Fig. 6. The binding of progranulin to Sortilin enhances Sortilin-LIFR proximity, leading to STAT3 hyperactivation in HStCs.

Immunoblot (A) and densitometric quantification (B) of pSTAT3/STAT3, relative to α-tubulin. Experiment was performed three times with similar results. Schematic (C) of recombinant full length (FL) progranulin, or progranulin lacking the C-term tail (Trunc PGRN) constructs, with an mCherry-StreptagII, and (D) illustration depicting Sortilin-mediated uptake of progranulin, via C-terminus end. E Schematic of experimental design for pre-treatment of LX2 cells with SCRAMBLE or SORT1 siRNA, and siGLO transfection reagent, prior stimulating with FL-progranulin. Representative images (F) and quantification (G) of FL-progranulin uptake (FL-PGRN+) in successfully transfected (siGLO+) LX2 cells. Scale bar, 40 µm. siRNA: SCRAMBLE, n = 48 cells. siRNA: SORT1, n = 47 cells. Distribution of data presented as violin plot. P value, two-tailed Mann–Whitney test. Immunoblot (H) and densitometric quantification of pSTAT3/STAT3 relative to GAPDH (loading control) (I) in LX2 cells pre-treated with SORT1 siRNA and stimulated with LIF in the presence or absence of progranulin, for 30 min. Successful receptor knockdown was validated by protein detection of sortilin. Experiment was performed three times with similar results. Representative images (J) and quantification (K) of the uptake of FL-progranulin (n = 51) and Trunc-progranulin (n = 35) (m-cherry; Red) in LX2 cells. Scale bar: 40 µm. Distribution of data presented as violin plots. P value, two-tailed Mann–Whitney test. Immunoblot (L) and densitometric quantification of pSTAT3/STAT3 relative to GAPDH (M) in LX2 cells stimulated with LIF in the presence or absence of FL- or Trunc-progranulin, for 30 min. Experiment was performed three times with similar results. N Illustration depicting the mechanism of duolink Proximity Ligation Assay. Two antibody-labelled proteins of interest, Sortilin and LIF receptor, generate a fluorescently detectable signal, visible as a single punctum, only when residing within 40 nm of each other. Representative fluorescence images (O) and quantification (P) of average number of puncta (PLA+) in LX2 cells stimulated with DMEM (n = 29), LIF (n = 29), FL-PGRN (n = 26), LIF + FL-PGRN (n = 25), Trunc-PGRN (n = 31), or LIF+Trunc-PGRN (n = 32). Scale bar: 20 µm. Error bars, SD. P value, one-way ANOVA with Tukey’s multiple comparisons. Source data are provided as a Source Data file.