Loss of RhoA promotes skin tumor formation. Supplementary Figure 1. Loss of RhoA does not impair F-actin organization.
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1 Supplementary Figure Legends Supplementary Figure 1. Loss of RhoA does not impair F-actin organization. a. Representative IF images of F-actin staining of big and small control (left) and RhoA ko tumors (right). Staining for F-actin, collagen IV, and nuclear DAPI was used. Scale bar: 20 μm. n=4. b. Representative images of F-actin staining by immunofluorescence in control (left) and pool of RhoA ko (right) keratinocytes. Scale bar: 50 μm. n=3. Supplementary Figure 2. Ultrastructural examination of the epithelial-stromal border. a. Images of representative semithin sections from control (left) and RhoA ko (right) tumors. Arrows indicate border between the epithelium (E) and the stroma (S). Note the intercellular space between the RhoA ko keratinocytes and the less defined epidermal-stromal border. Scale bar: 10 μm. b. Images of representative ultrathin sections from control (left) and RhoA ko (right) tumors. Arrows indicate a recognizable basement membrane at the epidermalstromal border. Black/white arrowheads indicate regions where the basement membrane is less defined, but a separation between epithelium (E) from the stroma (S) is evident. Black arrowheads indicate areas of clear disruption of the epithelialstromal border. The asterisks indicate amorphous material. In the absence of RhoA, the epidermal basement membrane and the epidermal-dermal border are less defined. Scale bar: 500 nm. 1
2 Supplementary Figure 3. Altered organization in RhoA-null skin tumors. a. Quantification of edgy contour line of small and big control and RhoA ko tumors. Staining for a6 integrin and nuclear DAPI was used. Scale bar: 20 μm. Control, n=9; RhoA ko, n=10. ***P<0.001, unpaired Student's t-test (two-tailed). b. Quantification of multiple basal cell layers of small and big control and RhoA ko tumors. Staining for a6 integrin and nuclear DAPI was used. Control, n=9; RhoA ko, n=10. ***P<0.001, unpaired Student's t-test (two-tailed). c. Quantification of scattered proliferative cells (Ki67 positive) of small and big control and RhoA ko tumors. Staining for a6 integrin, Ki67, and nuclear DAPI was used. Control, n=7; RhoA ko, n=7. **P<0.01, unpaired Student's t-test (two-tailed). d. Quantification of scattered suprabasal cells (keratin10) of small and big control and RhoA ko tumors. Staining for a6 integrin, keratin 10, and nuclear DAPI was used. Control, n=9; RhoA ko, n=10. ***P<0.001 and **P<0.01, unpaired Student's t-test (two-tailed). e. Quantification of E-cadherin expression of small and big control and RhoA ko tumors. Staining for E-cadherin, and nuclear DAPI was used. Control, n=5; RhoA ko, n=6. Unpaired Student's t-test (two-tailed). f. Quantification of areas with K10 positive expression of control and RhoA ko tumor epidermis. Staining for a6 integrin, keratin 10, and nuclear DAPI was used. Control, n=7; RhoA ko, n=10. Unpaired Student's t-test (two-tailed). g. Quantification of areas with reduced expression of E-cadherin of control and RhoA ko tumor epidermis. Staining for E-cadherin, and nuclear DAPI was used. Control, n=5; RhoA ko, n=6. **P<0.01, unpaired Student's t-test (two-tailed). 2
3 h. Quantification of ki67 positive cells of control and RhoA ko tumors. Staining for a6 integrin, Ki67, and nuclear DAPI was used. Control, n=7; RhoA ko, n=7. Unpaired Student's t-test (two-tailed). i. Representative IF images of edgy contour line of control (left) and RhoA ko (right) tumors. Staining for a6 integrin and nuclear DAPI was used. Scale bar: 50 μm. j. Representative IF images of multiple basal cell layers of control (left) and RhoA ko (right) tumors. Staining for a6 integrin and nuclear DAPI was used. k. Representative IF images of scattered proliferative cells (Ki67 positive) of control (left) and RhoA ko (right) tumors. Staining for a6 integrin, Ki67, and nuclear DAPI was used. l. Representative IF images of scattered suprasabal cells (keratin10) of control (left) and RhoA ko (right) tumors. Staining for a6 integrin, keratin 10, and nuclear DAPI was used. Arrowheads indicate scattered suprabasal cells among basal cells. m. Representative IF images of E-cadherin expression of control (left) and RhoA ko (right) tumors. Staining for E-cadherin, and nuclear DAPI was used. Arrowheads indicate areas with reduction expression of E-cadherin. Supplementary Figure 4. Altered proliferation and double nucleated cells in RhoAnull tumor-free skin.. a, b. Representative IHC (a) and quantification (b) of ki67 positive cells of control (left) and RhoA ko (right) DMBA/TPA-treated tumor-free skin. Staining for Ki67 was used. Control, n= 100 fields from 6 mice; RhoA ko, n 100 fields from 9 mice. ***P<0.001, unpaired Student's t-test (two-tailed). 3
4 c, d. Representative HE stainings (c) and quantification (d) of double-nucleated cells in control (left) and RhoA ko (right) DMBA/TPA-treated tumor-free skin. Arrowheads indicate double-nucleated cells. Scale bar: 10 μm. n=4. *P<0.05, unpaired Student's t-test (two-tailed). h, e. Representative IF images(h) and quantification (e) of DNA damage of control (left) and RhoA ko tumors (right). Staining for gh2ax, a6 integrin, and nuclear DAPI was used. Arrowheads indicate gh2ax positive cells. Scale bar: 20 μm. n=2. Supplementary Figure 5. Survival and apoptotic signalings are not affected by RhoA deletion in vivo. a, b. Representative blot and quantification of Western blot of lysates from control and RhoA ko DMBA/TPA-treated tumor-free back skin (a) and control and RhoA ko tumors for phospho-akt. a: Control, n=3; RhoA ko, n=4. b: n=6 from 3 mice. e: n=18 from 4 mice. Unpaired Student's t-test (two-tailed). c. Representative IF images of Caspase 3 expression of control (left) and RhoA ko tumors (right). Staining for Caspase 3, a6 integrin, and nuclear DAPI was used. Scale bar: 50 μm. Control, n=3; RhoA ko, n=5. Supplementary Figure 6. RhoA deletion induces invasion in vitro. a. In vitro invasion assay with control keratinocytes, subclone RhoA ko2, pool of RhoA ko keratinocytes, and subclone RhoA ko2 and pool of RhoA ko keratinocytes 48h after transfection with EGFP or RhoA-EGFP expressing plasmids is shown. Data are represented relative to control invading cells. n 3. ***P<0.001, **P<0.01, and *P<0.05, unpaired Student's t-test (two-tailed). 4
5 b, d. Representative blot and quantification of Western blot of lysates from control and RhoA ko immortalized keratinocytes for a6 integrin (b) and E-cadherin (d). Expression levels were normalized to actin and are represented relative to the corresponding control cells. b: n=4. d: n=3. *P<0.05, unpaired Student's t-test (twotailed). c. Invasion assay with control and pool of RhoA ko keratinocytes treated with blocking a6 integrin antibody or IgG. Data are represented relative to control invading cells. n=3 in duplicates. ***P<0.001, **P<0.01, and *P<0.05, unpaired Student's t-test (two-tailed). Supplementary Figure 7. Increased Rho signaling in RhoA ko keratinocytes. a. Representative blot and quantification of Western blot of lysates from control and pool of RhoA ko keratinocytes and pool of RhoA ko keratinocytes after 96h of transfection with EGFP or RhoA-EGFP expressing plasmids for phospho-mlc, phospho-cofilin and RhoA, as indicated. Expression levels were normalized to Actin and are represented relative to control keratinocytes. n=3. **P<0.01 and *P<0.05, unpaired Student's t-test (two-tailed). b. Representative blot and quantification of Western blot of lysates from control and subclone RhoA ko2 and subclone RhoA ko2 after 96h of transfection with EGFP or RhoA-EGFP expressing plasmids for phospho-mlc, phospho-cofilin and RhoA, as indicated. Expression levels were normalized to Actin and are represented relative to control keratinocytes. n 3. ***P<0.001 and *P<0.05, unpaired Student's t-test (twotailed). 5
6 c, d. Representative blot and quantification of Western blot of lysates from control and RhoA ko DMBA/TPA-treated tumor-free back skin (c), control and pool of RhoA ko keratinocytes, subclone RhoA ko1, and subclone RhoA ko2 for total MLC and total cofilin. Expression levels were normalized to actin and are represented relative to control keratinocytes. c: Control, n=3 mice; RhoA ko, n= 4 mice. d: n 3. *P<0.05, unpaired Student's t-test (two-tailed). Supplementary Figure 8. Increased Rho signaling in RhoA ko keratinocytes is necessary for in vitro invasion. a, b. Representative blot and quantification of Western blot of lysates from control and RhoA ko DMBA/TPA-treated tumor-free back skin (c), control and pool of RhoA ko keratinocytes, subclone RhoA ko1, and subclone RhoA ko2 for ROCK1and ROCK2. Expression levels were normalized to actin and are represented relative to control keratinocytes. a: Control, n=3 mice; RhoA ko, n= 4 mice. b: n 3. *P<0.05, unpaired Student's t-test (two-tailed). c, d. Invasion assay of control, pool of RhoA ko keratinocytes (c) or subclone RhoA ko2 (d) treated with Y27632 or Blebbistatin. Data are represented relative to control invading cells. n 4. **P<0.01 and *P<0.05, unpaired Student's t-test (two-tailed). e, f, h. Representative Western blot of lysates from control and subclone RhoA ko1 keratinocytes in the absence or presence of Y27632 (e, h) or blebbistatin (f, h) for 24h for phospho-mlc, phospho-cofilin, and a6 integrin as indicated. n 2. g. Representative bright-field images showing morphology of control (left) and subclone RhoA ko1 (right) in the absence or presence of Y27632 or blebbistatin for 24h. Scale bar: 50 μm. 6
7 Supplementary Figure 9. RhoB overcompensates the loss of RhoA under hyperproliferating conditions. a-c, f, i, j. Representative blot and quantification of Western blot of lysates from control and RhoA ko tumors (a), control and pool of RhoA ko keratinocytes (b), or subclone RhoA ko2 (c); pool of RhoA ko keratinocytes and subclone RhoA ko2 after 96h of transfection with EGFP or RhoA-EGFP expressing plasmids (b, c); pool of RhoA ko keratinocytes and RhoA/RhoB ko keratinocytes obtained by lentiviral transduction of CRISPR construct targeting RhoB into pool of RhoA ko keratinocytes (f); control keratinocytes after 96h of transfection with EGFP or RhoB-EGFP expressing plasmids (i, j) for RhoB, RhoC, phospho-mlc and phospho-cofilin, as indicated. Expression levels were normalized to actin and are represented relative to the corresponding control cells. a: Control, n=4 mice; RhoA ko, n= 10 mice. b, c, f, i, j: n 3. ***P<0.001, **P<0.01 and *P<0.05, unpaired Student's t-test (two-tailed). d, Representative Western blot and Rho-GTP pull down (PD) for RhoC of lysates from control and subclone RhoA ko1 keratinocytes. n=4. e. Representative blot and quantification of Western blot of total lysates (input) and membrane fraction (MF) of control keratinocytes and subclone RhoA ko1 for RhoB and TGFBRI. Expression levels were normalized to TGFBRI and are represented relative to the corresponding control cells. n=4. **P<0.01, unpaired Student's t-test (two-tailed). g, h. Representative blot and quantification of Western blot and Rho-GTP pull down for RhoB, phospho-mlc and phospho-cofilin of lysates from untreated or starved control and pool of RhoA ko keratinocytes. Expression levels were normalized to 7
8 actin and are represented relative to untreated conditions. g: n=5. h:.n 3 **P<0.01 and *P<0.05, unpaired Student's t-test (two-tailed). Supplementary Figure 10. RhoC does not overcompensate RhoA loss. a. Representative blot and quantification of Western blot of lysates from subclone RhoA ko2 and RhoA/RhoC ko keratinocytes for RhoB, phospho-mlc and phosphocofilin. Expression levels were normalized to actin and are represented relative to control cells. n 3. **P<0.01, unpaired Student's t-test (two-tailed). b. Invasion assay with subclone RhoA ko2 and RhoA/RhoC ko keratinocytes is shown. Data are represented relative to control invading cells. n=6. Unpaired Student's t- test (two-tailed). Supplementary Figure 11. RhoA and RhoB have redundant functions with respect to clonogenic growth. a. Representative Western blot showing efficient loss of RhoA and RhoB protein in primary keratinocytes. b. Clonogenic growth assay of control, RhoA ko, RhoB ko and RhoA/B ko primary keratinocytes. n 4. ***P<0.001 and *P<0.05, unpaired Student's t-test (two-tailed). c. Percentage of dead cells of control, RhoA ko, RhoB ko and RhoA/B ko primary keratinocytes after 24h of media exchange. n 4. **P<0.01 and *P<0.05, unpaired Student's t-test (two-tailed). Supplementary Figure 12. RhoA regulates lysosomal degradation of RhoB and GABARAP. 8
9 a, b. qrt-pcr analysis of RhoB and RhoC of control and pool of RhoA ko keratinocytes (a) or subclone RhoA ko2 (b). Expression levels were normalized to actin and are represented relative to control keratinocytes. n=3. *P<0.05, unpaired Student's t-test (two-tailed). c-h. Representative blot and quantification of Western blot for RhoB of lysates from control keratinocytes, subclones RhoA ko1 and RhoA ko2 treated with cyclohexamide (CHX) (c, f), chloroquine (CQ)(d, g), and starvation (e, h) at the indicated time points. Expression levels were normalized to actin and are represented relative to untreated conditions. For each treatment, all samples derived from the same experiment and gels/blots were processed in parallel. Different exposition times are shown for blots from control and RhoA ko1 cells. n 3. **P<0.01 and *P<0.05, unpaired Student's t-test (two-tailed). i, j. Representative blot and quantification of Western blot of lysates from control keratinocytes, subclones RhoA ko1 and RhoA ko2 for GABARAP and NDP52. Expression levels were normalized to actin and are represented relative to control cells. n=3. **P<0.01 and *P<0.05, unpaired Student's t-test (two-tailed). k. Representative blot and quantification of Western blot of lysates from control and pool of RhoA ko immortalized keratinocytes in the absence or presence of E64d/Pepstatin A for 1h for RhoB, GABARAP, and LC3B. Expression levels were normalized to actin and are represented relative to untreated conditions. n 5. **P<0.01 and *P<0.05, unpaired Student's t-test (two-tailed). Supplementary Figure 13. ATG5, ATG7, GABARAP in RhoB regulation and invasion. 9
10 a, d. Representative blot and quantification of Western blot and Rho-GTP pull down of lysates from control and pool of ATG5, ATG7, GABARAP ko keratinocytes for RhoB, phospho-mlc, GABARAP, ATG7, and RhoB-GTP, as indicated. Expression levels were normalized to actin and are represented relative to control cells. n 4. ***P<0.001 and **P<0.01, unpaired Student's t-test (two-tailed). b. Representative images of GFP staining by immunofluorescence in control and pool of ATG5, ATG7, GABARAP ko keratinocytes after 48h of transfection with RhoB-EGFP expressing plasmid. Scale bar: 20 μm. n=3. c. Invasion assay of control and pool of ATG5, ATG7, GABARAP ko keratinocytes is shown. Data are represented relative to control invading cells. n 4. *P<0.05, unpaired Student's t-test (two-tailed). Supplementary Figure 14. Loss of RhoA leads to accumulation of RhoB/GABARAP vesicles. a. Representative images of GFP, GABARAP and LAMP1 staining by immunofluorescence in untreated or 2h CQ-treated control and pool of RhoA ko keratinocytes after 48h of transfection with RhoB-EGFP expressing plasmid. Scale bar: 10 μm. n=3. Arrowheads indicate vesicles RhoB+, GABARAP-. Arrows indicate vesicles RhoB+, LAMP1+, GABARAP-. b. Quantification of GABARAP dots per cell in control and pool of RhoA ko keratinocytes in the absence or presence of CQ for 2h. n=3. ***P<0.001 and **P<0.01, unpaired Student's t-test (two-tailed). c. Quantification of cells with more than 15 double positive GABARAP/RhoB dots (left) and quantification of the fraction of RhoB overlapping with GABARAP 10
11 represented as relative Manders' Coefficients (right) in control and pool of RhoA ko keratinocytes in the absence or presence of CQ for 2h. n=3.. ***P<0.001, **P<0.01 and *P<0.05, unpaired Student's t-test (two-tailed). d. Quantification of the fraction of RhoB overlapping with LAMP1 represented as relative Manders' Coefficients in control and pool of RhoA ko keratinocytes in the absence or presence of CQ for 2h. n=3. **P<0.01, unpaired Student's t-test (twotailed). e. Quantification of the fraction of GABARAP overlapping with LAMP1 represented as relative Manders' Coefficients in control and pool of RhoA ko keratinocytes in the absence or presence of CQ for 2h. n=3. **P<0.01 and *P<0.05, unpaired Student's t- test (two-tailed). 11
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