Table 4. Top 20 correlations between GFP and LC3.
Top 20 correlations between GFP and LC3 in transfected cells |
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Plate | Well name | Gene | Median correlation | Standard deviation |
As an alternative readout for finding autophagy substrates/triggers we examined GFP/LC3 correlation. Note that p62 appears close to the top of the list, validating the approach (since autophagy adapters are known to be preferred substrates for autophagy, and to recruit LC3 to auto-aggregates). |
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To control for variation in transfection efficiency between DNA constructs we designed our analysis pipeline to only measure properties of transfected cells exhibiting GFP fluorescence. In this manner we were able to observe genuine signals induced by bacterial protein expression without being swamped by background noise from untransfected cells. The threshold for selecting cells as transfected was determined from samples taken from across untransfected wells, and was designed to use both a median intensity and mean absolute deviation intensity, to avoid under-selection of cells with either diffuse, or highly punctate signals. By this measure transfection efficiency ranged between 40-60% for LF82 genes, and 60% for empty pCMV-mEm-4GS vector. An average of 183 cells were present per site, with 90 successfully transfected cells for LF82 genes and 112 successfully transfected cells for control well sites. | ||||
Set1_Part1 | C09 | LF82_153 | 2.93 | 1.15 |
Set1_Part1 | C08 | LF82_152 | 2.75 | 1.51 |
Set3_Part2 | F08 | LF82_266 | 2.63 | 1.03 |
Set3_Part2 | G09 | p62 | 2.61 | 0.80 |
Set1_Part1 | E05 | LF82_326 | 2.57 | 1.44 |
Set3_Part2 | C05 | LF82_758 | 2.53 | 1.26 |
Set3_Part2 | E08 | LF82_121 | 2.50 | 1.12 |
Set1_Part1 | E06 | LF82_327 | 2.46 | 1.83 |
Set1_Part1 | D09 | LF82_250 | 2.44 | 1.61 |
Set1_Part1 | E04 | LF82_325 | 2.40 | 1.49 |
Set1_Part1 | F11 | LF82_603 | 2.27 | 1.47 |
Set1_Part1 | B08 | LF82_091 | 2.19 | 1.54 |
Set1_Part1 | D08 | LF82_248 | 2.13 | 1.47 |
Set1_Part1 | E09 | LF82_355 | 2.07 | 1.54 |
Set1_Part1 | F07 | LF82_537 | 2.05 | 1.50 |
Set3_Part2 | C06 | LF82_109 | 2.04 | 1.39 |
Set1_Part1 | E07 | LF82_350 | 2.01 | 1.34 |
Set3_Part2 | D05 | LF82_336 | 1.99 | 0.89 |
Set3_Part2 | D04 | LF82_0556 | 1.94 | 1.44 |
Set1_Part1 | F02 | LF82_434 | 1.94 | 1.53 |
Set1_Part1 | E08 | LF82_353 | 1.94 | 1.29 |