1 00:00:00,327 --> 00:00:06,580 In this tutorial, we will arrange the GSDMD and HSP90 band strips into this Figure. 2 00:00:06,580 --> 00:00:15,999 The upper strip contains the 53 and 30 kDa signals. HSP90 is the loading control below, with six matching sample columns. 3 00:00:15,999 --> 00:00:21,880 Open the project backup from the previous tutorial to continue working with these eight original images. 4 00:00:21,880 --> 00:00:28,926 First, select the GSDMD chemiluminescence image from the lower half of the membrane, labeled 1-2. 5 00:00:28,926 --> 00:00:32,618 The horizontal rows of protein signals are bands. 6 00:00:32,618 --> 00:00:37,934 Each vertical column represents one sample. This is called a lane. 7 00:00:37,934 --> 00:00:42,857 We will keep both the 53 and 30 kDa signals in the same crop. 8 00:00:42,857 --> 00:00:47,857 Drag a selection across all six lanes, then adjust the top and bottom edges. 9 00:00:47,857 --> 00:00:52,667 Both signals are now inside the crop, with a little background above and below. 10 00:00:52,667 --> 00:00:57,135 Click “Overlay link” to use the marker when placing molecular-weight labels. 11 00:00:57,135 --> 00:00:59,870 First, enter 53. 12 00:00:59,870 --> 00:01:02,346 Click “Click image to place”. 13 00:01:02,346 --> 00:01:05,494 Place 53 beside the upper signal. 14 00:01:05,494 --> 00:01:09,080 Then enter 30 and place it beside the lower signal. 15 00:01:09,080 --> 00:01:12,143 Return to the chemiluminescence image. 16 00:01:12,143 --> 00:01:15,593 Enter GSDMD as the protein name. 17 00:01:15,593 --> 00:01:17,694 Click “Save strip”. 18 00:01:17,694 --> 00:01:21,178 Both molecular-weight labels are saved with the crop. 19 00:01:21,178 --> 00:01:28,067 Next, select the HSP90 chemiluminescence image from the upper half of the same membrane, labeled 1-1. 20 00:01:28,067 --> 00:01:32,462 Again, select all six lanes and adjust the top and bottom edges. 21 00:01:32,462 --> 00:01:37,582 Enter HSP90 as the protein name, then enter 90 as the molecular weight. 22 00:01:37,582 --> 00:01:41,037 Place 90 beside the HSP90 signal. 23 00:01:41,037 --> 00:01:43,907 Save the HSP90 strip. 24 00:01:43,907 --> 00:01:47,280 Open “Figure builder”, then name the Figure. 25 00:01:47,280 --> 00:01:51,830 Expand “Layout and formatting” and check that the lane count is 6. 26 00:01:51,830 --> 00:01:55,545 Drag HSP90 into the empty strip position. 27 00:01:55,545 --> 00:01:59,872 Click GSDMD, then insert it below HSP90. 28 00:01:59,872 --> 00:02:03,208 Enter Sample as the label for the top row. 29 00:02:03,208 --> 00:02:09,799 Fill the six cells with Sample 1 through Sample 6. 30 00:02:09,799 --> 00:02:13,528 Drag GSDMD above HSP90. 31 00:02:13,528 --> 00:02:19,457 Expand “Layout and formatting” again. We will build an example treatment table. 32 00:02:19,457 --> 00:02:23,025 Select Sample, then add a condition row below it. 33 00:02:23,025 --> 00:02:25,669 Enter Treatment A as the label. 34 00:02:25,669 --> 00:02:29,863 Enter a minus sign in the first cell and a plus sign in the second. 35 00:02:29,863 --> 00:02:33,501 Select the plus sign and press Ctrl+C to copy it. 36 00:02:33,501 --> 00:02:36,454 Drag to select the next four cells. 37 00:02:36,454 --> 00:02:39,064 Press Ctrl+V to paste. 38 00:02:39,064 --> 00:02:42,374 The four selected cells are filled in one step. 39 00:02:42,374 --> 00:02:45,751 Add another condition row and label it Dose. 40 00:02:45,751 --> 00:02:49,535 Copy a minus sign, then fill all six cells with it. 41 00:02:49,535 --> 00:02:53,827 In the third through fifth cells, enter 2, 4, and 8. 42 00:02:53,827 --> 00:02:57,439 Add another condition row and label it Treatment B. 43 00:02:57,439 --> 00:03:02,897 Select the first five cells and paste minus signs, then enter a plus sign in the last cell. 44 00:03:02,897 --> 00:03:07,664 A plus sign means the treatment is present; a minus sign means it is absent. 45 00:03:07,664 --> 00:03:10,667 The numbers show the dose for each sample. 46 00:03:10,667 --> 00:03:13,832 Drag to select all six sample labels. 47 00:03:13,832 --> 00:03:16,954 Set the text angle to 30°. 48 00:03:16,954 --> 00:03:19,994 Select the GSDMD label. 49 00:03:19,994 --> 00:03:24,869 Set the GSDMD font size to 11, make it bold, and center it. 50 00:03:24,869 --> 00:03:29,044 Apply the same formatting to the HSP90 label. 51 00:03:29,044 --> 00:03:31,537 Open “Export preview”. 52 00:03:31,537 --> 00:03:37,054 All six samples, the three treatment rows, and the three molecular-weight labels are visible. 53 00:03:37,054 --> 00:03:40,828 Select PNG, then click “Save PNG”. 54 00:03:40,828 --> 00:03:45,297 Export SVG for further editing, or choose TIFF. 55 00:03:45,297 --> 00:03:48,242 Open the PNG you just saved. 56 00:03:48,242 --> 00:03:52,849 The angled sample labels and molecular-weight labels are preserved. 57 00:03:52,849 --> 00:03:56,808 Click the plus button on the left to create a second Figure. 58 00:03:56,808 --> 00:03:59,303 Name this Figure Treatment. 59 00:03:59,303 --> 00:04:02,893 Set the label column width to 64 pt. 60 00:04:02,893 --> 00:04:05,929 Enter Treatment as the first row label. 61 00:04:05,929 --> 00:04:09,703 Enter Control in the middle cell of the first three columns. 62 00:04:09,703 --> 00:04:13,263 Enter Drug in the middle cell of the last three columns. 63 00:04:13,263 --> 00:04:16,389 Add a row below and label it Replicate. 64 00:04:16,389 --> 00:04:20,310 Fill the first three cells with 1, 2, and 3. 65 00:04:20,310 --> 00:04:24,298 Fill the last three cells with 1, 2, and 3 as well. 66 00:04:24,298 --> 00:04:28,181 Drag GSDMD into the empty strip position. 67 00:04:28,181 --> 00:04:31,914 Insert HSP90 below GSDMD. 68 00:04:31,914 --> 00:04:36,508 Set the Control font size to 11, make it bold, and center it. 69 00:04:36,508 --> 00:04:39,659 Apply the same formatting to Drug. 70 00:04:39,659 --> 00:04:46,219 Select both protein labels. Set their font size to 11, make them bold, and center them. 71 00:04:46,219 --> 00:04:49,572 Drag to select the three cells for the Drug group. 72 00:04:49,572 --> 00:04:54,123 Click “Bottom edge” to add a bottom border to these three cells only. 73 00:04:54,123 --> 00:04:57,260 Open “Export preview” for this Figure. 74 00:04:57,260 --> 00:05:02,999 Control and Drug each have three replicates. The line marks only the Drug group. 75 00:05:02,999 --> 00:05:06,789 Select PNG, then click “Save PNG”. 76 00:05:06,789 --> 00:05:10,415 Save SVG and TIFF versions as well. 77 00:05:10,415 --> 00:05:13,815 Open the PNG of the second Figure. 78 00:05:13,815 --> 00:05:19,480 The group headings, replicate numbers, and molecular-weight labels are all preserved. 79 00:05:19,480 --> 00:05:25,452 Finally, click “Back up project” to save the original images, crops, and both Figures together. 80 00:05:25,452 --> 00:05:31,416 Use this project backup to organize the source data next, or continue editing later.