Fig 1.
Mycoplasma detection and removal.
(A) Top: Representative Western blot of InsP3R3 and PC2 expression after shRNA knockdown. Lanes to the left represent control; lanes to the right represent the knockdown condition. Actin was used as a loading control. Each lane represents a separate sample. Bottom: Quantified analysis of Western Blot. (B) Mycoplasma contamination was detected by PCR before (left lanes), but not after (right lanes) Plasmocin treatment. (C) Mycoplasma was removed from cell lines over 2 weeks by treatment with Plasmocin every other day. Following treatment, the cells lines were grown in antibiotic free media for an additional 2 weeks to promote the growth of any remaining mycoplasma.
Fig 2.
Cell proliferation in 2D cell cultures.
Mycoplasma positive and mycoplasma-negative cells were grown for 48 hours and assayed for cell proliferation with an MTT assay. Data represents an average of 8 replicates and the error bars represent the standard error of the mean. The respective p values are: LLC-PK1 = 0.996, InsP3R3 KD = 0.137, and PC2 KD = 0.76.
Fig 3.
Cyst formation is improved in 3D tissues without mycoplasma contamination.
Mycoplasma positive and mycoplasma-negative cells were grown in 3D tissues for 2 weeks at which time the tissues were fixed, sectioned, and stained for H&E. Note that the cysts are more diffuse in the mycoplasma-positive tissues. Scale bars = 200 μm.
Fig 4.
Cyst number is increased in 3D tissues without mycoplasma contamination.
(A) Mycoplasma positive and mycoplasma-negative cells were grown in 3D hydrogels for 2 weeks at which time the tissues were fixed and stained with Carmine for whole mount imaging. Note that cysts are more numerous in the mycoplasma negative tissues. Scale bars = 100 μm. (B) Quantified number of cysts across 4 different quadrants. (C) The diameter of the cysts is altered upon treatment of mycoplasma contamination. Data represents the average diameter and the error bars represent the standard error of the mean. * represents p>0.05, ** p>0.01, ***p>0.001, ****p>0.0001
Fig 5.
Mycoplasma-positive cysts have fewer proliferative cells.
(A). Cysts after 2 weeks in 3D cell culture were stained for an antibody against Ki-67, a marker of cell proliferation (green). Sections were counterstained with actin (using phalloidin red) and DAPI (to denote nuclei, blue). (B) Analysis of the number of cells (as assess by total number of nuclei) per cyst in a cross-sectional area. Analysis is the average of at least 2 different images from three different cyst preparations for each condition, and represents between 9–12 cysts. (C) Absolute number of Ki-67 cells per cyst. Analyses in B and C are the average of at least 2 images from three different cyst preparations for each condition, and represents between 9–12 cysts.
Fig 6.
Mycoplasma positive cysts lack organized tight junctions.
Cysts were stained for the tight junctional protein, ZO-1 (green), actin (using phalloidin, red), and nuclei (blue) after 2 weeks of 3D culture. Note that ZO-1 expression is reduced and actin is unorganized in mycoplasma positive cultures. Images are representative of at least two different at least 2 images from three different cyst preparations for each condition. Scale bars = 10 μm.