Hello researchers,

I am working on the differentiation of fibroblasts (FB) into myofibroblasts (MFB) using the L929 cell line. Following references, I attempted to induce differentiation with TGF-beta1.

The primary distinction between FB and MFB is that MFBs are characterized by the expression of α-smooth muscle actin. However, in my experiments, immunostaining for α-smooth muscle actin in L929-differentiated MFBs shows no detectable signal, even though Western blotting indicates the presence of α-smooth muscle actin.

The immunostaining protocol I used is a standard protocol that has worked successfully in our lab for other targets such as YAP and vinculin. I carefully reviewed all steps, from cell starvation prior to TGF-beta1 treatment, to the adhesion time of MFBs on fibronectin-coated surfaces (longer adhesion times are suggested to enhance α-smooth muscle actin expression), and the immunostaining process itself (including permeabilization and fixation). Despite these checks, the immunostaining still fails to show α-smooth muscle actin.

Additionally, I could not find any online references or images of α-smooth muscle actin immunofluorescence in L929-differentiated MFBs.

This leads me to question: Can TGF-beta1 successfully induce the differentiation of mouse L929 fibroblasts into myofibroblasts?

Any insights or suggestions would be greatly appreciated. Thank you.

for TGF-beta1

https://www.rndsystems.com/products/recombinant-human-tgf-beta-1-human-cell-expressed-protein_7754-bh

for α-smooth muscle actin

https://www.cellsignal.com/products/primary-antibodies/a-smooth-muscle-actin-d4k9n-xp-rabbit-mab/19245