EMBO/soda-nel huggingface dataset:
Loss of VGLL4 suppresses tumor PD-L1 expression and immune evasion (2018)
Figure 2

Figure 2. Disruption of VGLL4 expression suppresses PD-L1 expression.

(A) Relative mRNA levels of immune related genes in Vgll4-knockdown LLC or MB49 cells by qRT-PCR analysis. n=3, mean±SEM. * p<0.05, ** p<0.01, *** p<0.001, two-tailed Student's t-test.

(B) Knockdown of Vgll4 in MB49 cells decreased the surface expression of PD-L1 protein on control or shVgll4 MB49 cells measured by flow cytometry analysis with anti-mPD-L1 antibody. n=3, mean±SEM. *** p<0.001, two-tailed Student's t-test.

(C) Relative mRNA levels of immune related genes in Vgll4-knockdown A549 cells by qRT-PCR analysis. n=3, mean±SEM. ** p<0.01, *** p<0.001, two-tailed Student's t-test.

(D) Immunoblot analysis revealed the reduced expression of PD-L1 protein in VGLL4 knockdown A549, 16HBE and H358 cells compared with control cells.

(E) qRT-PCR analysis revealed a reduction of PD-L1 mRNA levels in VGLL4-knockdown 16HBE and H358 cells by siRNA. n=3, mean±SEM. ** p<0.01, *** p<0.001, two-tailed Student's t-test.

(F) Flow cytometry analysis of surface PD-L1 levels in control and VGLL4 knockdown A549 cells treated with IFNγ for the indicated times. n=3, mean±SEM. *** p<0.001, One-way ANOVA followed by Dunnett'stest.

(G) and (H) Immunoblot analysis revealed the reduced IFNγ-inducible PD-L1 expression in VGLL4 knockdown A549 (G) and 16HBE (H) cells.

(I) Immunoblot analysis of total cell lysates with the indicated antibodies revealed the attenuated IFNγ-inducible PD-L1 expression in VGLL4 knockout (KO) A549 cells generated by CRISPR/Cas9.

(J) Loss of VGLL4 dampened the activity of PD-L1 promoter reporter in a luciferase assay. A549 cells were transfected with human PD-L1-promoter luciferase plasmid and indicated siRNA, treated with IFNγ for 12 h, and subjected to a luciferase assay. n=3, mean±SEM. ** p<0.01, *** p<0.001, One-way ANOVA followed by Dunnett'stest.

(K) Overexpression of mouse PD-L1 in Vgll4 knockdown LLC cells restored the tumor growth in C57BL/6 mice. Tumor weights of shVgll4 or shVgll4-lenti-mPD-L1 LLC cells were determined 21 days after tumor cell transplantation into C57BL/6 mice (left panel). Expression of mPD-L1 was shown in the right panel by immunoblot analysis with anti-mPD-L1 antibody. n=10 tumors for each group. The solid line represents the average weight ± SEM. *** p<0.001, two-tailed Student's t-test.

(L) Knockdown of VGLL4 in A549 cells enhances T cell-meditated tumor cell killing. Activated T cells and A549 cells were co-cultured in 24-well plates for 4 days and then surviving tumor cells were visualized by crystal violet staining. Relative fold ratios of surviving cell intensities are shown in right panel. n=3, mean±SEM, ** p<0.01, two-tailed Student's t-test.
Loss of VGLL4 suppresses tumor PD-L1 expression and immune evasion (2018)
Figure 3

Figure 3. VGLL4 interacts with IRF2BP2 independent of TDU domains.

(A) The endogenous complex of VGLL4 and IRF2BP2 was detected by immunoprecipitation using anti-VGLL4 antibody and visualized by anti-IRF2BP2 antibody in A549 cells.

(B) The endogenous IRF2BP2 was immunoprecipitated by VGLL4-FLAG proteins in HEK293T cells.

(C) Interaction between VGLL4 and IRF2BP1 or IRF2BP2 was revealed by co-immunoprecipitation of differentially tagged proteins in HEK293T cells.

(D) C-terminus of IRF2BP2 interacted with VGLL4 by co-immunoprecipitation assay in HEK293T cells.

(E) Interaction of IRF2BP2 with VGLL1-4 was revealed by co-immunoprecipitation of differentially tagged proteins in HEK293T cells.

(F) TDU domains in VGLL4 are required for the interaction with TEADs by co-immunoprecipitation assay in HEK293T cells.

(G) and (H) TDU-deleted or HF4A VGLL4 mutations interacted with IRF2BP2 (G) and IRF2BP1 (H) by co-immunoprecipitation analysis in HEK293T cells.

(I) Expression of VGLL4-WT or VGLL4-HF4A enhances PD-L1 expression in A549 cells by immunoblot analysis. A549 cells were transfected with indicated plasmids and subjected to immunoblot with indicated antibodies.

(J) VGLL4-HF4A rescues PD-L1 expression in VGLL4-knockdown A549 cells. A549 cells were transfected with siRNA targeting to the 3'UTR of VGLL4 mRNA and followed by transduction with lenti-VGLL4-HF4A virus. Cell lysates were analyzed by western blot with indicated antibodies.

(K) Expression of VGLL4-HF4A rescues tumor growth of VGLL4-knockdown LLC cells in C57BL/6 mice. Control, Vgll4 knockdown or Vgll4 knockdown together with VGLL4-HF4A overexpressing LLC cells were transplanted into nude mice or C57BL6 mice. Tumor volumes were measured 15 days for nude mice and 21 days for C57BL/6 mice after tumor cell inoculation. n=8 tumors for each group. *** p<0.001, two-tailed Student's t-test. The solid line represents the average volume ± SEM.

(L) Expression of VGLL4-HF4A attenuates the T cell-meditated tumor cell killing. Activated T cells and A549 cells were co-cultured in 24-well plates for 4 days and Relative fold ratios of surviving cells are shown by measuring the intensities of surviving cells stained with crystal violet. n=3, mean±SEM.
Loss of VGLL4 suppresses tumor PD-L1 expression and immune evasion (2018)
Figure 4

Figure 4. VGLL4 protects proteasome-mediated IRF2BP2 degradation.

(A) and (B) Correlation between protein expression of VGLL4 and IRF2BP2 in 6 murine tumor cell lines (A) and human cell lines (B) with the indicated antibodies.

(C) Immunoblot analysis of A549 cells transfected with VGLL4-FLAG or VGLL4-HF4A plasmids revealed an increased IRF2BP2 protein levels compared with control cells.

(D) Deficiency of VGLL4 reduces IRF2BP2 protein levels. Immunoblot analysis of A549 and LLC cell lysates transfected with siRNA for control and VGLL4.

(E) The indicated cell lines were transfected with control or VGLL4 siRNA and subjected to immunoblot analysis with the indicated antibodies.

(F) qRT-PCR analysis of IRF2BP2 mRNA levels in control and siVGLL4 A549 cells. n=3, mean±SEM.

(G) A549 or 16HBE cells were transfected with control or VGLL4 siRNA and treated with MG132 for the indicated times and subjected to immunoblot analysis with the indicated antibodies. Relative IRF2BP2 expression levels were quantified in the right panel. n=3, mean±SEM.

(H) Lys48-linked ubiquitylation of IRF2BP2 is strongly inhibited when VGLL4 was overexpressed in HEK293T cells. HEK293T cells were transfected with indicated plasmids, treated with MG132 for 3 h before harvest, and subjected to immunoprecipitation and immunoblot analysis as indicated.

(I) Lys48-linked ubiquitylation of IRF2BP2 is increased in the absence of VGLL4 in HEK293T cells. HEK293T cells were transfected with indicated siRNA and plasmids, and subjected to immunoprecipitation and immunoblot analysis as indicated.
Loss of VGLL4 suppresses tumor PD-L1 expression and immune evasion (2018)
Figure 5

Figure 5. IFNγ stimulation elicits the release of IRF2 from PD-L1 promoter and the dynamic interaction of IRF2 with IRF2BP2.

(A). IRF2 inhibits IRF1 transcriptional activity. A549 cells transfected with human PD-L1 promoter luciferase reporter and indicated plasmids were analyzed using a luciferase assay. n=3, mean±SEM. *p<0.05, ** p<0.01, One-way ANOVA followed by Dunnett'stest.

(B) Knockdown IRF2 restores PD-L1 expression in VGLL4-knockdown A549 cells. Gene expression levels were analyzed by qRT-PCR in A549 cells transfected or transduced as indicated. n=3, mean±SEM. *p<0.05, *** p<0.001, One-way ANOVA followed by Dunnett'stest.

(C) Overexpression of IRF1 increases PD-L1 expression in VGLL4-knockdown A549 cells. A549 cells were transfected with VGLL4 siRNA or together with HA-IRF1 plasmid and total cell lysates were analyzed by western blot.

(D) IRF2 is released from PD-L1 promoter upon IFNγ stimulation. IRF2-3×HA knockin A549 cells were stimulated with IFNγ and subjected to ChIP-qPCR analysis in the PD-L1 promoter using control and HA antibodies. Normal mouse IgG was used as control. n=3, mean±SEM. *** p<0.001, One-way ANOVA followed by Tukey's test.

(E) Increased association of IRF2 with PD-L1 promoter in IRF2BP2 knockout (KO) A549 cells revealed by ChIP-qPCR analysis. Immunoblot showed the loss of IRF2BP2 in IRF2BP2 KO A549 cells in the lower panel. n=3, mean±SEM. * p<0.05, One-way ANOVA followed by Tukey's test.

(F) Dynamic association of IRF2 with IRF2BP2 during IFNγ stimulation by immunoprecipitation analysis using anti-HA antibody in IRF2-3×HA knockin A549 cells.

(G) and (H) Loss of IRF2BP2 attenuated IFNγ-inducible PD-L1 protein (G) and mRNA (H) expression revealed by immunoblot and qRT-PCR analysis respectively in A549 control and IRF2BP2 knockout cells. Quantification of PD-L1 protein levels in (G) is shown in the right panel. n=3, mean±SEM, * p<0.05, ** p<0.01, *** p<0.001, two-tailed Student's t-test.
Loss of VGLL4 suppresses tumor PD-L1 expression and immune evasion (2018)
Figure 6

Figure 6. YAP inhibits IFNγ-inducible PD-L1 and IRF1 expression.

(A) miR-130a represses VGLL4 and PD-L1 protein levels. A549 cells were transfected with control and miR-130a mimic and subjected to immunoblot analysis.

(B) and (C) Inhibition of miR-130a by its sponge enhances IFNγ-inducible PD-L1 expression. A549 cells were transduced with control or miR-130a sponge lentivirus and subjected to immunoblot (B) or qRT-PCR (C) analysis. n=3, mean±SEM, *** p<0.001, two-tailed Student's t-test.

(D) Decreased YAP target gene expression by inhibition of miR-130a using its sponge in A549 cells. n=3, mean±SEM, ** p<0.01, two-tailed Student's t-test.

(E) YAP5SA expression attenuates IFNγ-inducible PD-L1 expression. A549 cells were transduced with control, YAP-5SA or YAP-S94A lentivirus and treated with IFNγ for the indicated times and subjected to immunoblot analysis.

(F) qRT-PCR analysis of cell lysates from control, YAP-5SA or YAP-S94A expressing A549 cells treated with IFNγ for the indicated times. n=3, mean±SEM.

(G) Control or YAP-overexpressing LLC cells were transplanted into nude mice (upper panel) or C57BL/6 (lower panel) mice, and tumor-growth kinetics was measured at the indicated times. Tumor volumes at the end time points were analyzed and shown at the right panel. n=6 tumors for each group. The solid line represents the average weight ± SEM. n.s. p>0.05, *** p<0.001, two-tailed Student's t-test.

(H) Conservation of miR-130a sites in IRF1 3′UTR in vertebrates. miR-130a binding sites from IRF1 3′UTR of different species are aligned with human miR-130a.

(I) miR-130a regulates IRF1 3′UTR sensor activity. A549 cells were transfected with WT or mutant IRF1 3′UTR sensor plasmids together with miR-130a mimic. Sensor activities were determined by dual luciferase assay. n=3, mean±SEM. n.s. p>0.05, ** p<0.01, two-tailed Student's t-test.

(J) YAP5SA induces miR-130a expression and represses IRF1 expression. A549 cells were transduced with control or YAP-5SA lentivirus and subjected to qRT-PCR analysis. n=3, mean±SEM. ** p<0.01, *** p<0.001, two-tailed Student's t-test.

(K) Inhibition of miR-130a by its sponge enhances IFNγ-inducible IRF1 expression. A549 cells were transduced with control or miR-130a sponge lentivirus and subjected to qRT-PCR analysis. n=3, mean±SEM. ** p<0.01, *** p<0.001, two-tailed Student's t-test.

(L) Suppression of IFNγ-inducible PD-L1 expression by YAP5SA is compromised in miR-130a KO A549 cells. PD-L1 expression levels were analyzed by qRT-PCR in IFNγ-treated control or miR-130a KO A549 cells pre-transduced with lenti-YAP5SA virus. n=3, mean±SEM, ** p<0.01, *** p<0.001, One-way ANOVA followed by Tukey's test.
Loss of VGLL4 suppresses tumor PD-L1 expression and immune evasion (2018)
Figure 7

Figure 7. VGLL4 expression predicts cancer patient outcome and correlates with IRF2BP2 levels in non-small cell lung cancer.

(A) Representative immunohistochemical staining for VGLL4 and IRF2BP2 in human non-small cell lung cancer tissues. IHC score is indicated. Scale bar: 50 μm.

(B) Staining of VGLL4 and IRF2BP2 on human non-small cell lung cancer tissue arrays containing 71 samples was quantified as described in the Materials and Methods section. Correlation of VGLL4 and IRF2BP2 expressions was significant (Pearson correlation test; R = 0.6341, P < 0.001).

(C) Correlation analysis of VGLL4 and PD-L1 expression on human non-small cell lung cancer tissue arrays containing 71 samples.

(D) Kaplan-Meier survival analysis of patients with high (n=74) or low (n=130) VGLL4 mRNA levels from GSE31210, a lung cancer data set. Survival curves are calculated according to the Kaplan-Meier method.

(E) Model for the regulation of PD-L1 expression by VGLL4 and YAP. VGLL4 regulates PD-L1 expression through controlling IRF2BP2 protein stability. YAP represses IFNγ-inducible PD-L1 expression partially through miR-130a mediated inhibition of VGLL4 and IRF1 expression.

Expanded view figure legends
A critical period of pre-hearing spontaneous Ca2+ spiking is required for hair-bundle maintenance in inner hair cells (2022)
Figure 1

Figure 1: Basolateral membrane properties of IHCs overexpressing Kir2.1 channels

A, B, Maximum intensity projections of confocal z-stacks taken from the apical cochlear region of control (A) and littermate Kir2.1 overexpressing (B, Kir2.1-OE) mice at postnatal day 11. Inner hair cells (IHCs) were stained with antibodies against Kir2.1 (green) and the hair cell marker MYO7a (blue). At least 3 mice for each genotype were used. Scale bars: 10 μm.

C, D, Currents from IHCs of control (C, P9) and Kir2.1-OE (D, P10) pre-hearing mice. Currents were elicited by using depolarizing and hyperpolarizing voltage steps, with a nominal increment of 10 mV, from a holding potential of −84 mV. Test potentials are shown next to some of the traces. Note that the large inward rectifier Kir2.1 current is only present in the IHC of the Kir2.1-OE mouse (D). The outward current is primarily carried by a delayed rectifier current IK. IK1 identifies the small inwardly rectifying K+ current normally expressed in IHCs.

E, Steady-state current-voltage curves obtained from IHCs of control (P9-P11) and Kir2.1-OE (P9-P11) mice.

F, G, Size of the total steady-state outward (F, IK: Control 2.88 ± 1.07 nA, n = 8; Kir2.1-OE 2.25 ± 0.97 nA, n = 6) and inward (G, Control, IK1: 0.30 ± 0.05 nA, n = 8; Kir2.1-OE, IKir2.1: 3.13 ± 1.37 nA, n = 6) K+ currents measured at 0 mV and -124 mV, respectively. n.s: P = 0.2836.

H, Resting membrane potential (Vm) measured in IHCs from Control (−62.6 ± 3.8 mV, n = 7) and Kir2.1-OE (−73.5 ± 3.5 mV, n = 5).

I, Slope conductance of the current measured at around the respective resting Vm (Control 1.8 ± 0.4 nS, n = 8; Kir2.1-OE 25.5 ± 9.6 nA, n = 6).

Data information: In panels F-I, data are shown as means ± SD and the single cell value recordings (open symbols) are plotted with the average data. All statistical tests were performed using Student's t-test. The number of IHCs investigated is shown above the average data points (6 control and 3 Kir2.1-OE mice).
A critical period of pre-hearing spontaneous Ca2+ spiking is required for hair-bundle maintenance in inner hair cells (2022)
Figure 2

Figure 2. Kir2.1 overexpression prevents spontaneous, but not induced, Ca2+ action potentials in IHCs

A, B, Whole-cell recordings of Ca2+ action potential activity in apical-coil IHCs from P4 control (A) and Kir2.1-OE (B) mice in the presence of 1.3 mM Ca2+ in the extracellular solution and at body temperature. Note that IHCs from control mice (A) fire spontaneous action potentials (40 sec out of 142 sec recording time), while those from overexpressing Kir2.1 IHCs (B) require substantial current injection to elicit any spikes. For voltage-clamp data see also Fig EV1C-I. Data in Panel A,B and Fig EV1C-I were obtained from 8 control IHCs (7 mice) and 11 Kir2.1-OE IHCs (6 mice).

C, D, Calcium action potentials in IHCs from control (C) and Kir2.1-OE (D) mice during the second-postnatal week. IHC voltage responses were recorded during the application of a solution containing 1.3 mM Ca2+ (top panels) or 0.3 mM Ca2+ (bottom panels). The latter Ca2+ concentration (0.3 mM), which was used to mimic the estimated in vivo Ca2+ concentration in the endolymphatic compartment (Johnson et al., 2012), caused control IHCs, but not those from Kir2.1-OE mice, to elicit spontaneous action potentials (40 sec out of 56 sec recording time).

E, Diagram showing a cross-section of an immature organ of Corti. IHCs: inner hair cells; GER: greater epithelial ridge, which includes non-sensory cells surrounding the IHCs. Red arrows: indicate the propagation of ATP-induced Ca2+ waves from the GER towards the IHCs, which lead to their depolarization (Tritsch et al., 2007; Johnson et al., 2017; Wang et al., 2015).

F, G, Representative ∆F/F0 traces from the IHCs and GER of P7-P9 control (F) and Kir2.1-OE (G) mice in the presence of 0.3 mM Ca2+. Spontaneous ATP-dependent Ca2+ waves from the GER (green traces) were eliciting coordinated Ca2+ signals in the IHCs from both controls and Kir2.1-OE mice. For each genotype, two separate sets of recordings from 2 mice are shown (top and bottom right), with the top traces being linked to the images on the left: before [1], during [2] and after [3]) the generation of a large Ca2+ wave from the GER. For details about the frequency and duration of the Ca2+ waves, and the number of mice and recordings see Fig EV3. All recordings were obtained at body temperature. Traces are computed as pixel averages of regions of interest centred on IHCs.
A critical period of pre-hearing spontaneous Ca2+ spiking is required for hair-bundle maintenance in inner hair cells (2022)
Figure 3

Figure 3. Mechanoelectrical transduction in Kir2.1 overexpressing IHCs is normal during the first postnatal week

A, B, Saturating MET currents recorded from apical IHCs of P6 control (A) and Kir2.1-OE (B) mice in response to 50 Hz sinusoidal force stimuli to the hair bundles at membrane potentials of −124 and +96 mV. Driver voltage (DV) stimuli to the fluid jet are shown above the traces, with positive deflections of the DV being excitatory. The arrows indicate the closure of the transducer channel in response to inhibitory bundle stimuli at −124 and +96 mV.

C, Peak-to-peak MET current-voltage curves from P6-P7 apical-coil IHCs of 7 control (12 IHCs) and 3 littermates Kir2.1-OE mice (7 IHCs). Recordings were obtained by mechanically stimulating the hair bundles of IHCs at the same time as stepping their membrane potential from −124 mV to +96 mV in 20 mV increments. The two sets of data are not significantly different: P = 0.6320, 2-way ANOVA.

D, Maximum size of the MET current recorded at −124 mV (left panel) and +96 mV (right panel) in IHCs from both genotypes.

E, Resting open probability (Popen) of the MET current in IHCs from the two genotypes measured at −124 mV (left) and +96 mV (right). The resting open probability was calculated by dividing the resting MET current (the difference between the current level before the stimulus, indicated by the dashed line, and the current level at the negative phase of the stimulus when all channels are closed) by the maximum peak-to-peak MET current.

Data information: All comparisons in panels D and E are not significantly different between the two genotypes (D: −124 mV: P = 0.2269; +96 mV: P = 0.3620; E: −124 mV: P = 0.3766; +96 mV: P = 0.2846, t-test). In panels C-E, data are shown as means ± SD and the single cell value recordings (open symbols) are plotted behind the average data. The number of IHCs investigated is shown above the averaged data points from 7 control and 3 Kir2.1-OE mice.
A critical period of pre-hearing spontaneous Ca2+ spiking is required for hair-bundle maintenance in inner hair cells (2022)
Figure 4

Figure 4. Rapid disappearance of the MET current in Kir2.1 overexpressing IHCs during the second postnatal week

A, B, Saturating MET currents recorded from apical IHCs of P8 control (A) and Kir2.1-OE (B) mice. IHC hair bundles were stimulated as described in Figure 3.

C, Peak-to-peak MET current-voltage curves from P8-P9 apical-coil IHCs of 12 control (17 IHCs) and 13 littermates Kir2.1-OE mice (24 IHCs). The two sets of data are significantly different: P < 0.0001, 2-way ANOVA.

D, The maximum size of the MET current measured in IHCs at −124 mV (left panel) and +96 mV (right panel) from Kir2.1-OE was significantly reduced compared to that of control cells.

E, The resting open probability (Popen) of the MET current in IHCs was significantly increased in Kir2.1-OE compared to control cells at both −124 mV (left) and +96 mV (right).

F, G, Saturating MET currents recorded from apical IHCs of control (F, P10) and Kir2.1-OE (G, P11) mice. IHC hair bundles were stimulated as described in Figure 3.

H, Peak-to-peak MET current-voltage curves from P10-P11 apical-coil IHCs of 13 control (16 IHCs) and 4 littermates Kir2.1-OE mice (9 IHCs). The two sets of data are significantly different: P < 0.0001, 2-way ANOVA.

I, The maximum size of the MET current measured in IHCs at −124 mV (left panel) and +96 mV (right panel) from Kir2.1-OE is significantly reduced compared to that of control cells.

J, Example of FM1-43 uptake by IHCs from P11 control (top) and Kir2.1-OE (bottom) mice, showing the lack of fluorescence labelling in the latter, which is an indication of the lack of MET channels open at rest at this stage in the IHCs overexpressing the Kir2.1 channels. At least 3 mice for each genotype were used.

Data information: In panels D, E, I, data are shown as means ± SD and the single cell value recordings (open symbols) are plotted with the average data. The number of IHCs investigated is shown above the average data points from 12 control and 13 littermates Kir2.1-OE mice (panels D,E) and 13 control and 4 littermates Kir2.1-OE mice (panel I). Statistical tests in panels D, E, I was done using the t-test. The * defines the presence of statistical significance, with the P value shown above the data.
A critical period of pre-hearing spontaneous Ca2+ spiking is required for hair-bundle maintenance in inner hair cells (2022)
Figure 5

Figure 5. IHC bundle morphology progressively deteriorates in Kir2.1 overexpressing mice

A, B, Scanning electron microscope (SEM) images showing the IHC hair bundle structure in the apical coil of the cochlea of P11 control (A) and P8-P11 Kir2.1-OE (B) mice. Control IHCs and the large majority of P8 IHCs from Kir2.1-OE mice show a normal hair bundle structure composed of three rows of stereocilia: tall, intermediate and short (arrows). From about P9 in Kir2.1-OE mice, some IHCs start to lose the third row of stereocilia (arrowheads) and some already exhibit some fusion of the stereocilia (asterisk). These changes in hair-bundle structure became more prominent at P11. At least 3 mice for each genotype were used. In these panels and those below, asterisks are used to define some of the abnormal hair bundles.

C, D, SEM images of both IHCs and OHCs from the cochlea of P26 control (C, upper panel) and P26 Kir2.1-OE (D, upper panel) mice. Lower panels show a higher-magnification view of the hair bundle of IHCs from both genotypes, highlighting the profound disruption of the stereocilia in IHCs overexpressing Kir2.1 channels. At least 3 mice for each genotype were used.
A critical period of pre-hearing spontaneous Ca2+ spiking is required for hair-bundle maintenance in inner hair cells (2022)
Figure 6

Figure 6. Hair bundle proteins involved in stereociliary elongation are not affected in IHCs from Kir2.1-OE mice.

A,B, Maximum intensity projections of confocal z-stacks showing images of the hair bundles from apical-coil IHCs of P6 and P11 control (A) and Kir2.1-OE (B) mice immunostained with antibodies against MYOSIN VI (blue) and EPS8 (magenta). At least 3 mice for each genotype were used.

C,D, Confocal images of the hair bundles of P11 IHCs from control (C) and Kir2.1-OE (D) mice immunostained with antibodies against MYOSIN XV isoform 1 (blue) and WHIRLIN (magenta). In all panels (A-D), stereocilia are labelled with phalloidin (green). Note that despite the disrupted hair-bundle structure in the IHCs overexpressing Kir2.1 channels; the stereocilia retained a normal distribution of these bundle proteins. At least 3 mice for each genotype were used.