Journal: The Journal of Clinical Investigation

Article Title: Chromosomal 3q amplicon encodes essential regulators of secretory vesicles that drive secretory addiction in cancer

doi: 10.1172/JCI176355

Figure Lengend Snippet: ( A ) TurboID-GOLIM4 fusion construct (schema, top) localizes in the Golgi (confocal micrographs, bottom). H1299 cells were transfected with TurboID-GOLIM4 and costained with anti-Flag, anti-GM130, and DAPI (blue). Shown are single-channel and merged images. Scale bar: 50 μm. ( B ) Volcano plot of GOLIM4-associated proteins identified by TurboID-based proximity ligation assays. Results for each protein identified (data points) are expressed as a log 2 ratio (GOLIM4/CTL, x axis; P value, y axis). ( C ) IP/WB confirmation of ATP2C1 as a GOLIM4-associated protein in H1299 cells. ( D ) Confocal micrographs of endogenous ATP2C1 and GOLIM4 in H1299 cells. Cells were treated with nocodazole to disperse the Golgi. Line plot (under images) assesses colocalization of GOLIM4 and ATP2C1. Shown are the signal intensities ( y axis) and distances from the plasma membrane ( x axis). ( E ) Gene copy numbers (rows) in tumors (columns). HNSC, HN squamous cell carcinoma. P <0.001, significant co-occurrence, by 1-sided Fisher’s exact test. ( F ) Correlations between ATP2C1 mRNA levels and gene copy numbers in tumors (data points). Diploid, n = 109; gain, n = 311; amplification, n = 48. ( G ) Pearson’s correlation between GOLIM4 and ATPC1 mRNA levels in tumors (data points). ( H – J ) WB analysis of secreted protein levels in CM samples and cell lysates. H1299 cells were treated with a Ca 2+ chelator ( H ) or transfected with siRNAs against ATP2C1 ( I ) or Cab45 ( J ). ( K ) Number of APP + vesicles in H1299 cells transfected with indicated siRNAs. ( L ) Relative soft agar colony numbers generated by parental and GOLIM4 -KO (G4KO) H1299 cells following treatment with CM samples from siRNA-transfected H1299 cells. ( M ) Boyden chamber assays to quantify HUVEC and CAF recruitment by CM samples from siRNA-transfected H1299 cells. ( N ) qRT-PCR confirmation of target gene depletion by ATP2C1 shRNAs in H1299 cells. ( O and P ) Orthotopic tumor size ( O ) and distant metastases ( P ) per mouse (data points) generated by H1299 transfectants in N . ( Q ) WB confirmation of ATP2C1 reconstitution in ATP2C1 -KO cells by WT or D350A-mutant ATP2C1. Vec, empty vector. ( R and S ) Soft agar colony formation assay ( R ) and Boyden chamber migration assay ( S ) on cells generated in Q . Data indicate the mean ± SD from a single experiment incorporating biological replicate samples ( n = 3, unless otherwise indicated) and are representative of at least 2 independent experiments. * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001, by 1-way ANOVA ( F , K – P , R , and S ).

Article Snippet: 3724 and 2367), β-actin (no. 4970), GM130 (no. 12480), and Golgin-97 (no. 13192) from Cell Signaling Technology; ATP2C1 (13310-1-AP) APP (22952-1-AP), GGH (18070-1-AP), LOXL2 (11405-1-AP), PTX3 (12306-1-AP), GGA1 (25674-1-AP), BAG6 (26417-1-AP), UBL4A (14253-1-AP), TMED3 (21902-1-AP), SPP1 (22952-1-AP), and CLU (12289-1-AP) from Proteintech; GABBR1 (ab55051) from Abcam; and VSV-G (IE9F9) from Kerafast.

Techniques: Construct, Transfection, Ligation, Clinical Proteomics, Membrane, Amplification, Generated, Quantitative RT-PCR, Mutagenesis, Plasmid Preparation, Soft Agar Assay, Migration

Journal: The Journal of Clinical Investigation

Article Title: Chromosomal 3q amplicon encodes essential regulators of secretory vesicles that drive secretory addiction in cancer

doi: 10.1172/JCI176355

Figure Lengend Snippet: ( A ) Full-length and spliced (ΔE7) GOLIM4 isoforms. Exon 7 is located in the intraluminal STEM domain. TM, transmembrane domain; C, cytoplasmic domain. ( B ) IP/WB analysis of GOLIM4-KO H1299 cells reconstituted with full-length or ΔE7 GOLIM4. ATP2C1-binding activity was detected only in full-length GOLIM4-transfected cells. ( C ) WB analysis of secreted proteins in CM samples and cell lysates from parental and GOLIM4 -KO H1299 cells reconstituted with full-length or ΔE7 GOLIM4. α-Tubulin was used as a loading control. ( D ) Quantification of orthotopic tumor size (left) and distant metastases (right) per mouse (data points) generated by H1299 cells in C . ( E ) WB analysis of GOLIM4 levels in H23 cells transfected with full-length GOLIM4 or GOLIM4-ΔE7. ( F – H ) Boyden chamber migration and invasion assays ( F ), soft agar colony assays ( G ), and flank tumor growth assays ( H ) on cells in E . ( I ) qRT-PCR analysis of GOLIM4 isoforms in siRNA-transfected H1299 and H520 cells. Full-length GOLIM4 and GOLIM4-ΔE7 were included as controls. ( J ) IP/WB analysis of H1299 cells demonstrates that FOXF2 depletion attenuated the ATP2C1-binding activity of GOLIM4. ( K and L ) Boyden chamber migration assays ( K ) and relative cell density assays ( L ) on siRNA-transfected H1299 and H520 cells. Data indicate the mean ± SD from a single experiment incorporating biological replicate samples ( n = 3, unless otherwise indicated) and are representative of at least 2 independent experiments. *** P < 0.001, by 1-way ANOVA ( D , F – H , K , and L ).

Article Snippet: 3724 and 2367), β-actin (no. 4970), GM130 (no. 12480), and Golgin-97 (no. 13192) from Cell Signaling Technology; ATP2C1 (13310-1-AP) APP (22952-1-AP), GGH (18070-1-AP), LOXL2 (11405-1-AP), PTX3 (12306-1-AP), GGA1 (25674-1-AP), BAG6 (26417-1-AP), UBL4A (14253-1-AP), TMED3 (21902-1-AP), SPP1 (22952-1-AP), and CLU (12289-1-AP) from Proteintech; GABBR1 (ab55051) from Abcam; and VSV-G (IE9F9) from Kerafast.

Techniques: Binding Assay, Activity Assay, Transfection, Control, Generated, Migration, Quantitative RT-PCR

Journal: The Journal of Clinical Investigation

Article Title: Chromosomal 3q amplicon encodes essential regulators of secretory vesicles that drive secretory addiction in cancer

doi: 10.1172/JCI176355

Figure Lengend Snippet: ( A ) IP/WB analysis of H1299 cells detected HA-GOLPH3 in a GOLIM4-containing protein complex. ( B ) Confocal micrographs of H1299 cells transfected with HA-GOLPH3. Cells were treated with nocodazole to disperse the Golgi and costained with anti-HA and anti-ATP2C1. Scatter plot shows the percentages of HA-GOLPH3 that colocalized with ATP2C1 in each cell (data points). Scale bars: 50 μm. ( C ) WB analysis of secreted protein levels in CM samples and cell lysates following siRNA-mediated GOLPH3 depletion. Relative densitometric values under the gel lanes. α-Tubulin was used as a loading control. ( D ) Boyden chamber assays to quantify HUVEC and CAF recruitment by CM samples from siRNA-transfected H1299 cells. ( E ) GOLIM4 domain structure. KR residues are required to bind to GOLPH3. HA tag, luminal stem (STEM), and acidic domains are shown in the schema. ( F ) IP/WB analysis of GOLIM4 -KO H1299 cells reconstituted with HA-tagged WT (GOLIM4) or mutant (K11A;R12A) GOLIM4. ( G ) WB analysis of secreted proteins in CM samples and lysates from GOLIM4 -KO H1299 cells reconstituted with WT or mutant GOLIM4. ( H and I ) Boyden chamber migration assays ( H ) and flank tumor growth assays ( I ) on cells generated in G . ( J ) ATP2C1 and GOLPH3 are clients of the GOLIM4 scaffold. Data indicate the mean ± SD from a single experiment incorporating biological replicate samples ( n = 3, unless otherwise indicated) and are representative of at least 2 independent experiments. *** P < 0.001, by 2-tailed Student’s t test ( B ) or 1-way ANOVA ( D , H , and I ).

Article Snippet: 3724 and 2367), β-actin (no. 4970), GM130 (no. 12480), and Golgin-97 (no. 13192) from Cell Signaling Technology; ATP2C1 (13310-1-AP) APP (22952-1-AP), GGH (18070-1-AP), LOXL2 (11405-1-AP), PTX3 (12306-1-AP), GGA1 (25674-1-AP), BAG6 (26417-1-AP), UBL4A (14253-1-AP), TMED3 (21902-1-AP), SPP1 (22952-1-AP), and CLU (12289-1-AP) from Proteintech; GABBR1 (ab55051) from Abcam; and VSV-G (IE9F9) from Kerafast.

Techniques: Transfection, Control, Mutagenesis, Migration, Generated

Journal: The Journal of Clinical Investigation

Article Title: Chromosomal 3q amplicon encodes essential regulators of secretory vesicles that drive secretory addiction in cancer

doi: 10.1172/JCI176355

Figure Lengend Snippet: ( A and B ) WB analysis of GOLIM4 levels in lung cancer cells treated with Mn in a dose-dependent ( A ) or time-dependent ( B ) manner. Densitometric quantification of GOLIM4 (line graphs). Values normalized to PBS-treated cells. ( C ) Intra-Golgi Mn sensor M1 activity in lung cancer cells (data points) treated with Mn. ( D ) Single-channel and merged confocal micrographs of Mn sensor M1 (green) in siRNA-transfected H1299 cells treated with Mn. CellLight Golgi-RFP staining is shown in red. Scale bar: 100 μm. ( E ) Relative Mn sensor M1 intensity per cell (data points) in D . ( F ) WB analysis of H23 cells stably transfected with WT (ATP2C1) or D350A-mutant ATP2C1. ( G ) Relative Mn sensor M1 intensity per cell (data points) after Mn treatment of cells generated in F . ( H ) WB analysis of GOLIM4 levels in siRNA-transfected H1299 cells after Mn treatment. ( I ) WB analysis of H23 cells cotransfected with GOLIM4 and ATP2C1. ( J ) Relative densities of H23 cells in I treated with Mn. Data indicate the mean ± SD from a single experiment incorporating biological replicate samples ( n = 3, unless otherwise indicated) and are representative of at least 2 independent experiments. *** P < 0.001, by 2-tailed Student’s t test ( C , E , and J ) or 1-way ANOVA ( G ).

Article Snippet: 3724 and 2367), β-actin (no. 4970), GM130 (no. 12480), and Golgin-97 (no. 13192) from Cell Signaling Technology; ATP2C1 (13310-1-AP) APP (22952-1-AP), GGH (18070-1-AP), LOXL2 (11405-1-AP), PTX3 (12306-1-AP), GGA1 (25674-1-AP), BAG6 (26417-1-AP), UBL4A (14253-1-AP), TMED3 (21902-1-AP), SPP1 (22952-1-AP), and CLU (12289-1-AP) from Proteintech; GABBR1 (ab55051) from Abcam; and VSV-G (IE9F9) from Kerafast.

Techniques: Activity Assay, Transfection, Staining, Stable Transfection, Mutagenesis, Generated

Journal: Scientific Reports

Article Title: A Conditional Knockout Mouse Model Reveals a Critical Role of PKD1 in Osteoblast Differentiation and Bone Development

doi: 10.1038/srep40505

Figure Lengend Snippet: ( a , b ) IHC staining of PKD1 and osteoblastic markers OCN and OPN in femur from 4-week KO and control mouse. Positive osteoblasts were showed by black arrows. ( c ) Number of PKD1 positive, osteopontin positive (N.OPN + ) and osteocalcin positive (N.OCN + ) osteoblasts on the bone surface were measured as positive cells per millimeter of perimeter in sections (B.Pm). Measurements were presented as mean ± SD. * p < 0.05 versus respective control. The data in each group were analyzed using unpaired, two-tailed Student’s t-test. ( d ) Relative mRNA expression levels of osteoblast differentiation markers (ALP, OSX, Runx2, and col-α1) and osteoclast marker (TRAP) in long bone from wild-type and PKD1-KO female mice. The level of each marker gene expression was normalized to the level of the housekeeping gene. n > 3, WT and KO. * p < 0.05.

Article Snippet: For IHC, we incubated primary antibodies which recognized mouse PKD1 (Elabscience, 1:80, ESAP12972), OPN (bioworlde, 1:50, BS1264), OCN (proteintech, 1:50, 23418-1-AP) overnight at 4 °C.

Techniques: Immunohistochemistry, Two Tailed Test, Expressing, Marker

Journal: Scientific Reports

Article Title: A Conditional Knockout Mouse Model Reveals a Critical Role of PKD1 in Osteoblast Differentiation and Bone Development

doi: 10.1038/srep40505

Figure Lengend Snippet: ( a ) ALP staining and ALP-positive cell numbers of MC3T3-E1 and MG63 cells treated with 10 μM of CRT0066101(CRT) for 24 hours, positive numbers represent the mean ± SD of 3 wells (* p < 0.05). ( b ) MC3T3-E1 was transfected with siRNA of PKD1(si-PKD1) and negative control (si-CTL) for 48 hours. Osteoblastic markers (OPN and Runx2) and PKD1 expression were analyzed by Western blotting. The density of proteins bands was scanned and the values were normalized to control. ( c ) 0 day or 3 days differentiating MC3T3-E1 was treated with DMSO or 10 μM of CRT0066101 for 24 hours and then subjected to Western Blotting. ( d ) After days 3, 5, 8 differentiating, MC3T3-E1 cells were treated with CRT0066101 for 24 hours. Expression of PKD1 phosphorylation at ser916 and osteoblast regulators (OPN, Runx2 and OSX) were analyzed by Western Blotting. ( e ) Primary calvarial preosteoblast on the day of cell plating (day 0), on the 8 th and 12 th day of osteogenic differentiation were analyzed by Western blotting to detect expression of osteoblast markers Runx2, and OPN in WT or KO mice (n = 3). All Band densities were normalized to α-tublin content within each sample, and normalized to Day 0 at the far left WT group, control = 1.00. ( f , g ) PKD1, ser916 of PKD1phosphorylation, proliferative markers PCNA and Cyclin D1 were analyzed at the 0, 3 rd , 5 th and 7 th day growth in the MC3T3-E1 and MG63 cells. Representative Western Blots were shown. Uncropped western blot images corresponding to Fig. 6(b,c,d). Figure 6(e,f and g) were shown in .

Article Snippet: For IHC, we incubated primary antibodies which recognized mouse PKD1 (Elabscience, 1:80, ESAP12972), OPN (bioworlde, 1:50, BS1264), OCN (proteintech, 1:50, 23418-1-AP) overnight at 4 °C.

Techniques: Staining, Transfection, Negative Control, Expressing, Western Blot

Journal: Scientific Reports

Article Title: A Conditional Knockout Mouse Model Reveals a Critical Role of PKD1 in Osteoblast Differentiation and Bone Development

doi: 10.1038/srep40505

Figure Lengend Snippet: ( a ) After days 3, 5 differentiating, MC3T3-E1 cells were treated with CRT0066101 or vehicle for 24 hours and then underwent immunoblotting to detect PKD1, PKD1 phosphorylation activity, JAK1, STAT3 and phosphorylation (ser727). Photoshop analyzed the density of bands on western blot. For each marker band, values were normalized to control and the density of control was set at 1. ( b ) Western Blot analysis of differentiating primary calvarial preosteoblasts showed decreased expression of p38,P-p38,STAT3,P-STAT3-ser727, y705 and PCNA in KO cells (0, 12 th day). n = 3, WT or KO mouse. ( c ) MC3T3-EI cells were transfected with si-PKD1 and negative control (si-CTL), pcDNA PKD1 and empty vector plasmid for 60 hours to analyze osteoblast regulators (Runx2, OPN,OSX and OCN) and relative pathway markers (STAT3 and p38). ( d ) MC3T3-EI cells were transfected with si-STAT3 and negative control (si-CTL) for 60 hours. Uncropped western blot images corresponding to Fig. 7(a,b,c and d) were shown in .

Article Snippet: For IHC, we incubated primary antibodies which recognized mouse PKD1 (Elabscience, 1:80, ESAP12972), OPN (bioworlde, 1:50, BS1264), OCN (proteintech, 1:50, 23418-1-AP) overnight at 4 °C.

Techniques: Western Blot, Activity Assay, Marker, Expressing, Transfection, Negative Control, Plasmid Preparation