Fig 1.
Expression of AGAT and GAMT in HEK293T cells.
(A) HEK293T cells were transfected with the indicated plasmid. Cells were then lysed and analyzed by western blotting with the indicated antibody. N = 3. (B) Same lysates as in (A) were probed for the V5 tagged form of AGAT and GAMT by western blot. N = 3. (C) Cells transfected as in (A) were cultured in creatine-free media supplemented with amino acid precursors for 24 hours. Cells were lysed and the total cellular creatine was quantified. N = 4. Two-sided t-test * indicates P < 0.05. (D) Same experiment as in (C) plotting the total cellular protein measured. N = 4. n.s. indicates not significant for one-way ANOVA. Error bars show SEM.
Fig 2.
Total cellular expression of AGAT and GAMT in WT fibroblasts.
(A) WT control fibroblasts were infected with the indicated lentivirus. Cells were then lysed and analyzed by western blotting with the indicated antibody. N = 3. (B) Same lysates as in (A) were probed for the V5 tagged form of AGAT and GAMT by western blot. N = 3. (C) WT fibroblasts stably expressing the indicated transgenes were cultured in creatine-free media supplemented with amino acid precursors for 3 days. Cells were lysed and the total cellular creatine was quantified. N = 3. Two-sided t-test * indicates P < 0.05. (D) Same experiment as in (C) plotting the media creatine concentration measured. N = 3. Two-sided t-test ** indicates P < 0.01. (E) Same experiment as in (C) plotting the total cellular protein measured. N = 3. Two-sided t-test n.s. indicates not significant. Error bars show SEM.
Fig 3.
Localization of delivered AGAT and GAMT in WT fibroblasts.
Immunofluorescence (IF) of WT fibroblasts stably expressing AGAT + GAMT and empty vector infected controls imaged at 63x magnification. N = 4. Scale bars indicate 10 µm.
Fig 4.
Total cellular expression of AGAT and GAMT in CT1 mutant fibroblasts.
(A) Fibroblasts from a patient with CT1 loss-of-function (SLC6A8Δex10-11/y) were infected with the indicated lentivirus. Cells were then lysed and analyzed by western blotting with the indicated antibody. N = 3. (B) Same lysates as in (A) were probed for the V5 tagged form of AGAT and GAMT by western blot. N = 3. (C) The same CT1 loss-of-function fibroblasts stably expressing the indicated transgenes were cultured in creatine-free media supplemented with amino acid precursors for three days. Cells were lysed and the total cellular creatine was quantified. N = 3. Two-sided t-test P = 0.06. (D) Same experiment as in (C) plotting the media creatine concentration measured. N = 2. Two-sided t-test n.s. indicates not significant. (E) Same experiment as in (C) plotting the total cellular protein measured. N = 3. Two-sided t-test n.s. indicates not significant. Error bars show SEM. (F) A second line of CT1 loss-of-function fibroblasts (SLC6A8W556X/y) stably expressing the indicated transgenes were cultured in creatine-free media supplemented with amino acid precursors for three days. Cells were lysed and the total cellular creatine was quantified. N = 3. Two-sided t-test * indicates P < 0.05. (G) Same experiment as in (F) plotting the media creatine concentration measured. N = 3. Two-sided t-test * indicates P < 0.05. (H) Same experiment as in (F) plotting the total cellular protein measured. N = 3. Two-sided t-test n.s. indicates not significant. Error bars show SEM.
Fig 5.
Localization of delivered AGAT and GAMT in CT1 mutant (SLC6A8
Δex10-11/y) fibroblasts. IF of CT1 loss-of-function patient fibroblasts stably expressing AGAT + GAMT and empty vector infected controls imaged at 63x magnification. N = 4. Scale bars indicate 10 µm.
Fig 6.
Localization of delivered AGAT and GAMT in CT1 mutant (SLC6A8W556X/y) fibroblasts.
IF of a second line of CT1 loss-of-function patient fibroblasts stably expressing AGAT + GAMT and empty vector infected controls imaged at 63x magnification. N = 4. Scale bars indicate 10 µm.
Fig 7.
Growth characteristics of fibroblasts stably expressing AGAT and GAMT.
(A) A model depicting the two sources of intracellular creatine. Creatine is either imported via CT1 or synthesized from its amino acid precursors: L-arginine; glycine; and methionine. The latter is converted into SAM via MAT2. Created with BioRender.com. (B) Growth of WT fibroblasts stably expressing the indicated constructs in standard media (DMEM + 15% FBS + P/S). N = 6. Two-sided K-S test n.s. indicates not significant. (C) Growth of CT1 loss-of-function (SLC6A8Δex10-11/y) patient fibroblasts stably expressing the indicated constructs in standard media. N = 6. Two-sided K-S test **** indicates P < 0.0001. (D) Similar experiment to (C) except media was supplemented with creatine precursor amino acids to 1 mM above standard media. N = 6. Two-sided K-S test *** indicates P < 0.001. (E) Growth of CT1 loss-of-function (SLC6A8W556X/y) patient fibroblasts stably expressing the indicated constructs in standard media. N = 6. Two-sided K-S test * indicates P < 0.05. (F) Similar experiment to (E) except media was supplemented with creatine precursor amino acids. N = 6. Two-sided K-S test **** indicates P < 0.0001. Lines indicate a LOESS curve fit to the mean at each timepoint. Error bars show mean + /- SEM.