A 2D and 3D cell culture protocol to study O-GlcNAc in sphingosine-1-phosphate mediated fibroblast contraction

Summary O-linked β-N-acetylglucosamine (O-GlcNAc) is an abundant posttranslational modification involved in a wide range of signaling pathways, but its specific role in regulating biological processes remains unclear. This protocol describes approaches to understand O-GlcNAc’s role in fibroblast contraction. Specifically, cellular O-GlcNAc levels are controlled through treatment of fibroblasts with inhibitors in both 2D and 3D cultures. We then describe 2D contraction assay and 3D collagen gel contraction assay to analyze the effect of the modification on sphingosine-1-phosphate signaling for contraction. For complete details on the use and execution of this protocol, please refer to Pedowitz et al. (2021).


Highlights
Protocol describes approaches to test fibroblast contraction in 2D and 3D cell cultures Fibroblast contraction is induced by the signaling lipid sphingosine-1phosphate (S1P) Extent of S1Pinduced contraction is controlled by O-GlcNAc modification Protocol can be used to test a variety of established and potential contraction mediators SUMMARY O-linked b-N-acetylglucosamine (O-GlcNAc) is an abundant posttranslational modification involved in a wide range of signaling pathways, but its specific role in regulating biological processes remains unclear. This protocol describes approaches to understand O-GlcNAc's role in fibroblast contraction. Specifically, cellular O-GlcNAc levels are controlled through treatment of fibroblasts with inhibitors in both 2D and 3D cultures. We then describe 2D contraction assay and 3D collagen gel contraction assay to analyze the effect of the modification on sphingosine-1-phosphate signaling for contraction. For complete details on the use and execution of this protocol, please refer to Pedowitz et al. (2021).

BEFORE YOU BEGIN
The protocol below describes the method to study the effect of O-GlcNAc on sphingosine-1-phosphate (S1P) mediated 2D cell contraction in mouse embryonic fibroblast NIH3T3 cells and 3D cell contraction in primary human dermal fibroblast BJ-5ta cells. We have also used this protocol to look at signaling by lysophosphatidic acid (LPA) (Morales et al., 2021).
THAWING AND CULTURING NIH3T3 AND BJ-5TA CELLS b. Briefly thaw frozen cells at 37 C until only a small amount of ice remains. c. In a biological safety hood, transfer the cells into a 15 mL tube containing 10 mL of its appropriate pre-warmed culture medium. d. Centrifuge at 500 g for 5 min. e. Discard the supernatant without disturbing the cell pellet. f. Gently resuspend the cell pellet in the appropriate culture medium and transfer into a tissue culture plate. 3. Maintaining NIH3T3 or BJ-5ta cells a. Subculture cells every 2-3 days or until 80% confluent and replenish media every 3 days.
CRITICAL: Subculture NIH3T3 cells at least 3 times before continuing with contraction experiment and use before a maximum of 6 passages. Subculture BJ-5ta cells at least twice before experiment; however, do not use BJ-5ta cells that have been passaged more than 8 times.

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CRITICAL: ATCC recommends that BJ-5ta cells are grown in a 4:1 mixture of Dulbecco's medium and Medium 199 with supplements, however we have seen that BJ-5ta cells grow equally well in unmixed DMEM.

STEP-BY-STEP METHOD DETAILS
2D contraction assay using NIH3T3 cells

Timing: 2 days
This step contains information on plating NIH3T3 cells, and treatment of the cells with either inhibitor and sphingosine-1-phosphate to initiate contraction. Figure 1A shows a diagram of the layout of the different cell treatments.
1. Seed NIH3T3 cells at 1 3 10 5 cells per well in 12 wells of two 6-well cell culture dishes 8 h prior to treatment with either small molecule inhibitors. 2. After 8 h, treat a 6-well dish of NIH3T3 cells with either DMSO vehicle, 5SGlcNAc (200 mM), or Thiamet-G (10 mM). a. Allow cells to incubate for 16 h with 5SGlcNAc/DMSO or for 20 h with Thiamet-G/DMSO. 3. After the appropriate incubation, treat 1 well with DMSO as control and treat 5 wells of each plate with a S1P dose curve (0.05 mM, 0.1 mM, 0.5 mM, 1 mM, 5 mM) by pipetting it into the media (Figure 1A). Incubate the plate for 30 min. 4. After 30 min, take 5 representative images throughout each well by brightfield microscopy at 203 magnification. 5. Analyze the images by Adobe photoshop to find the change in cell area (see quantification protocol below).

Timing: 3 days
This step contains information on using a collagen gel contraction assay to evaluate the effect of inhibitors and sphingosine-1-phosphate on cell contraction in a tissue like environment. Figure 1B shows a diagram of the layout of the different cell treatments. This protocol was adapted from a procedure that can be found on the manufacturer's website: https://www.cellbiolabs.com/ cell-contraction-two-step-attached-model.
6. Suspend at least 7 3 10 6 BJ-5ta cells in fresh media at 2 3 10 6 cells per mL. 7. In a pre-chilled falcon tube, prepare the collagen solution on ice according to the 'Collagen solution' table above. a. First add the collagen solution and 53 medium and mix well by gently pipetting up and down. b. Add the neutralization solution and mix immediately.
CRITICAL: Keep everything on ice to prevent premature polymerization of the solution. Make the solution right before plating out for the experiment.
8. Make a 4:1 mixture of collagen stock solution to cells in a pre-chilled 15 mL falcon tube and mix well. a. To 12.936 mL of collagen solution, add 3.23 mL of BJ-5ta cells in media. b. Pipette up and down carefully to avoid bubbles. Do not vortex. 9. Plate 500 mL of the cell collagen solution into 30 wells in two 24-well dishes. 10. Incubate the cell-collagen matrix at 37 C with 5% CO 2 for 1 h to allow collagen polymerization to occur.
Note: It is difficult to determine whether polymerization is successful. Figure 7 shows a representative image of successful vs unsuccessful polymerization. Commonly, we discovered polymerization was unsuccessful when there was no contraction upon the addition of S1P.
11. After 1 h, add 1 mL of prewarmed media to each well.
CRITICAL: Add the media very gently to prevent the matrix from detaching from the plate. Using a pipet, press the pipet tip against the wall of the well and depress the plunger slowly ( Figure 2).
12. Incubate the matrix at 37 C with 5% CO 2 for 30 h. 13. After 30 h, supplement the wells with DMSO vehicle or with either 5SGlcNAc (200 mM) or Thiamet-G (10 mM). a. Treat 15 wells with DMSO vehicle and 15 wells with 5SGlcNAc/Thiamet-G. 14. Incubate for an additional 16 h. 15. After incubation, carefully aspirate the liquid media out of the well and replace the media with serum-free media containing S1P (0.1-5 mM) or no S1P as a control.

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a. Treat 6 wells with each concentration of S1P: 3 wells that were pre-treated with DMSO and 3 wells that were pre-treated with 5SGlcNAc. b. Treat one row of 6 wells with DMSO as control. 16. Initiate contraction by gently releasing the sides and the bottom of the matrix from the plate using a 10 mL pipet tip. 17. Image the wells using a ChemiDoc XRS+ molecular imager (Bio-Rad) immediately after release (t = 0). Alternatively, images can be taken with any type of imager or the matrix can be measured with a ruler. 18. Incubate the matrix at 37 C for 30 min and then image the wells again (t = 30). 19. Quantify the change in diameter of each matrix using ImageJ (see quantification protocol below).

EXPECTED OUTCOMES
The modulation of O-GlcNAc levels through treatment with inhibitors can be confirmed by western blotting (Figure 3). For the 2D contraction assay, NIH3T3s with normal levels of O-GlcNAc undergo contraction at higher concentrations of S1P. However, NIH3T3s treated with OGT inhibitor 5SGlcNAc with lower levels of O-GlcNAc displayed increased sensitivity to lower concentrations of S1P ( Figure 4A). Conversely, NIH3T3s that were treated with OGA inhibitor Thiamet-G and had higher O-GlcNAc levels were more resistant to S1P-mediated contraction compared to control DMSO treated cells ( Figure 4B). Similarly in the 3D contraction assay with BJ-5ta cells, the matrices that were pretreated with 5SGlcNAc were significantly more sensitive to S1P-mediated contraction than the control ( Figure 5).

QUANTIFICATION AND STATISTICAL ANALYSIS
2D contraction assay using NIH3T3 cells Quantify the contraction phenotype by taking the mean G SEM of the relative culture plate area covered by cells in four randomly selected frames per well. Analyze the images using Adobe Photoshop ( Figure 6). Use the Magic Wand tool to select ''empty'' sections of the image that do not contain cells and subtract this pixel value from total pixels. Normalize this value by using the control DMSO treated well allowing for quantification of the difference in area taken up by cells before and after contraction. Perform at least 2 biological replicates and determine statistical significance by using a 2-way ANOVA test followed by Sidak's multiple comparisons test.
3D contraction assay using BJ-5ta cells Measure contraction in each well by using ImageJ. Specifically, measure the collagen matrix diameter by taking the average of two perpendicular diameter measurements of each matrix. Average these two measurements to calculate the average diameter of the collagen matrix. Then subtract the average diameter of the matrix after contraction (t = 30) from the average diameter before contraction (t = 0) to find the extent of contraction. Determine statistical significance by using a 2way ANOVA test followed by Sidak's multiple comparisons test.

LIMITATIONS
This current protocol has been optimized for the analysis of sphingosine-1-phosphate mediated contraction in NIH3T3 cells in 2D contraction and in BJ-5ta cells in 3D contraction. We have also used this protocol to investigate lysophosphatidic acid (LPA) mediated contraction. The use of another pro-contractile signaling molecule or cell line would have to be further optimized. In addition, NIH3T3 fibroblast cells were not able to be used in the 3D contraction assay, as we experienced issues with polymerization with S1P.

TROUBLESHOOTING
Problem 1 Too many dead cells and cell debris in NIH3T3 cells after treatment with inhibitors ( Figure 7; 2D Contraction assay using NIH3T3 cells, step 2). Following overnight incubation, blots were washed in TBST (Cell Signaling, 3 3 10 min) and incubated with HRPconjugated secondary antibody (1:10,000 dilution) in fresh blocking buffer for 1 h at RT then washed again with TBST. Blots were developed using ECL reagents (Bio-Rad) and the ChemiDoc XRS+ molecular imager.

Potential solution
The NIH3T3 cell culture may not be in good condition. Cells may die if they are overgrown or if nutrients are depleted. Make sure not to overgrow cells and to wait at least 2 passages after thawing before treatment with inhibitors.

Problem 2
The cell collagen solution does not polymerize after 2 days of incubation (Figure 8; 3D contraction assay using BJ-5ta cells, step 10).  . O-GlcNAc controls the sensitivity of human dermal fibroblasts to S1P-mediated contraction in 3D collagen matrices BJ-5ta cells were plated with a collagen solution and allowed to polymerize before being pre-treated with either DMSO or 5SGlcNAc. The matrices were then released from the plate and contraction was initiated by the addition of S1P. Images were taken after release of the matrix then after 30 min of incubation with S1P.

Potential solution
The concentration of cells used in the collagen solution may be too low. Increase the amount of concentration of cells used. In addition, try incubating the cell collagen solution for an additional day, for a total of 3 days, to promote polymerization. Also, collagen is sensitive to changes in pH which can occur due cell death or contamination.

Problem 3
Running out of cell collagen solution when plating out 500 mL into 30 wells (3D contraction assay using BJ-5ta cells, step 9).

Potential solution
Because the solution is very viscous, some volume may be lost during pipetting. Next time, try plating out a smaller volume in each well. For example, plating 450 mL of cell collagen solution should still polymerize properly in the well for the experiment.

Problem 4
Some wells polymerize during the 3D contraction assay and some wells do not (3D contraction assay using BJ-5ta cells, step 10).

Potential solution
This can occur if the cell collagen solution is not mixed thoroughly enough. The concentration of cells will not be homogenous, allowing some wells to polymerize while other wells fail. When creating the cell collagen solution, use a serological pipette controller to pipette up and down and mix the solution well. Do not vortex, and mix carefully to avoid bubbles. Because of the viscosity of the solution, some volume may be lost.

RESOURCE AVAILABILITY
Lead contact Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Matthew Pratt (matthew.pratt@usc.edu).

Materials availability
This protocol did not generate new unique reagents.

Data and code availability
This protocol did not generate datasets or code.