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PMC12738729
PHLDA1 silencing in IMR-32 human neuroblastoma cells results in ABCB1 overexpression, augments chemoresistance and leads to increased growth of tumors
PHLDA1-silenced (shP) and control (shC) IMR-32 cells were treated with 30 nM doxorubicin (dox) or water (Ø) for 48 h, protein lysates were subsequently obtained, and western blot analysis was performed in three independent experiments.
PMC12738729
PHLDA1 silencing in IMR-32 human neuroblastoma cells results in ABCB1 overexpression, augments chemoresistance and leads to increased growth of tumors
Representative immunoblots are shown (*- unspecific band).
PMC12738729
PHLDA1 silencing in IMR-32 human neuroblastoma cells results in ABCB1 overexpression, augments chemoresistance and leads to increased growth of tumors
An ATP luminescence test was performed to confirm the cytotoxic effect of doxorubicin (c).
PMC12738729
PHLDA1 silencing in IMR-32 human neuroblastoma cells results in ABCB1 overexpression, augments chemoresistance and leads to increased growth of tumors
The number of control cells treated with water was set to 1.
PMC12738729
PHLDA1 silencing in IMR-32 human neuroblastoma cells results in ABCB1 overexpression, augments chemoresistance and leads to increased growth of tumors
The samples were run in triplicate.
PMC12738729
PHLDA1 silencing in IMR-32 human neuroblastoma cells results in ABCB1 overexpression, augments chemoresistance and leads to increased growth of tumors
The data are shown as the means (± SEM) of three independent experiments.
PMC12738729
PHLDA1 silencing in IMR-32 human neuroblastoma cells results in ABCB1 overexpression, augments chemoresistance and leads to increased growth of tumors
Statistical significance was assessed by one-way ANOVA, followed by post hoc Tukey’s test (*p < 0.05, exact p = 0.022 for shP water vs. shC water; ***p < 0.001, exact p = 0.00043 for shC water vs. shC doxorubicin; p = 0.000024 for shP doxorubicin vs. shC doxorubicin).
PMC12738729
PHLDA1 silencing in IMR-32 human neuroblastoma cells results in ABCB1 overexpression, augments chemoresistance and leads to increased growth of tumors
Another functional test, an indirect test of ABCB1 protein activity, involved incubating shP and shC cells with doxorubicin (Fig. 2b, c; Supplementary Fig. S7-S10 online).
PMC12738729
PHLDA1 silencing in IMR-32 human neuroblastoma cells results in ABCB1 overexpression, augments chemoresistance and leads to increased growth of tumors
The P21 protein is strongly elevated in many types of cells exposed to doxorubicin, including human IMR-32 neuroblastoma cells.
PMC12738729
PHLDA1 silencing in IMR-32 human neuroblastoma cells results in ABCB1 overexpression, augments chemoresistance and leads to increased growth of tumors
Therefore, in this test, the protein served as a marker for the cellular response to this chemotherapeutic agent.
PMC12738729
PHLDA1 silencing in IMR-32 human neuroblastoma cells results in ABCB1 overexpression, augments chemoresistance and leads to increased growth of tumors
PHLDA1-silenced cells (shP) and control cells (shC) were incubated with complete medium containing 30 nM doxorubicin (or water as the drug diluent) for 48 h, after which the levels of P21, ABCB1 and PHLDA1 were determined (Fig. 2b).
PMC12738729
PHLDA1 silencing in IMR-32 human neuroblastoma cells results in ABCB1 overexpression, augments chemoresistance and leads to increased growth of tumors
Doxorubicin induced high P21 expression solely in shC cells (Fig. 2b).
PMC12738729
PHLDA1 silencing in IMR-32 human neuroblastoma cells results in ABCB1 overexpression, augments chemoresistance and leads to increased growth of tumors
Additionally, a significant reduction in ATP levels, by more than 50%, was detected in the doxorubicin-treated shC cells, indicating a strong cytotoxic effect of the chemotherapeutic agent in the control IMR-32 cells (Fig. 2c; Supplementary Fig. S10 online).
PMC12738729
PHLDA1 silencing in IMR-32 human neuroblastoma cells results in ABCB1 overexpression, augments chemoresistance and leads to increased growth of tumors
In contrast, the relative ATP levels in shP cells after doxorubicin treatment remained unchanged, indicating that PHLDA1-silenced cells gained resistance to doxorubicin.
PMC12738729
PHLDA1 silencing in IMR-32 human neuroblastoma cells results in ABCB1 overexpression, augments chemoresistance and leads to increased growth of tumors
These findings indicate that the ABCB1 efflux pump operates efficiently in PHLDA1-silenced cells.
PMC12738729
PHLDA1 silencing in IMR-32 human neuroblastoma cells results in ABCB1 overexpression, augments chemoresistance and leads to increased growth of tumors
Furthermore, we aimed to investigate the response of CHP-134 cells in which PHLDA1 was silenced to doxorubicin to examine whether a similar regulatory effect could be observed in yet another neuroblastoma cell line.
PMC12738729
PHLDA1 silencing in IMR-32 human neuroblastoma cells results in ABCB1 overexpression, augments chemoresistance and leads to increased growth of tumors
To assess the cytotoxicity of doxorubicin following PHLDA1 silencing in CHP134, the intracellular ATP content was again measured.
PMC12738729
PHLDA1 silencing in IMR-32 human neuroblastoma cells results in ABCB1 overexpression, augments chemoresistance and leads to increased growth of tumors
The decreased cytotoxicity of doxorubicin was observed following PHLDA1 silencing in the S6 and S17 clones of CHP-134 neuroblastoma cells compared with that in the control cells, and a statistically significant decrease in ATP levels was detected only at the highest concentration tested (150 nM) (Supplementary Fig. S11 online).
PMC12738729
PHLDA1 silencing in IMR-32 human neuroblastoma cells results in ABCB1 overexpression, augments chemoresistance and leads to increased growth of tumors
Finally, IMR-32 cells overexpressing PHLDA1, after stable transfection with a plasmid vector, presented lower ABCB1 levels, which correlated with their increased susceptibility to 30 nM doxorubicin (Supplementary Fig. S12, S13 online).
PMC12738729
PHLDA1 silencing in IMR-32 human neuroblastoma cells results in ABCB1 overexpression, augments chemoresistance and leads to increased growth of tumors
Therefore, we can conclude that PHLDA1 affected levels of the ABCB1 transporter in IMR-32 and CHP-134 neuroblastoma cells, which impacted on their sensitivity to doxorubicin.
PMC12738729
PHLDA1 silencing in IMR-32 human neuroblastoma cells results in ABCB1 overexpression, augments chemoresistance and leads to increased growth of tumors
Athymic nude mice were injected subcutaneously (s.c.)
PMC12738729
PHLDA1 silencing in IMR-32 human neuroblastoma cells results in ABCB1 overexpression, augments chemoresistance and leads to increased growth of tumors
with shP or shC human neuroblastoma IMR-32 cells to determine the impact of PHLDA1 silencing on xenograft tumor growth and morphology.
PMC12738729
PHLDA1 silencing in IMR-32 human neuroblastoma cells results in ABCB1 overexpression, augments chemoresistance and leads to increased growth of tumors
The silencing of PHLDA1 was monitored before injecting the cells into the mice (Supplementary Fig. S14, S15 online).
PMC12738729
PHLDA1 silencing in IMR-32 human neuroblastoma cells results in ABCB1 overexpression, augments chemoresistance and leads to increased growth of tumors
Tumors derived from shP cells showed many differences compared to those from shC cells.
PMC12738729
PHLDA1 silencing in IMR-32 human neuroblastoma cells results in ABCB1 overexpression, augments chemoresistance and leads to increased growth of tumors
Macroscopically, tumors derived from shC cells presented light pink coloration, whereas tumors derived from shP cells presented intensely red coloration, indicating a greater blood supply and extravasation (Fig. 3a; Supplementary Fig. S16 online).
PMC12738729
PHLDA1 silencing in IMR-32 human neuroblastoma cells results in ABCB1 overexpression, augments chemoresistance and leads to increased growth of tumors
Indeed, numerous extravasations were observed in tumors derived from shP cells, which were absent in tumors derived from shC cells (Supplementary Fig. S17 online).
PMC12738729
PHLDA1 silencing in IMR-32 human neuroblastoma cells results in ABCB1 overexpression, augments chemoresistance and leads to increased growth of tumors
Another observed difference was that the cells in the shP tumors were packed thicker than those in the control tumors were (Figs. 3c and 4; Supplementary Fig. S17, S18 online).
PMC12738729
PHLDA1 silencing in IMR-32 human neuroblastoma cells results in ABCB1 overexpression, augments chemoresistance and leads to increased growth of tumors
Neuroblastoma tumors with PHLDA1 silencing tended to have greater masses than tumors derived from control cells did (Fig. 3b; Supplementary Table S1a, b online).
PMC12738729
PHLDA1 silencing in IMR-32 human neuroblastoma cells results in ABCB1 overexpression, augments chemoresistance and leads to increased growth of tumors
This change was clearly visible, although it was statistically significant only for the means of one of the experiments.
PMC12738729
PHLDA1 silencing in IMR-32 human neuroblastoma cells results in ABCB1 overexpression, augments chemoresistance and leads to increased growth of tumors
Microscopic observations revealed that neuroblastoma tumors derived from implanted shP and shC cells display the typical packed, small round cell tumor architecture, which is characteristic of NB tumors (Fig. 3c; Supplementary Fig. S17 online).
PMC12738729
PHLDA1 silencing in IMR-32 human neuroblastoma cells results in ABCB1 overexpression, augments chemoresistance and leads to increased growth of tumors
They exhibited a nonhomogeneous structure.
PMC12738729
PHLDA1 silencing in IMR-32 human neuroblastoma cells results in ABCB1 overexpression, augments chemoresistance and leads to increased growth of tumors
These studies demonstrated that PHLDA1 silencing significantly affected the morphology of neuroblastoma tumors.
PMC12738729
PHLDA1 silencing in IMR-32 human neuroblastoma cells results in ABCB1 overexpression, augments chemoresistance and leads to increased growth of tumors
The cells within the shP tumors also had more morphologically altered nuclei, indicating frequent mitotic disruptions.
PMC12738729
PHLDA1 silencing in IMR-32 human neuroblastoma cells results in ABCB1 overexpression, augments chemoresistance and leads to increased growth of tumors
Apoptotic cells, identified on the basis of morphological features and the expression of cleaved PARP-1 and cleaved caspase-3, were statistically significantly more common in shC tumors than in shP tumors (Fig. 3c, d; Supplementary Fig. S19, S20 online; Supplementary Table S2a-f online).
PMC12738729
PHLDA1 silencing in IMR-32 human neuroblastoma cells results in ABCB1 overexpression, augments chemoresistance and leads to increased growth of tumors
This finding was consistent with our in vitro observations, where more dead cells were present in the microscopic field of view in the shC group during trypan blue staining for cell counting (for passages and experiments).
PMC12738729
PHLDA1 silencing in IMR-32 human neuroblastoma cells results in ABCB1 overexpression, augments chemoresistance and leads to increased growth of tumors
Fig. 3Impact of PHLDA1 gene silencing on NB tumors grown in vivo in immunocompromised mice.
PMC12738729
PHLDA1 silencing in IMR-32 human neuroblastoma cells results in ABCB1 overexpression, augments chemoresistance and leads to increased growth of tumors
Photographic images of xenograft neuroblastoma tumors grown from PHLDA1-silenced (shP) and control cells (shC) in the second experiment (a).
PMC12738729
PHLDA1 silencing in IMR-32 human neuroblastoma cells results in ABCB1 overexpression, augments chemoresistance and leads to increased growth of tumors
Photographs of tumors from two complete experiments are presented in Supplementary Fig. S16 online.
PMC12738729
PHLDA1 silencing in IMR-32 human neuroblastoma cells results in ABCB1 overexpression, augments chemoresistance and leads to increased growth of tumors
Comparison of the weights of extracted shC and shP tumors in the first and second experiments (b).
PMC12738729
PHLDA1 silencing in IMR-32 human neuroblastoma cells results in ABCB1 overexpression, augments chemoresistance and leads to increased growth of tumors
The data are presented as the means of 8 tumors per group (± SEM).
PMC12738729
PHLDA1 silencing in IMR-32 human neuroblastoma cells results in ABCB1 overexpression, augments chemoresistance and leads to increased growth of tumors
The exact tumor weights are shown in the Supplementary Table S1a, b online.
PMC12738729
PHLDA1 silencing in IMR-32 human neuroblastoma cells results in ABCB1 overexpression, augments chemoresistance and leads to increased growth of tumors
Statistical significance between the groups was measured with two-tailed Student’s t tests for independent samples. ***
PMC12738729
PHLDA1 silencing in IMR-32 human neuroblastoma cells results in ABCB1 overexpression, augments chemoresistance and leads to increased growth of tumors
p < 0.001, exact p = 0.00016; ns – not statistically significant.
PMC12738729
PHLDA1 silencing in IMR-32 human neuroblastoma cells results in ABCB1 overexpression, augments chemoresistance and leads to increased growth of tumors
Microscopy images of xenograft neuroblastoma tumors grown from shP and shC cells in immunocompromised mice.
PMC12738729
PHLDA1 silencing in IMR-32 human neuroblastoma cells results in ABCB1 overexpression, augments chemoresistance and leads to increased growth of tumors
The tissue slices were stained with Meyer’s hematoxylin and eosin (left column), cleaved PARP-1 (middle column), and cleaved caspase-3 (right column) (c).
PMC12738729
PHLDA1 silencing in IMR-32 human neuroblastoma cells results in ABCB1 overexpression, augments chemoresistance and leads to increased growth of tumors
Enlarged fragments of the images containing apoptotic cells identified in HE-stained sections on the basis of morphological features, cleaved PARP-1- and cleaved caspase-3-positive cells are indicated with white rectangles and are shown as inserts.
PMC12738729
PHLDA1 silencing in IMR-32 human neuroblastoma cells results in ABCB1 overexpression, augments chemoresistance and leads to increased growth of tumors
Scale bars, 100 μm.
PMC12738729
PHLDA1 silencing in IMR-32 human neuroblastoma cells results in ABCB1 overexpression, augments chemoresistance and leads to increased growth of tumors
Mean (± SEM) number of apoptotic cells in the shC and shP groups detected on the basis of morphological features via HE staining, cleaved PARP-1, and cleaved caspase-3 immunostaining, respectively (see Supplementary Table S2a-f online for the counting results from a given tumor sample).
PMC12738729
PHLDA1 silencing in IMR-32 human neuroblastoma cells results in ABCB1 overexpression, augments chemoresistance and leads to increased growth of tumors
Statistical significance was assessed via the two-tailed unpaired Student’s t test for independent samples, *p < 0.05, exact p = 0.043 for apoptotic cells; ***p < 0.001, exact p = 9.05 × 10 for cleaved PARP-1-positive cells; ***p < 0.001, exact p = 1.7 × 10 for cleaved caspase-3-positive cells (d).
PMC12738729
PHLDA1 silencing in IMR-32 human neuroblastoma cells results in ABCB1 overexpression, augments chemoresistance and leads to increased growth of tumors
Impact of PHLDA1 gene silencing on NB tumors grown in vivo in immunocompromised mice.
PMC12738729
PHLDA1 silencing in IMR-32 human neuroblastoma cells results in ABCB1 overexpression, augments chemoresistance and leads to increased growth of tumors
Photographic images of xenograft neuroblastoma tumors grown from PHLDA1-silenced (shP) and control cells (shC) in the second experiment (a).
PMC12738729
PHLDA1 silencing in IMR-32 human neuroblastoma cells results in ABCB1 overexpression, augments chemoresistance and leads to increased growth of tumors
Photographs of tumors from two complete experiments are presented in Supplementary Fig. S16 online.
PMC12738729
PHLDA1 silencing in IMR-32 human neuroblastoma cells results in ABCB1 overexpression, augments chemoresistance and leads to increased growth of tumors
Comparison of the weights of extracted shC and shP tumors in the first and second experiments (b).
PMC12738729
PHLDA1 silencing in IMR-32 human neuroblastoma cells results in ABCB1 overexpression, augments chemoresistance and leads to increased growth of tumors
The data are presented as the means of 8 tumors per group (± SEM).
PMC12738729
PHLDA1 silencing in IMR-32 human neuroblastoma cells results in ABCB1 overexpression, augments chemoresistance and leads to increased growth of tumors
The exact tumor weights are shown in the Supplementary Table S1a, b online.
PMC12738729
PHLDA1 silencing in IMR-32 human neuroblastoma cells results in ABCB1 overexpression, augments chemoresistance and leads to increased growth of tumors
Statistical significance between the groups was measured with two-tailed Student’s t tests for independent samples. ***
PMC12738729
PHLDA1 silencing in IMR-32 human neuroblastoma cells results in ABCB1 overexpression, augments chemoresistance and leads to increased growth of tumors
p < 0.001, exact p = 0.00016; ns – not statistically significant.
PMC12738729
PHLDA1 silencing in IMR-32 human neuroblastoma cells results in ABCB1 overexpression, augments chemoresistance and leads to increased growth of tumors
Microscopy images of xenograft neuroblastoma tumors grown from shP and shC cells in immunocompromised mice.
PMC12738729
PHLDA1 silencing in IMR-32 human neuroblastoma cells results in ABCB1 overexpression, augments chemoresistance and leads to increased growth of tumors
The tissue slices were stained with Meyer’s hematoxylin and eosin (left column), cleaved PARP-1 (middle column), and cleaved caspase-3 (right column) (c).
PMC12738729
PHLDA1 silencing in IMR-32 human neuroblastoma cells results in ABCB1 overexpression, augments chemoresistance and leads to increased growth of tumors
Enlarged fragments of the images containing apoptotic cells identified in HE-stained sections on the basis of morphological features, cleaved PARP-1- and cleaved caspase-3-positive cells are indicated with white rectangles and are shown as inserts.
PMC12738729
PHLDA1 silencing in IMR-32 human neuroblastoma cells results in ABCB1 overexpression, augments chemoresistance and leads to increased growth of tumors
Scale bars, 100 μm.
PMC12738729
PHLDA1 silencing in IMR-32 human neuroblastoma cells results in ABCB1 overexpression, augments chemoresistance and leads to increased growth of tumors
Mean (± SEM) number of apoptotic cells in the shC and shP groups detected on the basis of morphological features via HE staining, cleaved PARP-1, and cleaved caspase-3 immunostaining, respectively (see Supplementary Table S2a-f online for the counting results from a given tumor sample).
PMC12738729
PHLDA1 silencing in IMR-32 human neuroblastoma cells results in ABCB1 overexpression, augments chemoresistance and leads to increased growth of tumors
Statistical significance was assessed via the two-tailed unpaired Student’s t test for independent samples, *p < 0.05, exact p = 0.043 for apoptotic cells; ***p < 0.001, exact p = 9.05 × 10 for cleaved PARP-1-positive cells; ***p < 0.001, exact p = 1.7 × 10 for cleaved caspase-3-positive cells (d).
PMC12738729
PHLDA1 silencing in IMR-32 human neuroblastoma cells results in ABCB1 overexpression, augments chemoresistance and leads to increased growth of tumors
The xenograft shP tumors were stiff, possibly because of hemorrhages within them and the presence of extracellular matrix (ECM) abnormalities.
PMC12738729
PHLDA1 silencing in IMR-32 human neuroblastoma cells results in ABCB1 overexpression, augments chemoresistance and leads to increased growth of tumors
In that context, the collagen network was examined in shP and shC cells.
PMC12738729
PHLDA1 silencing in IMR-32 human neuroblastoma cells results in ABCB1 overexpression, augments chemoresistance and leads to increased growth of tumors
Masson-Goldner and van Gieson trichrome staining revealed that tumors derived from shP cells contained more collagen fibers and that the fibers were denser than the fibers of tumors derived from shC cells were (Fig. 4; Supplementary Fig. S18 online).
PMC12738729
PHLDA1 silencing in IMR-32 human neuroblastoma cells results in ABCB1 overexpression, augments chemoresistance and leads to increased growth of tumors
Fig. 4PHLDA1 silencing augments the collagen net density within tumor tissue.
PMC12738729
PHLDA1 silencing in IMR-32 human neuroblastoma cells results in ABCB1 overexpression, augments chemoresistance and leads to increased growth of tumors
Microscopy images of xenograft neuroblastoma tumors grown from PHLDA1-silenced (shP) and control cells (shC) in immunocompromised mice.
PMC12738729
PHLDA1 silencing in IMR-32 human neuroblastoma cells results in ABCB1 overexpression, augments chemoresistance and leads to increased growth of tumors
The tissue slices were stained with Masson-Goldner trichrome or van Gieson trichrome.
PMC12738729
PHLDA1 silencing in IMR-32 human neuroblastoma cells results in ABCB1 overexpression, augments chemoresistance and leads to increased growth of tumors
Scale bars – 100 μm.
PMC12738729
PHLDA1 silencing in IMR-32 human neuroblastoma cells results in ABCB1 overexpression, augments chemoresistance and leads to increased growth of tumors
The arrows indicate collagen fibers stained with Masson-Goldner trichrome or van Gieson trichrome.
PMC12738729
PHLDA1 silencing in IMR-32 human neuroblastoma cells results in ABCB1 overexpression, augments chemoresistance and leads to increased growth of tumors
PHLDA1 silencing augments the collagen net density within tumor tissue.
PMC12738729
PHLDA1 silencing in IMR-32 human neuroblastoma cells results in ABCB1 overexpression, augments chemoresistance and leads to increased growth of tumors
Microscopy images of xenograft neuroblastoma tumors grown from PHLDA1-silenced (shP) and control cells (shC) in immunocompromised mice.
PMC12738729
PHLDA1 silencing in IMR-32 human neuroblastoma cells results in ABCB1 overexpression, augments chemoresistance and leads to increased growth of tumors
The tissue slices were stained with Masson-Goldner trichrome or van Gieson trichrome.
PMC12738729
PHLDA1 silencing in IMR-32 human neuroblastoma cells results in ABCB1 overexpression, augments chemoresistance and leads to increased growth of tumors
Scale bars – 100 μm.
PMC12738729
PHLDA1 silencing in IMR-32 human neuroblastoma cells results in ABCB1 overexpression, augments chemoresistance and leads to increased growth of tumors
The arrows indicate collagen fibers stained with Masson-Goldner trichrome or van Gieson trichrome.
PMC12738729
PHLDA1 silencing in IMR-32 human neuroblastoma cells results in ABCB1 overexpression, augments chemoresistance and leads to increased growth of tumors
IMR-32 shP cells used to generate tumors were checked for the occurrence of stable PHLDA1 silencing via western blot analysis (Supplementary Fig. S14, S15 online).
PMC12738729
PHLDA1 silencing in IMR-32 human neuroblastoma cells results in ABCB1 overexpression, augments chemoresistance and leads to increased growth of tumors
Tumors derived from shP cells presented a lower intensity of immunohistochemical (IHC) staining with anti-PHLDA1 antibodies (Fig. 5a, b; Supplementary Fig. S21 online; Supplementary Table S3 online).
PMC12738729
PHLDA1 silencing in IMR-32 human neuroblastoma cells results in ABCB1 overexpression, augments chemoresistance and leads to increased growth of tumors
After injection and development into tumors in mice, shP cells presented some level of PHLDA1, but the silencing still persisted.
PMC12738729
PHLDA1 silencing in IMR-32 human neuroblastoma cells results in ABCB1 overexpression, augments chemoresistance and leads to increased growth of tumors
In shP slices, the mean number of positively stained cells was lower (13 cells per field view) than that in shC slices (24.8 cells per field view; see Fig. 5a, b; Supplementary Table S3 online); however, statistical significance was not reached.
PMC12738729
PHLDA1 silencing in IMR-32 human neuroblastoma cells results in ABCB1 overexpression, augments chemoresistance and leads to increased growth of tumors
The PHLDA1 level in shP tumors was stably downregulated, compared to tumors derived from shC cells.
PMC12738729
PHLDA1 silencing in IMR-32 human neuroblastoma cells results in ABCB1 overexpression, augments chemoresistance and leads to increased growth of tumors
In shC cells, the staining intensity was greater.
PMC12738729
PHLDA1 silencing in IMR-32 human neuroblastoma cells results in ABCB1 overexpression, augments chemoresistance and leads to increased growth of tumors
In both shP and shC tumors, PHLDA1 localization was cytoplasmic, however in shC tumors localization of the protein was observed as more intense staining foci.
PMC12738729
PHLDA1 silencing in IMR-32 human neuroblastoma cells results in ABCB1 overexpression, augments chemoresistance and leads to increased growth of tumors
Fig. 5Expression and localization of the PHLDA1 and ABCB1 proteins in tumors grown in vivo from PHLDA1-silenced and control human IMR-32 cells (a).
PMC12738729
PHLDA1 silencing in IMR-32 human neuroblastoma cells results in ABCB1 overexpression, augments chemoresistance and leads to increased growth of tumors
Representative microphotographs visualizing immunostaining of shC and shP tumors are shown.
PMC12738729
PHLDA1 silencing in IMR-32 human neuroblastoma cells results in ABCB1 overexpression, augments chemoresistance and leads to increased growth of tumors
Nuclei were counterstained with Meyer’s hematoxylin.
PMC12738729
PHLDA1 silencing in IMR-32 human neuroblastoma cells results in ABCB1 overexpression, augments chemoresistance and leads to increased growth of tumors
A positive control for anti-PHLDA1 antibodies was presented in the mouse kidney.
PMC12738729
PHLDA1 silencing in IMR-32 human neuroblastoma cells results in ABCB1 overexpression, augments chemoresistance and leads to increased growth of tumors
A positive control for anti-ABCB1 antibodies was presented on the mouse kidney and mouse liver.
PMC12738729
PHLDA1 silencing in IMR-32 human neuroblastoma cells results in ABCB1 overexpression, augments chemoresistance and leads to increased growth of tumors
Scale bars – 50 μm.
PMC12738729
PHLDA1 silencing in IMR-32 human neuroblastoma cells results in ABCB1 overexpression, augments chemoresistance and leads to increased growth of tumors
Arrows indicate PHLDA1-positive or ABCB1-positive cells, respectively.
PMC12738729
PHLDA1 silencing in IMR-32 human neuroblastoma cells results in ABCB1 overexpression, augments chemoresistance and leads to increased growth of tumors
The percentages of positively stained cells are shown in the graphs below (as means ± SEM, see the Supplementary Table S3 online for the counting results from a given tumor sample).
PMC12738729
PHLDA1 silencing in IMR-32 human neuroblastoma cells results in ABCB1 overexpression, augments chemoresistance and leads to increased growth of tumors
Statistical significance was assessed via the two-tailed unpaired Student’s t test.
PMC12738729
PHLDA1 silencing in IMR-32 human neuroblastoma cells results in ABCB1 overexpression, augments chemoresistance and leads to increased growth of tumors
No statistically significant differences in PHLDA1-positive or ABCB1-positive cell numbers were found between shC and shP tumors (b, c).
PMC12738729
PHLDA1 silencing in IMR-32 human neuroblastoma cells results in ABCB1 overexpression, augments chemoresistance and leads to increased growth of tumors
Expression and localization of the PHLDA1 and ABCB1 proteins in tumors grown in vivo from PHLDA1-silenced and control human IMR-32 cells (a).
PMC12738729
PHLDA1 silencing in IMR-32 human neuroblastoma cells results in ABCB1 overexpression, augments chemoresistance and leads to increased growth of tumors
Representative microphotographs visualizing immunostaining of shC and shP tumors are shown.
PMC12738729
PHLDA1 silencing in IMR-32 human neuroblastoma cells results in ABCB1 overexpression, augments chemoresistance and leads to increased growth of tumors
Nuclei were counterstained with Meyer’s hematoxylin.
PMC12738729
PHLDA1 silencing in IMR-32 human neuroblastoma cells results in ABCB1 overexpression, augments chemoresistance and leads to increased growth of tumors
A positive control for anti-PHLDA1 antibodies was presented in the mouse kidney.
PMC12738729
PHLDA1 silencing in IMR-32 human neuroblastoma cells results in ABCB1 overexpression, augments chemoresistance and leads to increased growth of tumors
A positive control for anti-ABCB1 antibodies was presented on the mouse kidney and mouse liver.
PMC12738729
PHLDA1 silencing in IMR-32 human neuroblastoma cells results in ABCB1 overexpression, augments chemoresistance and leads to increased growth of tumors
Scale bars – 50 μm.
PMC12738729
PHLDA1 silencing in IMR-32 human neuroblastoma cells results in ABCB1 overexpression, augments chemoresistance and leads to increased growth of tumors
Arrows indicate PHLDA1-positive or ABCB1-positive cells, respectively.