Supplementary Figures and Material
1
2
Supplementary Figure 1. Confocal microscopy composition of a micropipetting experiment. In
this case, WT and Bbs4-/- Actin-GFP transfected cells were subjected to a step pressure of 7cm
H2O. From these images the temporal change in aspiration length of the cell into the micropipette
was measured and used to calculate the Equilibrium modulus (see Material and Methods for more
information). No statistically significant differences were found between the Equilibrium
Modulus of WT and Bbs4-/- cells.
3
Supplementary Figure 2. Depolymerisation of Bbs4 null cells leads to a delay in recovery of Factin filament polymerisation. Black scale bar is 20 m. White scale bar is 40 m.
polymerisation. Scale bar 20 m.
4
Supplementary Figure 3. Colocalization of Bbs8 and Bbs9 with F-actin.
5
Supplementary Figure 4. BBS8 and BBS9 are expression and localisation is not affected in the
Bbs4 -/- cells. A-B. Immunofluorescence of BBS9 (green) in non-confluent cells in WT and Bbs4
-/-
cells showing no difference in their localisation around the edge of the cell. C-D. BBS8
expression in non-confluent cells in WT and Bbs4
pattern and intensity in WT and in Bbs4
-/-
-/-
cells. BBS8 is expressed with the same
cells. Scale bar 20 m C-D. Scale bar 10 m. Blue
channel is DAPI.
6
Supplementary Figure 5. BBS8 and BBS9 are expression and localisation is not affected in the
Bbs6 -/- cells. A-B. Immunofluorescence of BBS9 (green) in non-confluent cells in WT and Bbs6
-/-
cells showing no difference in their localisation around the edge of the cell. C-D. BBS8
expression in non-confluent cells in WT and Bbs6
-/-
cells. BBS8 is expressed with the same
pattern and intensity in WT and in Bbs6 -/- cells. C-D. Scale bar 10 m. Blue channel is DAPI.
7
Supplementary Figure 6. A. Number of Focal adhesions in WT vs Bbs4- /- cells and Bbs8-shRNA
vs control cells. B. Distance between the FAs and the membrane in Bbs4- /- and Bbs8-shRNA. *:
p-value<. 0.05: ***, p-value,0.001
8
Supplementary Figure 7. C3 treatment restores the actin cytoskeleton and cilia length in
Bbs4 null cells. A-B. Cilia length of WT and Bbs4 -/- cells after C3 transferase treatment (2 g/ml
for 3 hours). (A). Cilia staining of WT and Bbs4
-/-
cells. Cells were stained for cilia (Acetylated
tubulin, red), basal bodies (gamma tubulin, green) and DAPI (blue). (B) Measurement of cilia
length showed that Bbs4 -/- cells have shorter cilia than WT (1.558 ± 0.03 N=101 vs 2.553 ± 0.07
N=81). When the cells are treated with C3 transferase, WT cells have longer cilia than the
untreated ones, (4.697 ± 0.13 N=72 vs 2.553 ± 0.07 N=81). The same phenotype was observed
with Bbs4
-/-
treated and untreated cells (4.636 ± 0.14 N=72 vs 1.558 ± 0.032 N=101: p-
value<0.0001). There is no significant difference in the length of the cilia between treated WT
9
cells and treated Bbs4
-/-
cells.. ***, p-value<0.0001). ns, not significant). Scale Bar 5 m. (C)
Phalloidin-rhodamine stained serum starved cells treated for 5 hours with a 2 g/ml of C3
transferase. Confluent treated WT confluent cells show a reduction of actin filaments, consistent
with the reduction RhoA activity. The same effect can be observed in the Bbs4
-/-
cells, where the
actin aggregates are not present after the treatment. Observe than in treated non-confluent cells
are much more sensitive to C3 transferase and the whole actin cytoskeleton is collapsing in both
cell lines. Scale Bar 20 m. (D). RhoA activity is reduced in C3 transferase treated cells. WT and
Bbs4 -/- cells RhoA hyperactivity of RhoA was reduced after 5 hours of treatment.
10
Supplementary Figure 8. Bbs8 and Bbs9 Antibody Western Blot controls. A. Control and
Bbs8 knockdown cell protein extraction Western blots tested to check the specificity of the Bbs8
antibody. The cells treated with Bbs8 shRNA show a reduction of the 61 kDa band corresponding
to BBS8. A GAPDH antibody was used as loading control for this experiment. B. Bbs9 antibody
western blots from different cell lines protein extracts, including IMCD3 and 3T3 cells lines used
in most of the experiments. A 99 kDa specific band is the one expected for BBS9. C. Gene
expression profile of primary renal cells. In order to check the homogeneity of the cell cultures
we check the expression of epithelial and mesenchimal renal markers. All three primary cell lines
11
(WT, Bbs6 -/- and Bbs4 -/-) have the same profile of gene expression. The primary cells are
expressing Aquaporin-2, an epithelial collecting duct marker but not Uromodulin (also known as
Tamm-Horsfall protein, expressed in cells lining the thick ascending limb of Henle's loop) or
Slc12a3 (also known as Na-Cl cotransporter, expressed in the distal convoluted tubule). All three
primary kidney lines also shown Vimentin expression, linked with a mesenchimal cell lineage.
IMCD3 cells show expression of Uromodulin and Aquoporin-2 but not of Vimentin. These
results show that our primary renal cells are homogeneous in their cellular population
composit
smooth muscle actin; I, IMCD3; 6, Bbs6 -/-; 4, Bbs4 -/-; WT, Wild-type; +, Positive control
whole kidney cDNA; -, negative control.
12
13
Supplementary Figure 9. bbs6 and bbs9 morphants treated with Y27632. 4dpf bbs6 and bbs9
morphants present a characteristic eye reduction, pronephric cysts and curly body. When embryos
were treated with 100 nM of Y27632 the phenotype is partially rescued. WT embryos Not treated
n=52, bbs6 morphants embryos. Not treated n=59, bbs9 morphants embryos Not treated n=51,
WT treated embryos n=43, bbs6 morphants embryos treated n=48, bbs9 morphants embryos
treated n=43 Scale Bar 300 m.
14
Supplementary Figure 10. Recovery of the somite width in bbs6 and bbs9 Y27632 treated
morphants.
A. Flat mounted Phalloidin stained 4dpf zebrafish embryos. B. Quantification of somitic width.
There is a small but statistically significant reduction of the somite width in the Wt treated
15
embryos (71.8 m ± 2.13 N+7 vs 66.5 m ± 1.10 N=11: p-value=0.027). bbs6 and bbs9 treated
morphants recover partially the loss of somite width (bbs6 mo 30.0 m ± 1.19 N=17 vs bbs6 mo
treated 43.0 m ± 0.668 N=9: p-value<0.0001; bbs9 mo 43.9 m ± 0.914 N=11 vs bbs9 mo
treated 49.6 m ± 0.849 N=10). C. The angle formed by the somites in the bbs6 and bbs9
morphants is bigger than the angle found in controls embryos (98.9 o ± 3.12 N=7) than in the
bbs6 (139 o ± 1.45 N=17) and bbs9 (147 o ± 1.38 N=10) morphants. In contrast with the width the
somite angle cannot be rescued by the Y27632 treatment (bbs6 mo 139 o ± 1.45 N=17 vs bbs6
mo treated 137 o ± 1.75 N=9, p-value 0.3650; bbs9 mo 147 o ± 1.38 N=11 vs bbs9 mo treated 146
o
± 1.14 N=10, p-value 0.4833)
16
17
Supplementary Figure 11. Rhodopsin expressing cells are increased in bb6 treated
morphants.
4 dpf Transgenic zebrafish expressing gfp under Rhodopsin promoter were injected with bbs6
morpholino and treated with Y27632. Confocal sections of showed that the Y27632 treatment
didn’t affect the normal expression of Rhodopsin in the WT embryos, with a strong ventral
expression. When bbs6 morpholino was injected a strong reduction of GFP was detected. After
Y27632 treatment of bbs6 mo, there was a recovery of GFP expression with the previously
observed eye diameter expansion. v; ventral.
18
Supplemental Tables
Raw data-Absorbances of the G-LISAs Assays and Normalised figures
RhoA_Bbs4_Assay
Photometric1 Bbs4
Plate 1: plate 1
Value
A
B
C
D
E
F
G
H
Sample
A
B
C
D
E
F
G
H
1
0.4683
0.4935
0.4775
0.5251
2
1.0636
0.9684
0.9356
1.0385
1
Wt
Wt
Wt
Wt
Raw Data
WT
0.4683
0.4935
0.4775
0.5251
1/4
2/4
3/4
4/4
2
bbs4 1/4
bbs4 2/4
bbs4 3/4
bbs4 4/4
bbs4
1.0636
0.9684
0.9356
1.0385
Wt Average
Blank average
3
0.1515
0.1274
0.8645
0.7925
3
Blank_Assay 1/2
Blank_Assay 2/2
Ctrl_0001 1/2
Ctrl_0001 2/2
after blank subtraction
WT
bbs4
0.3288
0.9241
0.3540
0.8289
0.3380
0.7961
0.3856
0.8990
0.3516
4
4
Normalized for Wt Average
bbs4
2.6283
2.3575
2.2642
2.5569
0.1515
0.1274
0.1395
19
RhoA_Bbs6 _Assay
Photometric1 Bbs6
Plate 1: plate 1
Value
A
B
C
D
E
F
G
H
Sample
A
B
C
D
E
F
G
H
1
0.5264
0.5302
0.5602
0.5403
2
0.9664
0.9857
1.0249
0.9735
1
Wt
Wt
Wt
Wt
1/4
2/4
3/4
4/4
Raw Data
WT
bbs4
0.5264
0.9664
0.5302
0.9857
0.5602
1.0249
0.5403
0.9735
Blank average
2
bbs6 1/4
bbs6 2/4
bbs6 3/4
bbs6 4/4
3
0.0925
0.1325
0.9037
0.8328
3
Blank_Assay 1/2
Blank_Assay 2/2
Ctrl_0001 1/2
Ctrl_0001 2/2
after blank subtraction
WT
bbs4
0.4139
0.8539
0.4177
0.8732
0.4477
0.9124
0.4278
0.8610
Wt Average
0.4268
4
4
Normalized for Wt Average
bbs6
2.0007
2.0459
2.1378
2.0173
0.0925
0.1325
0.1125
20
RhoA_bbs8_shRNA_Assay
Photometric1 bbs8_shRNA
Plate 1: plate 1
Value
A
B
C
D
E
F
G
H
Sample
A
B
C
D
E
F
G
H
Wt
Wt
Wt
Wt
1
0.4683
0.5201
0.4964
0.5002
2
1.1825
1.2136
1.1973
0.9552
1
2
bbs8_0001 1/4
bbs8_0001 2/4
bbs8_0001 3/4
bbs8_0001 4/4
1/4
2/4
3/4
4/4
Raw Data
WT
bbs8_shRNA
0.4683
1.1825
0.5201
1.2136
0.4964
1.1973
0.5002
0.9552
Wt Average
Blank average
3
0.1152
0.0952
0.7628
0.7204
3
Blank_Assay 1/2
Blank_Assay 2/2
Ctrl_0001 1/2
Ctrl_0001 2/2
after blank subtraction
WT
bbs8_shRNA
0.3631
1.0773
0.4149
1.1084
0.3912
1.0921
0.3950
0.8500
0.3911
Normalized for Wt Average
bbs8_shRNA
2.7545
2.8341
2.7924
2.1734
0.1152
0.0952
0.1052
21
RhoA_Y27632_treatment
Photometric1_Y27632_Inh
Plate 1: plate 1
Value
A
B
C
D
E
F
G
H
1
0.2003
0.2578
0.2350
0.2073
Sample
A
B
C
D
E
F
G
H
2
0.0658
0.1230
0.1610
0.0658
1
Wt
Wt
Wt
Wt
1/4
2/4
3/4
4/4
3
0.3633
0.3959
0.4370
0.3650
2
Wt_Inh 1/4
Wt_Inh 2/4
Wt_Inh 3/4
Wt_Inh 4/4
Raw Data
Wt
Wt_Y27632 bbs4 -/bbs4 -/- Y27632
0.2003
0.0658
0.3633
0.1627
0.2578
0.1230
0.3959
0.1577
0.2350
0.1610
0.4370
0.1893
0.2073
0.0658
0.3650
0.1572
4
0.1627
0.1577
0.1893
0.1572
3
bbs4_null
bbs4_null
bbs4_null
bbs4_null
1/4
2/4
3/4
4/4
5
0.0125
0.0170
0.5200
0.5320
4
bbs4_Inh 1/4
bbs4_Inh 2/4
bbs4_Inh 3/4
bbs4_Inh 4/4
5
Blank_Assay 1/2
Blank_Assay 2/2
Ctrl_0001 1/2
Ctrl_0001 2/2
after blank subtraction
Wt
Wt_Y27632 bbs4 -/bbs4 -/- Y27632
0.1855
0.0510
0.3485
0.1479
0.2430
0.1082
0.3811
0.1429
0.2202
0.1462
0.4222
0.1745
0.1925
0.0510
0.3502
0.1424
Wt_ Average
0.2103
Normalized for Wt Average
Wt_Y27632
bbs4 -/bbs4 -/- Y27632
0.2425
1.6572
0.7033
0.5145
1.8122
0.6795
0.6952
2.0076
0.8298
0.2425
1.6652
0.6771
Blank Average
0.0125
0.0170
0.0148
22
RhoA_C3_Treatment
Photometric1 C3_treatment
Plate 1: plate 1
Value
A
B
C
D
E
F
G
H
1
0.5501
0.5081
0.5396
0.4934
Sample
A
B
C
D
E
F
G
H
2
0.1477
0.2033
0.1698
0.1547
1
Wt
Wt
Wt
Wt
1/4
2/4
3/4
4/4
3
1.0874
1.0140
1.1592
1.1413
2
Wt_Inh 1/4
Wt_Inh 2/4
Wt_Inh 3/4
Wt_Inh 4/4
Raw Data
Wt
Wt_C3
bbs4 -/bbs4 -/- C3
0.5501
0.1477
1.0874
0.2209
0.5081
0.2033
1.0140
0.2300
0.5396
0.1698
1.1592
0.1884
0.4934
0.1547
1.1413
0.1604
4
0.2209
0.2300
0.1884
0.1604
3
bbs4_null
bbs4_null
bbs4_null
bbs4_null
1/4
2/4
3/4
4/4
5
0.0250
0.0302
0.8560
0.8720
4
bbs4_Inh 1/4
bbs4_Inh 2/4
bbs4_Inh 3/4
bbs4_Inh 4/4
5
Blank_Assay 1/2
Blank_Assay 2/2
Ctrl_0001 1/2
Ctrl_0001 2/2
after blank subtraction
Wt
Wt_C3
bbs4 -/bbs4 -/- C3
0.5225
0.1201
1.0598
0.1933
0.4805
0.1757
0.9864
0.2024
0.5120
0.1422
1.1316
0.1608
0.4658
0.1271
1.1137
0.1328
0.4952
Normalized for Wt Average
Wt_C3
bbs4 -/bbs4 -/- C3
0.2425
2.1402
0.3903
0.3548
1.992
0.4088
0.2872
2.2851
0.3247
0.2566
2.249
0.2682
Blank Average
0.0250
0.0302
0.0276
23
Supplementary Material
Primer sets used for RT-PCRs:
N-cadherin: 5′CACCCAACATGTTTACAATCAACAATGAGAC3′ and
5′CTGCAGCAACAGTAAGGACAAACATCCTATT3
5′CACGAGTAACAAATCAAAGC3′.
Vimentin: 5’CCAGCGCTCCTACGATTCAC3’ and
5’TCTACCTTCTCGTTGGTGCG3’
Aquaporin2: 5’CTCCGGTCCATAGCGTTCTC5’ and
5’GAAGGAGACATGGCAACCCA3’
Uromodulin: 5’AGATCCAGGTGAAGGCTTGC3’ and
5’CTGTCCCACAGGGACCATTC3’
Slc12a3: 5’AGGGCTTGGGAGAATGAAGC3’ and
5’AGGAGGGTGAGACCTCCATC3’.
:
24