Sustained intestinal epithelial monolayer wound closure after transient application of a FAK-activating small molecule

Access this dataset on Dryad:  https://doi.org/10.5061/dryad.qnk98sfqp

We compared the effects of shorter M64HCl treatment in vitro to continuous treatment for 24 hours on monolayer wound closure. We then investigated how long FAK activation and downstream ERK1/2 activation persist after two hours of M64HCl treatment in Caco-2 cells. M64HCl concentrations immediately after washing measured by mass spectrometry confirmed that M64HCl had been completely removed from the medium while intracellular concentrations had been reduced by 95%. Three-hour and four-hour M64HCl (100 nM) treatment promoted epithelial sheet migration over 24 hours similar to continuous 24-hour exposure. 100nM M64HCl did not increase cell number. Exposing cells twice with 2-hr exposures of M64HCl during a 24-hour period had a similar effect. Both FAK inhibitor PF-573228 (10 µM) and ERK kinase (MEK) inhibitor PD98059 (20 µM) reduced basal wound closure in the absence of M64HCl, and each completely prevented any stimulation of wound closure by M64HCl. Rho kinase inhibitor Y-27632 (20 µM) stimulated Caco-2 monolayer wound closure, but no further increase was seen with M64HCl in the presence of Y-27632. M64HCl (100 nM) treatment for 3 hours stimulated Rho kinase activity.  M64HCl decreased F-actin in Caco-2 cells. Furthermore, a two-hour treatment with M64HCl (100 nM) stimulated sustained FAK activation and ERK1/2 activation for up to 16 and hours 24 hours, respectively. The following dataset was generated using different experiments such as Ultra-performance liquid chromatography-mass spectrometry (UPLC-MS) analysis for M64HCl concentrations in cells and media, Crystal Violet Assay, Wound closure Assay, Flow Cytometry, Rho kinase activity assay, Immunofluorescence, and Western Blotting.

Dataset For Fig 1

·       M64HCl concentration was measured in the media and within the cells before and after washing

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·       There are 5 conditions measured which are 0h, 2h, AW-0min, AW-30 min, and AW-60 min. There are 3 replicates per each condition. “0h” is the zero hours right after the addition of the 100nM M64HCl concentration media to the cells, “2h” is the 2 hours after the addition of 100nM M64HCl concentration media to the cells, right before washing, “AW-0 min” is measured right after the cells were washed 3 times (1 min each) with PBS, “AW-30 min” is measured after washing and incubating in normal media for 30 minutes, and “AW-60 min” is measured after washing and incubating in normal media for one hour. “nM” is the M64HCl Concentration for each replicate of each condition in nanomolar, “AVE (nM)” is the average concentration of M64HCl for the 3 replicates, “SD” is the standard deviation and “SE” is the Standard Error.

Dataset for Fig 2

·       Caco-2 cells were wounded and treated with H20 or M64HCl (100nM) for 1h, 2h, 3h, 4h, and 24.

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·       The wound areas are measured using Image J at 0h and 24h time points.  “n” = the number of wound replicates, “Wound closure” = 0h-24h wound areas, “% area” = [(0h-24h wound area)/0h wound area] x 100, and “Norm” = the wound closure % area normalized to the average % area of the control (H2O).

·       The summary table contains the n for each treatment condition, consisting of the control (H2o) and each M64HCl time point treatment. “Average” is the average wound closure % area of all the replicates for each treatment, “SD” is the standard deviation for all the replicates for each treatment, “SE” is the standard error, and “TTest” is the P value significance test used.

·       Another summary table was added for the NORM instead of the % area.

Dataset for Fig 3

·       Caco-2 cells were seeded in 12 well plates. Half the wells were treated with H2O (control) and the other half with 100nM M64HCl for 24 hours. The cells were then stained with crystal violet.

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·       The assay was done on 3 plates (A, B, and C), and the crystal violet absorbance was measured.

·       Plate A showed 4 replicates for each treatment and Plates B and C showed 6 replicates.

·       The “Mean control” is the average of all the control replicates in each plate. Each replicate for the M64HCl and the control treatment is normalized to the mean control and then multiplied by 100 to show the percentage.

·       The mean and the standard error for all the replicates for the control and M64HCl in each plate are then calculated. The TTest for p-value significance is also calculated.

·       The data from the 3 plates for each treatment is then compiled into one table where the mean and the standard error are calculated. The TTest is also calculated.

Dataset for Fig 4

·       Caco-2 cells were wounded and treated with H2O (control) or 100nM M64HCl in media without FBS for 24hrs.

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·       The wound closure areas are measured using Image J at 0h and 24h time points. “Wound closure %” = [(0h-24h wound area)/0h wound area] x 100.

·       The values in the tables that show N/A indicate that the wound closure areas are not available due to technical problems with the wound in the assay

·       The summary table shows “n” which is the total number of each wound replicate for each of the two treatments (control and 100nM M64HCl). “Mean” is the average wound closure % for all the replicates for each treatment, and “SE” is the Standard error. TTest is done for the P-value to determine the significance.

Dataset for Fig 5

·       Caco-2 cells were wounded and treated with H2O (control) or M64HCl 100nM different conditions.

·       The first group is wounded and treated with Control (H2O) for 24 hours (H2O- 24h), while the second group is treated with M64HCl 100nM for 24 hours as well (M64HCl-24h).

·       The third group is treated with H2O for 2 hours, washed with PBS three times, and incubated with fresh media for 10 hours. Then treated again with H2O for another two hours, washed, and replaced again with fresh media for 10 hours (H2O(2h+10h) *2). The fourth group is similar to the third one but instead of being treated with H2O, it was treated with 100nM M64HCl. (M64HCl(2h+10h) *2)

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·       The wound areas are measured using Image J at 0h and 24h time points.  “n” = the number of wound replicates, “Wound closure” = 0h-24h wound areas, “% area” = [(0h-24h wound area)/0h wound area] x 100, and “Norm” = the wound closure % area normalized to the average % area of the control (H2O).

·       The summary table contains the n for each treatment condition, consisting of the control (H2O-24h), M64HCl 24h, H2O(2h+10h) *2, and M64-HCl(2h+10h) *2. “Average” is the average wound closure % area of all the replicates for each treatment, “SD” is the standard deviation for all the replicates for each treatment, and “SE” is the standard error.

·       Another summary table was added for the NORM instead of the % area.

Dataset for Fig 6

·       Caco-2 cells were wounded and treated with H2O (control), M64HCl 100nM, PF (FAK inhibitor), PF+M64HCl, PD (ERK inhibitor), PD+M64HCl, Y-27632 (Rho Kinase), Y-27632+M64HCl.

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·       The wound closure areas are measured using Image J at 0h and 24h time points. “Wound closure %” = [(0h-24h wound area)/0h wound area] x 100.

·       The values in the table that show “N/A” indicate that the wound closure areas are not available due to technical problems with the wound in the assay.

Dataset for Fig 7

·       Caco-2 cells were treated with H2O (control) or M64HCl and rho kinase activity was measured

·       The table shows the normalized Rho kinase activity for the control and M64HCl treatment.

Dataset for Fig 8 (A)_062223-F-actin confocal

·       Caco2 cells were stained with Alexa Fluor 647 Phalloidin as a measure of F-actin and DAPI and visualized on a Leica DMi8 Stellaris or Olympus FV3000 confocal microscope. 

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·       Fiji ImageJ (NIH) was used to determine mean fluorescence and the plot profile fluorescence over the “Whole” image. In this file, there are raw data values for the “Area” being measured for the whole image, the “Mean”, “Min”, “Max” grey values, the integrated density (“IntDen”), the raw integrated density and the calculated CTCF which is equal to the IntDen – (Area*(Average (Bkgd 1 Mean +Bkgd 2 Mean + Bkgd 3 Mean/3))).  The CTCF values were used to plot the graph in Fig 8 A.  (The cell 1, cell 2, and cell 3 values were not used in the final graph in Fig 8.)

·       Mean = mean grey value; Min = minimum grey value; Max = maximum grey value; IntDen = integrated density; Raw IntDen = raw integrated density; bkgd = background; whole = whole image; cell = individual cell values. 

Dataset for Fig 8 (B) Intensity over rectangle

·       Caco2 cells were stained with Alexa Fluor 647 Phalloidin as a measure of F-actin and DAPI and visualized on a Leica DMi8 Stellaris or Olympus FV3000 confocal microscope. 

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·       Fiji ImageJ (NIH) was used to determine mean fluorescence and the plot profile fluorescence over the length of a selected rectangle region from 8.701um from the migrating edge to 100 um into the migrating sheet. The fraction of maximum fluorescence was calculated as previously described by Patel18.  Each tab represents one image taken from different samples (eg. 5A or 5B) dash the replicate number image from that slide.  Then within each tab, there were 5 rectangle measurements that were taken (1-1 through 1-5) and a background rectangle measurement that was taken.  Columns A, C, E, G, I, and K are the distance lengths in um where the Gray values were measured.  The Gray values are in columns B, D, F, H, J, and L.  The average background (bkgd) gray value was determined and is in cell L2.  Columns N-R calculates the fraction of maximum fluorescence which is equal to (Gray value – average bkgd gray value) / (4095-average bkgd gray value).  The value 4095 is the maximum gray value measured.  The distance values and the fraction of maximum fluorescence values were then plotted to make the graph in Fig 8B. 

·       Bkgd = background.

Dataset for Fig 9

·       Caco-2 cells were treated with H2O (0h) or 100nM M64HCl for 2 hours and then washed with PBS and collected at different time points after washing (2h, 3h, 4h, 8h,12h,16h, 24h)

·       Western blotting is done for all the different time points. PFAK397 and FAK proteins are analyzed.

·       PFAK397 is normalized to the FAK protein of the same sample in the same blot for each condition in the set and then the PFAK397/FAK for each time point is normalized to the Control (0h)

·       The values in the table that show “N/A” indicate that the data are not available due to technical problems with the sample or the blot.

·       n= the number of replicates for each time point, Ave= the average of all the normalized PFAK397/FAK replicates for each condition, SD= the standard deviation, and SE= the Standard error.

Dataset for Fig 10

·       Caco-2 cells were treated with H2O (0h) or 100nM M64HCl for 2 hours and then washed with PBS and collected at different time points after washing (2h, 3h, 4h, 8h,12h,16h, 24h)

·       Western blotting is done for all the different time points. PERK1/2 and ERK1/2 proteins are analyzed.

·       PERK1/2 is normalized to the ERK1/2 protein of the same sample in the same blot for each condition in the set and then the PERK1/2/ERK1/2 for each time point is normalized to the Control (0h)

·       The values in the table that show “N/A” indicate that the data are not available due to technical problems with the sample or the blot.

·       n= the number of replicates for each time point, Ave= the average of all the normalized PERK1/2/ERK1/2 replicates for each condition, SD= the standard deviation, and SE= the Standard error.

Dataset for Fig 11

·       Caco-2 cells were treated with Control, 100nM M64HCl, PF, PF+100nM M64HCl, PD, PD+100nM M64HCl for 24 hours.

·       Western blotting is done for all the conditions. PFAK397 and FAK proteins are analyzed.

·       PFAK397 is normalized to the FAK protein of the same sample in the same blot for each condition in the set and then the PFAK397/FAK for each condition is normalized to the Control.

·       The values in the table that show “N/A” indicate that the data are not available due to technical problems with the sample or the blot. While N/A highlighted in red indicates that the value was removed for being an outlier.

·       Average is the mean of all the normalized PFAK397/FAK replicates for each condition, SD is the standard deviation, SE is the Standard error, and TTest is the test used to calculate the P-value to check for significance.

Dataset for Fig 12

·       Caco-2 cells were treated with Control, 100nM M64HCl, PF, PF+100nM M64HCl, PD, PD+100nM M64HCl for 24 hours.

·       Western blotting is done for all the conditions. PERK1/2 and ERK1/2 proteins are analyzed.

·       PERK1/2 is normalized to the ERK1/2 protein of the same sample in the same blot for each condition in the set and then the PERK1/2/ERK1/2 for each condition is normalized to the Control.

·       The values in the table that show “N/A” indicate that the data are not available due to technical problems with the sample or the blot.

·       Average is the mean of all the normalized PERK1/2/ERK1/2 replicates for each condition, SD is the standard deviation, SE is the Standard error, and TTest is the test used to calculate the P-value to check for significance.

Dataset for Fig 13

·       Caco-2 cells were treated with Control, 1, PF, or PD for 1 hour.

·       Two samples for each condition were loaded into each Gel.

·       Western blotting is done for all the conditions. PERK1/2 and ERK1/2 proteins are analyzed.

·       We added the two PERK1/2 replicates for each condition on the same blot to each other. We did this for each of the 3 conditions (Control, PF, PD)

·       We added the two ERK1/2 replicates for each condition on the same blot to each other. We did this for each of the 3 conditions (Control, PF, PD)

·       The summation of the two PERK1/2 replicates for each condition on the same blot is normalized to the summation of the two ERK1/2 replicates for each condition on the same blot as well. This has been done for all the 10 blots.

·       The PERK1/2/ERK1/2 for each condition is then normalized to the Control.

·       The values in the table that show “N/A” indicate that the data are not available due to technical problems with the sample or the blot.

·       Average is the mean of all the normalized PERK1/2/ERK1/2 replicates for each condition, SD is the standard deviation, SE is the Standard error, and TTest is the test used to calculate the P-value to check for significance.