1：Experimental Design and Method Selection:

The study used a reductionist approach with primary RGC cultures to test whether Wnt signaling acts directly on RGCs to induce neurite growth and identify intrinsic mechanisms. Wnt3a was used to stimulate Wnt signaling due to its endogenous expression and known effects on axonal regrowth.

2：Sample Selection and Data Sources:

Retinal ganglion cells (RGCs) were isolated from 10-11 day-old wildtype mouse pups using a two-step immunopanning protocol with positive and negative selection to obtain almost pure (>95%) RGC cultures.

3：List of Experimental Equipment and Materials:

Equipment included Zeiss Axiovert 200 fluorescent microscope, Leica Microsystems confocal microscope, Eppendorf Realplex 2 Mastercycler. Materials included papain (Worthington Biochemical Corp), anti-macrophage antibody (Accurate Chemical), CD90.2/Thy1.2 hybridoma supernatant, Neurobasal/B27 media (Thermo Fisher Scientific), Wnt3a (R&D Systems, Inc.), Necrostatin-1s (Millipore), primary antibody rabbit anti-βIII-tubulin (Abcam), secondary antibodies (anti-rabbit Alexa 546, Life Technologies), DAPI, Absolutely RNA Nanoprep Kit (Agilent Technologies), SuperScript III First-Strand Synthesis SuperMix (Life Technologies), PowerUp SYBR Green Master Mix (ThermoFisher).

4：2/Thy2 hybridoma supernatant, Neurobasal/B27 media (Thermo Fisher Scientific), Wnt3a (R&D Systems, Inc.), Necrostatin-1s (Millipore), primary antibody rabbit anti-βIII-tubulin (Abcam), secondary antibodies (anti-rabbit Alexa 546, Life Technologies), DAPI, Absolutely RNA Nanoprep Kit (Agilent Technologies), SuperScript III First-Strand Synthesis SuperMix (Life Technologies), PowerUp SYBR Green Master Mix (ThermoFisher). Experimental Procedures and Operational Workflow:

4. Experimental Procedures and Operational Workflow: RGCs were plated and treated with Wnt3a, PBS (vehicle control), or Necrostatin-1s for 2 days. For immunocytochemistry, cells were fixed, permeabilized, blocked, incubated with primary and secondary antibodies, and counterstained with DAPI. Neurite length and number were quantified from immunostained images. For gene expression analysis, total RNA was isolated, cDNA synthesized, and QPCR performed with specific primers for Ripk1 and Ripk

5：Data Analysis Methods:

Neurite measurements were performed by investigators masked to treatments. QPCR data were analyzed using the delta-delta Ct method normalized to a reference gene. Statistical analysis used ANOVA or Student’s t-test with GraphPad Prism, with p < 0.05 considered significant.