In vitro study in mast cells:
Mast cell lines were purchased from (Kerafast-LUVA-Human Mast Cell Line,
(EG1701-FP, MA, USA). Suspended cells were grown to confluence in six
well culture plate and were exposed to 10ng/ml & 20ng/ml concentration
of IgE (Abcam-Native human IgE protein (ab65866, MA, USA) for 16
hours in serum free medium (STEM PRO-34 nutrient supplement (Gibco
#10641-025, MA, USA); At the end of 16 hours, cells were centrifuged at
3000g, washed in 1X-PBS. A) The gene expressions were analysed by RT-PCR
(Biorad-Touch real time PCR, CA, USA) for the enzymes. B) Lysed using
RIPA buffer. The lysate after protein estimation (Pierce™ BCA Protein
Assay Kit Cat No: 23225, MA, USA) was used for western blot analysis
(30 μg/ul) to assess endogenous Histone Deacetylase (HDAC) expression
using Antibody Sampler Kit (#9928, Cell signalling Technology, MA,USA)
using the corresponding antibodies namely, HDAC1, HDAC2, HDAC4, HDAC6
and 7 (representing Zinc dependent class I, class IIa and IIb ) as well
as the Beta actin (β-Actin Antibody (AC-15) sc-69879, Santa Cruz
Biotechnology, CA, USA) used as the loading control. Chemidoc-XRS+
(Biorad, CA, USA) was used for gel documentation and image lab tool
(Biorad, CA, USA) was used for estimating the pixel density of the
bands.
An in vitro study to evaluate the altered sphingolipid metabolism
in mast cells that infiltrate conjunctival cells was done. Mast cells
were treated with IgE that showed an increased expression of both S1P1R
and S1P3R (p<0.05). Protein expression of HDAC (1and 6) were
increased significantly (p<0.05). Increased S1P1R and 3R
expressions that internalize S1P seem to target HDAC for epigenetic
modulation in mast cells (Supp Fig 2).