PRG3 knock-down reduces the formation of small neurites. (A) Primary neurons were transfected with PRG3 shRNA or control shRNA at DIV 1 and morphologically analyzed at DIV 4. Determination of branching points according to branching order revealed no differences between the two groups (n = 80). (B) Axon length measurement, in which the longest neurite of the transfected neuron was measured (n = 32). (C) Confocal images of representative transfected primary neurons showed a decreased quantity of small neurites in PRG3 shRNA-expressing neurons compared to controls. Scale bar: 5 μm. (D) All neurites within one neuron were grouped between lengths of 2–5 μm and 5–10 μm and are presented as subgroups. PRG3 knock-down decreased the number of neurites between 2–5 μm (p = 0.0006) but not in the number of neurites with a length between 5–10 μm. We co-transfected primary neurons with a PRG3 rescue plasmid together with the PRG3 shRNA. The double transfected neurons show neither a reduction of neurites with a length between 2–5 μm nor neurites with a length between 5–10 μm. (E) PRG3 knock-down did not affect the average neurite length per neuron as revealed by comparing PRG3 shRNA expression neurons to controls or to the PRG3 shRNA resistant construct. (F) Confocal images of representative transfected primary neurons showed an increased quantity of small neurites in PRG3-eGFP-expressing neurons compared to controls. Scale bar: 5 μm. (G) PRG3 overexpression leads to an increase in the number of neuritis with a length between 2–5 μm (p <= 0.0489). No effect was detectable in the number of neuritis with a length between 5–10 μm. (H) The average neurite length for each neuron was unchanged for PRG3-eGFP overexpressing cells and respective controls.