Fig 2: Loss of UBQLN1 and UBQLN2 increases cell viability, clonogenic potential and combined loss of UBQLN1 and UBQLN2 exhibits additive effect on cell viability, clonogenic potential and induces EMT in A549 cells. Loss of UBQLN1 and UBQLN2 increases cell migration in A549 and HOP62 cells and combined loss of UBQLN1 and UBQLN2 exhibits additive effect on cell migration and cell invasion in A549 cells. (A) Cell viability assay in A549 cells. Cells (500,000) were seeded on 60mm plate and were transfected with either non targeting (si_NT) siRNA or positive control Kif11 (si_Kif11), two different siRNAs targeting UBQLN1 (si_U1 and si_U1-2), two different siRNAs targeting UBQLN2 (si_U2-5 and si_U2-6) and also combining two different siRNAs of UBQLN1 and UBQLN2 (si_U1+si_U2-5, si_U1-2+si_U2-6). After 24 hrs of transfection, cells were trypsinized and 1000 cells were reseeded in 96 well plates and cell viability was assessed for consecutive 4 days. Relative number of cells were compared by performing students’ t-test. * = significance compared to NT (*P < 0.05; **P < 0.005). (B) Clonogenic assay in A549 cells. Cells were prepared as described in A. After 24 hrs of transfection, cells were trypsinized and 1000 cells were reseeded on 6 well plates in triplicates. Following the 10 days, cells were fixed and stained. (C) Quantification of relative number of colonies formed per 1000 number of cells seeded in B. Number of colonies formed were compared by performing students’ t-test. * = significance compared to NT (*P < 0.05; **P < 0.005). (D) Western blot analysis of proteins involved in EMT. Cells were prepared as described in A. After 72 hrs of transfection, cells were harvested and analyzed for protein involved in EMT. * - UBQLN1; + - UBQLN2. (E) Cell migration assay/scratch assay in A549 and HOP62 cells. Representative bar graph showing percentage wound closure following loss of either UBQLN1, UBQLN2 or combined loss of UBQLN1 and UBQLN2. Cells were prepared as described in A. 24 hrs post siRNA transfections a pipette tip was used to scratch the dish to make a “wound”. Cells were examined after wound has been formed and successively for 24hr and 48hr post wound formation, photographed and quantified. Wound closure was analyzed using a Two-way ANOVA followed by a Tukey post-hoc test. * = significance compared to NT, $ = significance compared to U1, ^ = significance compared to U2. One symbol denotes a p-value < 0.05, two symbols denote a p-value < 0.005. (F) Boyden chamber cell migration and invasion assay in A549 cells. Cells were prepared as described in A and 24 h after siRNA transfection cells were seeded into Boyden chambers without or with matrigel. The lower chamber contained media with serum, whereas the upper chamber containing the cells was without serum. 48 h later cells on the underside of the membrane were fixed and stained. (G) Quantification of relative number of cells migrated or invaded through matrigel. Migration and invasion was analyzed using a one way ANOVA followed by a Tukey post-hoc test. * = significance compared to NT, $ = significance compared to U1, ^ = significance compared to U2. One symbol denotes a p-value < 0.05, two symbols denote a p-value < 0.005.

Fig 3: shUbqln1 mES cells Differentiation into Neuronal or Epithelial-like cells. Schematic presentation of UBQLN1 and UBQLN2 having different cell type specific functions. (A) Schematic of doxycycline inducible UBQLN1 shRNA differentiation into mouse embryonic stem cell neuronal (mESN) and mouse embryonic stem cell epithelial (mESE) cells. (B) Representative microscopy images of unmanipulated mES cells and mES cells following doxycycline induction. (C) Representative t-Distributed Stochastic Neighbor Embedding (t-SNE) plots of unmanipulated neuronal/differentiated neuronal and unmanipulated epithelial/differentiated epithelial cells. Neuronal cells revealed identical transcriptional profile throughout the cell populations (green vs black spots) while loss of UBQLN1 found to alter differentiation of epithelial cells (blue vs red spots). (D) Schematic presentation of UBQLN1 and UBQLN2 having different cell type specific functions. UBQLNs has N-terminal UBL domain (ubiquitin-like), C-terminal UBA domain (ubiquitin-associated) and STI regions in the middle. Ubiquilin2 has an additional collagen-like domain between the fourth STI and UBA domains.

Fig 4: RNAseq comparisons of overlapping genes differentially expressed upon UBQLN1 and UBQLN2 knockdown in A549 and SK-N-BE(2) cells. (A) Representative Venn diagram showing comparisons of overlapping genes differentially expressed upon UBQLN1 and UBQLN2 knockdown in A549 cells following RNAseq analysis. (B) Representative list of top 10 genes down-regulated specifically by siUBQLN1. (C) Representative list of top 10 genes down-regulated specifically by siUBQLN2. (D) Representative list of top 10 genes up-regulated specifically by siUBQLN1. (E) Representative list of top 10 genes up-regulated specifically by siUBQLN2. (F) Representative bar graph showing confirmation of genes that were significantly altered following loss of UBQLN1 in A549 and HOP62 cells. (G) Representative Venn diagram showing comparisons of overlapping genes differentially expressed upon UBQLN1 and UBQLN2 knockdown in SK-N-BE(2) cells following RNAseq analysis. (H) Representative Venn diagram showing comparisons of overlapping genes differentially expressed upon UBQLN1 and UBQLN2 knockdown in A549 and SK-N-BE(2) cells following RNAseq analysis.

Fig 5: RNAseq comparisons of overlapping genes differentially expressed upon UBQLN1 knockdown in differentiated iPSC-WNT and mESC-EGFR cells. (A) Representative Venn diagram showing comparisons of overlapping genes differentially expressed upon UBQLN1 knockdown in unmanipulated iPSC and differentiated iPSC-WNT epithelial and iPSC-EGF neuronal cells following RNAseq analysis. (B) Representative list of overlapping 17 genes that were significantly altered following UBQLN1 knockdown in unmanipulated iPSC, differentiated iPSC-WNT epithelial and iPSC-EGF neuronal cells following RNAseq analysis. (* = found in A549 UBQLN1/UBQLN2 knockdown common signature). (C) Representative Venn diagram showing comparisons of overlapping genes differentially expressed upon UBQLN1 knockdown in unmanipulated mESC, differentiated mESC-WNT epithelial and mESC-EGF neuronal cells following RNAseq analysis. (D) Representative list of overlapping 21 genes that were significantly altered following UBQLN1 knockdown in unmanipulated mESC, differentiated mESC-WNT epithelial and mESC-EGF neuronal cells following RNAseq analysis.