In “AAV-mediated gene therapy for Choroideremia: Preclinical studies in personalized
in vitro models”, by Vasireddy et al (DOI: 10.1371/journal.pone.0061396), two panels
in Fig 1 contained immunoblot images that were spliced together by the authors for
the sake of re-ordering lanes and omitting unnecessary data on the published figure.
The authors apologize for not acknowledging these manipulations in figure preparation
in the original Figure legend.
In the corrected version of Fig 1, we present the same data as those published in
the original article. However, panels (IIi) and (III) have been modified so that black,
vertical lines clearly indicate where lanes have been spliced together during figure
preparation. To demonstrate the validity of the data in the affected panels, we present
the original gel images in Supporting Figs 1 and 2. The figure legends clearly indicate
which lanes from the original gels are included in the published figure. See the original
article for detailed Methods and a discussion of the Results. We have also replicated
the Fig 1 immunoblot experiments in full (including the transfections and lysate preparations)
but placed the samples in the correct order in the new gels. Those unmanipulated blot
images are presented as Fig 2 of this Correction.
10.1371/journal.pone.0129982.g001
Fig 1
Generation and Characterization of AAV2.
hCHM. I). Schematic of the AAV proviral plasmid carrying human CHM. under the control
of the cytomegalovirus enhancer chicken beta actin (eCBA) promoter. ITR: Inverted
terminal repeats; Ori: Replication origin; KanR: Kanamycin resistance gene. II) i)
Immunoblot and ii) fluorescent analysis reveals REP-1 protein in CHO cells transfected
with pAAV2.hCHM. Lane A: Transfected cell (25 μg protein), B: Control (untransfected)
cells, C- protein marker. Immunocytochemical analysis revealed the localization of
REP-1 to the cytosolic region (II-ii-B; Green). No REP1 is observed in control cells
(II-ii-A). Nuclei are stained with DAPI and appear blue. Scale bar is 50 μM. III)
Immunoblot analysis of CHO cells infected with 1E3-2E5 viral genomes (vg) of AAV2.
hCHM shows an increase in REP-1 protein (indicated by arrow) proportional to the titer.
Positive (+ve) control: pAAV2. hCHM-transfected CHO cell lysate. Black lines in panels
IIi and III indicate where lanes from original immunoblots have been spliced together
during figure preparation (original legend is reproduced from the published PLOS ONE
article).
10.1371/journal.pone.0129982.g002
Fig 2
Revised Fig 1, with additional immunoblots made using a complete replication of the
experiment described in the original Fig 1 (including the transfections and lysate
preparations) but with samples loaded in the correct consecutive order on the gel.
I). Schematic of the AAV proviral plasmid carrying human CHM under the control of
the cytomegalovirus enhancer chicken beta actin (eCBA) promoter. ITR: Inverted terminal
repeats; Ori: Replication origin; KanR: Kanamycin resistance gene. II) i) Immunoblot
and ii) fluorescent analysis reveals REP-1 protein in CHO cells transfected with pAAV2.hCHM.
Lane A: Transfected cell (25 μg protein), B: Control (untransfected) cells, C- protein
marker (SeeBlue Plus2, Invitrogen, Grand Island, NY). Immunocytochemical analysis
revealed the localization of REP-1 to the cytosolic region (II-ii-B; Green). No REP1
is observed in control cells (II-ii-A). Nuclei are stained with DAPI and appear blue.
Scale bar is 50 μM. III) Immunoblot analysis of CHO cells infected with 1E3-2E5 viral
genomes (vg) of AAV2. hCHM (lanes 2–5) show an increase in REP-1 protein (indicated
by arrow) proportional to the titer. Lane 1 is a negative control containing lysate
from uninfected CHO cells. Positive (+ve) controls: pAAV2. hCHM-transfected CHO cell
lysates (lanes 9, 10). Lanes 6 and 8 were not loaded. Lane 7 contains the SeeBlue
Plus 2 protein marker.
Supporting Information
S1 Fig
Raw, unaltered immunoblot image (using the human REP-1-specific antibody, antibody,
2F1) used to make Fig 1IIi.
Lane 12 contains a protein marker (SeeBlue Plus2, Invitrogen, Grand Island, NY), lanes
2–6 and 9–10 show results of loading 25 μg of CHO cell lysate after replicate transfections
with pAAV2.hCHM, and lane 1 was blank. Lanes 7, 8 and 11 show untreated control CHO
cell lysates. Lysates from cells that were floating after transfection are shown in
lanes 3 and 6. **Lanes 4, 7, and 12 were presented in Fig 1IIi lanes A, B, and C,
respectively.
(TIF)
Click here for additional data file.
S2 Fig
Raw, unaltered immunoblot image (using the human REP-1-specific antibody, antibody,
2F1) used to make Fig 1III.
Lanes 1, 3, 4, and 6 contained lysates of CHO cells infected with 1E4, 1E5, 2E5 and
1E3 vg of AAV2.hCHM, respectively. Lane 5 is a positive control (pAAV2.hCHM-transfected
CHO cell lysate). Lane 2 was not used and lane 7 was lysate from untreated CHO cells.
Lanes 8 and 9 contained samples from an unrelated experiment and lane 10 contained
the SeeBlue Plus2 protein marker. **Lanes 6, 1, 3, 4, 5, 7, 10 were presented in Fig
1III lanes 1, 2, 3, 4, 5, 6, 7, respectively in order to present the immunoblot results
according to increase in AAV2.hCHM titer. The irrelevant lanes (original lanes 2,
8, and 9) were not shown in the original Fig 1III.
(TIF)
Click here for additional data file.