1. Volume 26, Issue 5, Pages 738-753 (November 2014)
TRPM3 and miR-204 Establish a Regulatory Circuit that Controls Oncogenic Autophagy in Clear Cell Renal Cell Carcinoma 
Daniel P. Hall, Nicholas G. Cost, Shailaja Hegde, Emily Kellner, Olga Mikhaylova, Yiwen Stratton, Birgit Ehmer, William A. Abplanalp, Raghav Pandey, Jacek Biesiada, Christian Harteneck, David R. Plas, Jarek Meller, Maria F. Czyzyk-Krzeska 
Cancer Cell 
Volume 26, Issue 5, Pages (November 2014)
DOI: /j.ccell
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2. Cancer Cell 2014 26, 738-753DOI: (10.1016/j.ccell.2014.09.015)
Copyright © 2014 Elsevier Inc. Terms and Conditions

3. Figure 1
The TRPM3 Channel Is Overexpressed in ccRCC and Regulates the Growth of Xenograft Tumors
(A) Box-and-whisker plot showing the quantification of the tumor/kidney ratio of TRPM3 protein levels in tumors with wild-type (WT) or inactive VHL. The boxes represent lower and upper quartiles separated by the median (thick horizontal line), and the whiskers extend to the minimum and maximum values. Means ± SD of each distribution are indicated by closed dots and crosses on the whiskers, respectively.
(B) Representative western blot showing the expression of TRPM3 protein in lysates of ccRCCs (T) with deleted VHL compared with adjacent kidneys (K). Different bands detected by the antibody may correspond to different splice variants of TRPM3 (Ensembl) or posttranslational modifications.
(C) Representative images of TRPM3 immunocytochemistry in sections of two different ccRCCs and normal kidney.
(D) Bar graph showing the incidence and weight of tumors formed in orthotopic xenografts by VHL(−) cells with endogenous (endog.) TRPM3, TRPM3KD (shRNA I and III), and TRPM3DN. The tumor weight includes the kidney in which the tumor was growing. NK, average weight of normal kidney. Control cells with endogenous TRPM3 were stably transfected with scrambled pLKO.1 vector.
(E) Hematoxylin and eosin (H&E) staining of representative sections of xenograft tumors formed by 786-O VHL(−) cells shown in (D). The clear separation of tumors from kidneys in the case of tumors formed by cells with TRPM3KD(shRNA III) or TRPM3DN is indicated by a black dashed line.
(F) Representative sections from tumors formed by 786-O VHL(−) cells shown in (D) stained for Ki67 and bar graph quantification of Ki67-positive nuclei compared with the total number of nuclei.
(G) Box-and-whisker plot showing the subcutaneous growth of tumors formed by 786-O VHL(−) cells with endogenous TRPM3 or TRPM3R cells. Open circles represent points that are outside of the 1.5 interquartile range from the box. Day 1 corresponds to the time point 2.5 weeks after injections.
(H) Examples and incidence of tumors formed by 786-O VHL(−) cell lines with the indicated status of TRPM3. Scale bar, 1 cm.
(I) Weight of the collected tumors formed by 786-O VHL(−) cell lines with the indicated status of TRPM3.
(J) Representative H&E staining of sections from tumors shown in (H).
(K) Representative sections from subcutaneous tumors in (H) stained for Ki67 and bar graph quantification of Ki67-positive nuclei compared with the total number of nuclei.
Unless indicated otherwise indicated, all scale bars are 50 μm, and error bars indicate SEM. See also Figure S1.
Cancer Cell  , DOI: ( /j.ccell )
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4. Figure 2
VHL Inhibits TRPM3 Expression through Direct Targeting by miR-204
(A) Western blot showing levels of TRPM3 protein in VHL(−) cells and in the Caki-1 cell line with endogenous VHL.
(B) Western blot showing TRPM3 protein levels in 786-O and A498 cells with reconstituted VHL (VHL(+)) compared with isogenic VHL(−) cells.
(C) Western blot showing the effects of anti-miR-204 on the expression of TRPM3 protein in the indicated VHL(+) RCC cells.
(D) Western blot showing the effects of pre-miR-204 on the expression of TRPM3 protein in the indicated VHL(−) RCC cells.
(E) Effects of miR-204 on the activity of the luciferase (Luc) reporter construct containing the WT or mutated miR-204 binding site (mut) 3′ UTR of TRPM3 in 786-O VHL(−) cells. The ratio of luciferase activity in cells transduced with wild-type miR-204 to the activity in cells transduced with a nontargeting lentivirus control is shown. Error bars indicate SEM.
(F) Representative western blot showing the expression of TRPM3 protein (top) in nine kidneys with low and high levels of miR-204 (bottom).
(G) Regression analysis showing the negative correlation between the normalized TRPM3 protein level and the normalized miR-204 level in human kidneys. Each dot represents a sample, and the solid line represents the linear regression fit, with the Pearson correlation coefficient (r) as well as the slope and intercept of the fitted line shown in the upper left corner of the box.
See also Figure S2.
Cancer Cell  , DOI: ( /j.ccell )
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5. Figure 3 CAV1 Is a miR-204 Target and Regulates Expression of TRPM3
(A) Western blot showing levels of CAV1 protein in the indicated cell lines.
(B) Expression of CAV1 protein and mRNA using western blot (left) and qRT-PCR (right) in 786-O and A498 VHL(+) cells compared with the isogenic VHL(−) cells. Error bars indicate SD. IB, immunoblot.
(C) Western blot showing the effects of pre-miR-204 on the expression of CAV1 protein in VHL(−) cells.
(D) Western blot showing the effects of anti-miR-204 on the expression of CAV1 protein in VHL(+) cells.
(E) Effects of miR-204 on the activity of a luciferase reporter containing the WT or mutated miR-204 binding site chimeric 3′ UTR of CAV1 in 786-O VHL(−) cells. Error bars indicate SEM.
(F) Effects of CAV1KD on the expression levels of TRPM3 protein in 786-O VHL(−) cells.
(G) Effects of stable overexpression of FLAG-tagged CAV1 on TRPM3 protein levels in two different pools (P1 and P2) of 786-O VHL(+) cells. Endog., endogenous.
(H) Regression analysis demonstrating the positive correlation between normalized TRPM3 and normalized CAV1 protein levels in human ccRCCs. The analysis was performed as in (G).
(I) Representative western blot showing protein levels of TRPM3 and CAV1 in 14 human ccRCCs.
See also Figure S3.
Cancer Cell  , DOI: ( /j.ccell )
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6. Figure 4 The TRPM3 Channel Regulates Autophagy
(A) Bar graph quantification of the number of autophagic vesicles (top) and the cytoplasmic area (bottom) in TEM sections of O VHL(−) control (stably transfected with scrambled pLKO.1 vector [Scr]) or TRPM3KD cells. In this and all subsequent figures, if not indicated otherwise, “TRPM3KD” refers to knockdowns using shRNA I.
(B) Western blot and bar graph quantification showing effects of TRPM3KD on the accumulation of LC3A/LC3B-II in VHL(−) cells. The dashed line shows the level of LC3-II accumulation in control cells, which is accepted as 1.
(C) Western blots showing the effects of TRPM3DN on the accumulation of LC3A/B-II in 786-O VHL(−) cells. P1 and P2 are two separate pools of cells.
(D) Western blot showing the rescue of LC3A/LC3B-II accumulation upon reconstitution of TRPM3R in VHL(−) TRPM3KD cells.
(E) Western blot showing the effects of TRPM3KD on the accumulation of LC3A/LC3B-II in VHL(+) cells.
(F) Immunostaining for LC3B puncta in sections from respective orthotopic xenografts of 786-O VHL(−) cells with the indicated status of TRPM3. Scale bar, 20 μm.
(G) Immunostaining for LC3B puncta in sections from subcutaneous xenografts of 786-O VHL(−) cells with the indicated status of TRPM3. Scale bar, 20 μm.
(H) Incidence and weight of tumors formed in orthotopic xenografts by VHL(−) TRPM3KD cells with re-expressed FLAG-LC3B. Note that TRPM3KD(shI) cells did not form tumors, similar as shown in Figure 1D.
In all panels, error bars indicate SEM. See also Figure S4.
Cancer Cell  , DOI: ( /j.ccell )
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7. Figure 5
TRPM3 Regulates Autophagy through the CAMKK2/AMPK/ULK1 Pathway
(A) Bar graph quantification of ATG-16L puncta detected by immunofluorescence staining in 786-O VHL(−) control and TRPM3KD cells. Error bars indicate SEM.
(B) Bar graph quantification of ATG5-ATG12 puncta detected by immunofluorescence staining in 786-O VHL(−) control and TRPM3KD cells. Error bars indicate SEM.
(C) Western blot showing steady-state expression levels of ATG16L protein and ATG5-ATG12 conjugate in 786-O VHL(−) control and TRPM3KD cells.
(D) Western blot showing the effects of TRPM3KD on the protein expression and phosphorylation of AMPKα and ULK1 in VHL(−) cells.
(E) Western blot showing the effects of TRPM3R on the protein expression and phosphorylation of AMPKα and ULK1 in 786-O VHL(−) TRPM3KD cells.
(F) Western blot showing the effects of TRPM3KD on the protein expression and phosphorylation of AMPKα and ULK1 in VHL(+) cells.
(G) Western blot showing the effects of the constitutively active CAMKK2 on the accumulation of LC3A/LC3B-II and phosphorylation of AMPKα and ULK1 in 786-O VHL(−) TRPM3KD cells. EV, empty virus.
(H) Western blot showing the effects of CAMKK2 shRNA on the accumulation of LC3A/LC3B-II and phosphorylation of AMPK and ULK1 in 786-O VHL(−) cells.
(I) Western blot showing the effects of the constitutively active AMPKα2 on the accumulation of LC3A/LC3B-II and phosphorylation of AMPK and ULK1 in 786-O TRPM3KD cells. EV, empty virus.
(J) Western blot showing the effects of AMPKα1/α2 shRNAs on the accumulation of LC3A/LC3B-II and phosphorylation of AMPK and ULK1 in 786-O VHL(−) cells.
See also Figure S5.
Cancer Cell  , DOI: ( /j.ccell )
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8. Figure 6 TRPM3 Regulates Autophagy through miR-214
(A) Normalized levels of miR-214 in 786-O VHL(−) control, TRPM3KD, and TRPM3R cells.
(B) Normalized levels of miR-214 in 786-O VHL(−) control, TRPM3KD, and VHL(+) cells.
(C) Normalized levels of miR-214 in VHL(−) RCC cell lines and the VHL(+) Caki-1 cell line.
(D) Effects of overexpression of TRPM3WT or TRPM3DN on the steady-state levels of miR-214 in the indicated cells.
(E) Effects of TRPM3KD on normalized levels of miR-214 in VHL(+) cells.
(F) Effects of treatment with the indicated concentrations of pre-miR-214 and anti-miR-214 on the accumulation of LC3A/LC3B in starved VHL(−) cells. All cells were treated with CQ.
(G) Effects of anti-miR-214 (lentivirus, lenti) treatments on the accumulation of LC3A/LC3B in starved 786-O VHL(−) TRPM3KD cells. In the case of siRNA, only CQ-treated cells are shown.
(H) Effects of lentiviral anti-miR-214 and pre-miR-214 treatments on the accumulation of LC3A/B in starved 786-O VHL(+) cells.
(I) Effects of pre-miR-214 on the activity of luciferase reporter constructs containing LC3A or LC3B 3′ UTRs with either WT or mutated miR-214 binding sites in 786-O VHL(−) cells.
(J) Effects of constitutively active CAMKK2 and AMPKα2 on the accumulation of miR-214 in starved 786-O VHL(−) TRPM3KD cells.
(K) Effects of CAMKK2, AMPKα1/2, ULK1, and PIK3C3 knockdowns on the accumulation of miR-214 in starved 786-O VHL(−) cells.
(L) Effects of anti-miR214 on the accumulation of LC3A and LC3B in 786-O VHL(−) cells treated with STO-609.
In all panels, n represents a number of individual experiments in triplicate. If n < 3, error bars indicate SD. If n ≥ 3, error bars indicate SEM. See also Figure S6.
Cancer Cell  , DOI: ( /j.ccell )
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9. Figure 7
The Effects of TRPM3KD Are Reproduced by MFA, a Small Molecule Blocker of TRPM3
(A) Box-and-whisker plot showing the growth of subcutaneous xenograft tumors formed by the indicated VHL(−) cells in mice treated with MFA, IDM, or vehicle. Open circles represent points that are outside of the 1.5 interquartile range from the box.
(B) Representative sections of 786-O tumors from each treatment group stained for Ki67 and bar graph quantification performed as in Figure 1F. Scale bar, 100 μM.
(C) Effects of treatment with MFA on the expression of endogenous TRPM3 measured by western blot and by qRT-PCR in VHL(−) cells.
(D) Effects of MFA treatments of 786-O and A498 VHL(−) cells on the accumulation of LC3A/LC3B-II and phosphorylation of ULK1-Ser317. R, ratio of p-ULK1-Ser317 to total ULK1.
(E) Effects of MFA on accumulation of miR-214 in 786-O and A498 VHL(−) cells.
In all panels, error bars indicate SEM. See also Figure S7.
Cancer Cell  , DOI: ( /j.ccell )
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10. Figure 8
Model Summarizing the Proposed Regulatory Network Controlling Oncogenic LC3A/LC3B Autophagy
Black lines, oncogenic pathways; gray lines, tumor-suppressing pathways; asterisk, work reported by Mikhaylova et al. (2012).
Cancer Cell  , DOI: ( /j.ccell )
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