MF20 (red) for si-Control and si-SIX1/2/4 conditions. C Fusion index was calculated for the conditions shown in B. D-G Myogenic markers for (D) myogenic commitment, D early and late differentiation, E fast fiber-types, F slow fiber-types and G regenerative were assessed. For fusion index, there were three fields captured with three biological replicates. Each experimental siRNA group (n = 3) was normalized to a negative si-Control with the mean and standard deviation depicted. Asterisks demonstrate statistical significance between the control vs experimental group using an unpaired two tailed t-test (ns = not significant; *p < 0.05)

Fig 5: Low level of DUX4 expression in myoblasts are unaffected by SIX1/2/4 knockdown. FSHD1 (54–2) were transfected with SIX1, SIX2, and SIX4 siRNAs in proliferating myoblasts 72 h before harvesting. qPCR results show relative mRNA expression for A and C DUX4, A-C DUX4 targets and D siRNAs targeting each respective SIX target. E-F Effects on SIX1 and SIX2 targets (PGK1, SLC4A7 and EYA1) were analyzed in E myoblasts and F myotubes. All conditions were carried out in triplicates with the mean and standard deviation depicted. Each experimental group (n = 3) was normalized to a negative si-Control with the mean and standard deviation depicted. Asterisks demonstrate statistical significance between the control vs experimental group using an unpaired two tailed t-test (ns = not significant; *p < 0.05)