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Figueroa-Hall et al. TLR4-mediated signaling in CHME-5 cells

Confocal microscopy variance (ANOVA) with Dunnett’s or Tukey’s multiple
The Leica SPE Scanhead RYBV confocal microscope comparison tests were used, unless otherwise stated
was used to capture images for brightfield, and in the results section. Significance was determined at
immunofluorescence and all images were processed P < 0.05.
in Leica Application Suite Advanced Fluorescence.
For brightfield imaging, the 488 laser (55%) was RESULTS
used to capture images with the 100× oil objective in
unstimulated and LPS-stimulated cells, which were CHME-5 cells are not of human origin (STR
fixed with 4% PFA for 10 min at 25 °C. For fluorescent genotyping of CHME-5 cells)
imaging, cells were prepared as described in the STR Genotyping was performed to validate the claim
immunocytochemistry section and images captured that CHME-5 cells are not of human origin. STR
with the 100× oil objective using fluorescent channels genotyping confirmed that CHME-5 cells are devoid
for DAPI (350 nm), TLR4 (Rhodamine-555 nm), and of any human genomic DNA [Supplementary Figure 1].
CD68 (Texas Red-647 nm). TLR4 channel (red) gain
and laser was set to 925 and 33%, CD68 channel LPS-induced activation of NF-κB p65 in
(green) gain and laser was set to 975 and 33%, CHME-5 cells
and DAPI channel (blue) gain and laser was set to Activation of NF-κB p65 is a crucial step for nuclear
839 and 15%. These settings were applied to all translocation and transcription of pro-inflammatory
confocal images. One random field-of-view (FOV) mediators. Phosphorylation of NF-κB p65 was used
was obtained for each group at a resolution of 1024 × as a measure of NF-κB activation. Immunoblot
1024 pixels. Eighty images were then taken through analysis indicated that LPS increased nuclear levels
the depth (Z) of 6.63 µm for each FOV. This value of phospho-NF-κB p65 [Figure 1A]. Further analysis
of 6.63 μm was used to provide equal X, Y, and Z with a Student’s t-test revealed that the increase was
dimensions, providing standardization of collecting statistically significant, P < 0.02 [Figure 1B].
depth image sets.
LPS is not toxic
Three dimensional reconstruction The MTT assay was used to determine the extent to
Confocal image sets for each fluorescent channel which LPS stimulation affects cell viability. ANOVA
were converted to 8-bit and processed in image J revealed that LPS (0.001-10 μg/mL) at 30 min did not
using Isosurface within the Bone J plugin (http:// significantly (P = 1.00) affect viability of CHME-5 cells
bonej.org/). TLR4 three dimensional (3D) mesh was [Figure 1C].
produced with a resample rate of 2 and a threshold of
20. CD68 3D mesh was generated with a resample LPS-induced NF-κB p65 binding activity
rate of 2 and threshold of 23. DAPI 3D mesh was For transcription of pro-inflammatory mediators to
constructed with a resample rate of 2 and a threshold occur, the NF-κB p65 subunit must bind to nuclear
of 30. 3D meshes for all fluorescent channels were consensus sequences. NF-κB p65 activation was also
imported into Blender (blender.org) and scaled determined by assessing NF-κB p65 binding activity
uniformly. Artifacts due to apparent non-specific and in nuclear extracts. Tubulin and histone 3 expression
non-cellular labeling were deleted in some channels were measured as internal controls to confirm cellular
for better imaging. Lighting, cameras and mesh- compartmentalization in cytoplasmic and nuclear
materials were created to image each scene. The fractions [Supplementary Figure 2]. ANOVA and
respective brightfield image for each FOV was then uncorrected Fisher’s LSD tests indicated that NF-κB
placed at the base of each 3D reconstruction and p65 binding activity was significantly increased at 10
scaled to fit. Images were rendered using Blender (P < 0.04) and 90 min (P < 0.01) [Figure 1D]. Binding
Cycles Render at 3840 × 3840 pixel resolution. activity at 30 and 180 min remained at basal levels.

Statistical analysis LPS-mediated IκBα phosphorylation in
Image J Software was used to obtain the mean CHME-5 cells
grey intensity of all immunoblots and the mean was Inhibitor kappa b alpha (IκBα) is the negative regulator
taken for each time point. GraphPad Prism 7.0 was for NF-κB and thus must be activated for release of
used for transformation, quantification, and graphing NF-κB into the nucleus. Phosphorylation of IκBα in
of all data. For statistical analysis of mRNA and cytoplasmic lysates was used as a measure of IκBα
protein expression, binding activity, cell viability, activation [Figure 2A]. ANOVA and Dunnett’s multiple
and immunofluorescence, one-way analysis of comparison tests revealed LPS significantly increased
Neuroimmunology and Neuroinflammation ¦ Volume 4 ¦ October 19, 2017 223