Journal: bioRxiv

Article Title: L1CAM signaling through planar cell polarity generates SOX2 + metastatic progenitors in lung adenocarcinoma

doi: 10.1101/2025.08.22.671773

Figure Lengend Snippet: a , Scatter plot of accumulated enrichment score and negative log-transformed false discovery rate (FDR) of pathways associated with the L1CAM + /SOX2 + tumoroid cell cluster defined by scRNA-seq. The pathway with the highest total enrichment score and the lowest FDR is labeled in red. b , Scatter plot showing the expanded list of individual WNT/PCP-related terms. c , Schematic representations of L1CAM and PCP complex components at a cell-cell junction. d , Heatmap displaying the average expression of core PCP components in the KP tumoroid cell clusters defined in . The clusters are ranked left to right according to the average L1cam expression level. e , L1CAM and CELSR1 IF staining in KP tumoroid-derived cell monolayer. The merged image is magnified for visualization ( red box ). Scale bar, 10 μm. f , L1CAM and CELSR1 IF staining of lung metastasis one week after tail vein injection of H23 LUAD cells into athymic mice. Scale bar, 10 μm. g , Co-immunoprecipitation of L1CAM and the PCP component CELSR1 in H23 LUAD cell lysates. h , Quantification of L1CAM/CELSR1 PLA dots per cell in H23 LUAD cells. Negative PLA control, n = 25 cells; L1CAM/CELSR1 PLA, n = 31 cells. **** P < 0.0001. i , Control and L1CAM-knockout (KO) H23 cell monolayers were subjected to cytokeratin, CELSR1, and SOX2 IF staining. Scale bar, 20 μm. j , Fraction of SOX2 + cells quantified in control versus L1CAM-knockout H23 cell monolayers. Mean ± S.D. Control, n = 43 cells; L1CAM KO, n = 58 cells. * P = 0.0488. k , Western immunoblotting analysis of control and L1CAM-knockdown ( shL1CAM ) PDXs after incubating the cells with 1 μM bafilomycin A for 24 h. l , H&E staining of lung sections of athymic mice after tail-vein inoculation of L1CAM-knockout H23 cells with or without SOX2 overexpression. Scale bar, 20 μm. m , n , Box and whisker plots showing the number ( m ) and percent area ( n ) of metastatic lesions per lung in the experiments of panel ( l ). n = 5 for L1CAM KO-1; 10 for L1CAM KO-2. * P = 0.0317; ** P = 0.0012 in ( m ). ** P = 0.0079; *** P = 0.0002 ( n ). Data are shown as a box (median ± 25-75%) and whisker (maximum to minimum values) plot ( h , m , n ). Statistical significance was assessed using the two-tailed Mann-Whitney test ( h , j , m , n ).

Article Snippet: Once membranes were blocked with Intercept Tris-buffered saline (TBS) blocking buffer (LICOR Biosciences Cat# 927-60001) for 1 h at room temperature, they were probed with antibodies against c-Jun (Cell Signaling Technology Cat# 9165, RRID: AB_2130165), c-Jun(pS73) (Cell Signaling Technology Cat# 9164, RRID: AB_330892), CHD1 (Cell Signaling Technology Cat# 4351, RRID: AB_11179073), β-actin (Sigma-Aldrich Cat# A1978, RRID: AB_476692), L1CAM (BioLegend Cat# 826701, RRID: AB_2564904), Sox2 (Cell Signaling Technology Cat# 3579, RRID: AB_2195767), CELSR1 (Bethyl Cat# A304-432A, RRID: AB_2620626), and RTF1 (Cell Signaling Technology Cat# 14737, RRID: AB_2798594) in blocking buffer for overnight at 4°C.

Techniques: Transformation Assay, Labeling, Expressing, Staining, Derivative Assay, Injection, Immunoprecipitation, Control, Knock-Out, Western Blot, Knockdown, Over Expression, Whisker Assay, Two Tailed Test, MANN-WHITNEY

Journal: bioRxiv

Article Title: L1CAM signaling through planar cell polarity generates SOX2 + metastatic progenitors in lung adenocarcinoma

doi: 10.1101/2025.08.22.671773

Figure Lengend Snippet: a , WNT/PCP pathway scoring as the top hit among highly enriched genes in L1CAM + /SOX2 + KP tumoroid cells. Top 3000 ranked differentially expressed genes in cluster #12 versus the rest of cell population in day-7 KP tumoroids were analyzed to determine uniquely enriched biological pathways from the Gene Ontology (GO), Reactome, and PANTHER databases. 30 pathways shared among these three analyses were concatenated into three related processes. 13 of these pathways were from the WNT/PCP-related process and had the lowest accumulated false discovery rate (FDR). Within the WNT/PCP-related processes, the PCP and non-canonical WNT signaling pathways showed the highest enrichment score. b , Heatmap displaying the average expression of core PCP components in the primary tumor clusters defined in . The clusters are ranked left to right according to the average L1cam expression level. c , L1CAM and CELSR1 IF staining in KP tumoroids. The magnified region is indicated by red or white boxes . Scale bar, 10 μm. d , Image analysis workflow for detecting the colocalization of L1CAM and CELSR1 at cell-cell junctions using a steerable filter to extract curvilinear image features. e , Computer vision-driven detection and segmentation of L1CAM and CELSR1 at cell-cell junctions through steerable filtering. The thickness of segmented junctions was dilated for visualization purposes. Scale bar, 1 μm. f , L1CAM and CELSR1 PLA fluorescence in H23 LUAD cells. The PLA signals are shown as red dots with nuclei counterstaining. Scale bar, 10 μm. g , L1CAM western immunoblotting analysis in parental and L1CAM knockout H23 cells. h , Western immunoblotting analysis of SOX2 overexpression in H23 LUAD cells upon CRISPR/Cas9-mediated L1CAM knockout, clones 1 and 2. i. L1CAM and CELSR1 IF staining in HEK293T cells engineered to overexpress L1CAM versus control. Scale bar, 10 μm. j , L1CAM and CELSR1 segmentation analysis at cell-cell junctions in L1CAM overexpressing HEK293T cells. The region of higher magnification is indicated by a red box . k , Quantification of CELSR1 at cell-cell junctions in control or L1CAM overexpressing HEK293T cells. Control, n = 3406 junctions; L1CAM OE, n = 6508 junctions. **** P < 0.0001. l , L1CAM and FZD6 IF staining in control or L1CAM overexpressing HEK293T cells. Scale bar, 10 μm. m , Western immunoblotting analysis of SOX2 overexpression in CHD1 knockout H23 cells. n , Box and whisker plots showing the size of metastatic lesions per lung in SOX2 overexpressing, CHD1 knockout H23 cells. n = 9 for CHD1 KO-1; 10 for CHD1 KO-2. ns, P = 0.2805; * P = 0.0415. Data are shown as a box (median ± 25-75%) and whisker (maximum to minimum values) plot ( k , n ). Statistical significance was assessed using the one-way analysis of variance followed by the Tukey test ( k ) or two-tailed Mann-Whitney test ( n ).

Article Snippet: Once membranes were blocked with Intercept Tris-buffered saline (TBS) blocking buffer (LICOR Biosciences Cat# 927-60001) for 1 h at room temperature, they were probed with antibodies against c-Jun (Cell Signaling Technology Cat# 9165, RRID: AB_2130165), c-Jun(pS73) (Cell Signaling Technology Cat# 9164, RRID: AB_330892), CHD1 (Cell Signaling Technology Cat# 4351, RRID: AB_11179073), β-actin (Sigma-Aldrich Cat# A1978, RRID: AB_476692), L1CAM (BioLegend Cat# 826701, RRID: AB_2564904), Sox2 (Cell Signaling Technology Cat# 3579, RRID: AB_2195767), CELSR1 (Bethyl Cat# A304-432A, RRID: AB_2620626), and RTF1 (Cell Signaling Technology Cat# 14737, RRID: AB_2798594) in blocking buffer for overnight at 4°C.

Techniques: Protein-Protein interactions, Expressing, Staining, Fluorescence, Western Blot, Knock-Out, Over Expression, CRISPR, Clone Assay, Control, Whisker Assay, Two Tailed Test, MANN-WHITNEY

Journal: Scientific Reports

Article Title: The nucleoplasmic interactions among Lamin A/C-pRB-LAP2α-E2F1 are modulated by dexamethasone

doi: 10.1038/s41598-021-89608-3

Figure Lengend Snippet: The cellular quantities of LLRE complex components are dex modulated. ( A ) Typical western blots of total E2F1, pRB and LAP2α protein amounts. ( C ) AT cells showed a larger amount of LAP2α than WT (Mann–Whitney test p < 0.01, n = 8). Dex altered the pRB (in E ) and E2F1 (in G ) amounts only in AT 648 hT sample (Wilcoxon test p < 0.05, n = 8). ( B ) Representative western blots of nuclear E2F1, pRB and LAP2α. ( D ) Also in the nuclear compartment LAP2α is more abundant in the AT sample than in the WT sample (Mann–Whitney test p < 0.05, n = 8), and dex reduced the amount of LAP2α only in AT 648 hT cells (Wilcoxon test p < 0.05, n = 8). ( F ) Nuclear pRB was higher in AT than in the WT cell line (Mann–Whitney test p < 0.01, n = 8). Dex increased the nuclear amount of pRB in both samples (Wilcoxon test, p < 0.05 n = 8). ( H ) The nuclear amount of E2F1 was more elevated in AT than WT cells (Mann–Whitney test p < 0.001, n = 8), and dex considerably reduced its quantity only in AT 648 hT cells (Wilcoxon test, p < 0.001 n = 8). Original images in the series of figures labeled S8O in supplementary information.

Article Snippet: The antibodies listed below were purchased from commercial sources: anti Lamin A/C (Cell Signaling Technology CST #4777 and Diatheva #ANT0072), FITC-conjugated anti Lamin A/C (CST #8617), anti LAP2α (BETHYL, A304-839A-M), anti phospho S22 Lamin A/C (CST #13448), anti phospho S404 Lamin A/C was kindly provided by Prof. Marmiroli , anti pRB (CST #9309, BETHYL #A302-433A-T), anti E2F1 (Santa Cruz Biotechnology #sc-251, BETHYL #A300-766A-M).

Techniques: Western Blot, MANN-WHITNEY, Labeling

Journal: Scientific Reports

Article Title: The nucleoplasmic interactions among Lamin A/C-pRB-LAP2α-E2F1 are modulated by dexamethasone

doi: 10.1038/s41598-021-89608-3

Figure Lengend Snippet: PLA assay outcomes showed that dex altered the interactions between the LLRE elements. ( A ) Dex decreased the extent of nuclear interaction between Lamin A/C and pRB both in WT hT and AT 648 hT cells (Welch test p < 0.001). ( B ) Dex increased the extent of nuclear interaction between Lamin A/C and E2F1 in the WT sample (Welch test p < 0.05) and in the AT 648 hT sample (Welch test p < 0.001). ( C ) No changes were detected in the interaction between pRB and E2F1.

Article Snippet: The antibodies listed below were purchased from commercial sources: anti Lamin A/C (Cell Signaling Technology CST #4777 and Diatheva #ANT0072), FITC-conjugated anti Lamin A/C (CST #8617), anti LAP2α (BETHYL, A304-839A-M), anti phospho S22 Lamin A/C (CST #13448), anti phospho S404 Lamin A/C was kindly provided by Prof. Marmiroli , anti pRB (CST #9309, BETHYL #A302-433A-T), anti E2F1 (Santa Cruz Biotechnology #sc-251, BETHYL #A300-766A-M).

Techniques:

Journal: Scientific Reports

Article Title: The nucleoplasmic interactions among Lamin A/C-pRB-LAP2α-E2F1 are modulated by dexamethasone

doi: 10.1038/s41598-021-89608-3

Figure Lengend Snippet: LLRE complex composition on E2F1 binding site of THBS1 promoter. THBS1 is specifically upregulated by dex only in AT 648 hT cells and likely transcribed by E2F1. In the E2F1 consensus binding site the amounts of ( A ) Lamin A/C, ( B ) LAP2α, ( C ) pRB and ( D ) E2F1 are reported for all samples. The E2F1 improved transcription of THBS1 only in AT 648 hT. This improvement seemed to be dependent on the increase in E2F1 itself, an increase in LAP2α and a concomitant decrease in pRB.

Article Snippet: The antibodies listed below were purchased from commercial sources: anti Lamin A/C (Cell Signaling Technology CST #4777 and Diatheva #ANT0072), FITC-conjugated anti Lamin A/C (CST #8617), anti LAP2α (BETHYL, A304-839A-M), anti phospho S22 Lamin A/C (CST #13448), anti phospho S404 Lamin A/C was kindly provided by Prof. Marmiroli , anti pRB (CST #9309, BETHYL #A302-433A-T), anti E2F1 (Santa Cruz Biotechnology #sc-251, BETHYL #A300-766A-M).

Techniques: Binding Assay

Journal: Scientific Reports

Article Title: The nucleoplasmic interactions among Lamin A/C-pRB-LAP2α-E2F1 are modulated by dexamethasone

doi: 10.1038/s41598-021-89608-3

Figure Lengend Snippet: LLRE complex composition at the E2F1 binding site of the BRCA1 promoter. BRCA1 is specifically downregulated by dex only in AT 648 hT cells, and likely transcribed by E2F1. At the E2F1 consensus binding site the amounts of ( A ) Lamin A/C, ( B ) LAP2α, ( C ) pRB and ( D ) E2F1 are reported for all samples. In this context, the only reasonable explanation is the inhibitory repressor effect of Lamin A/C on the LLRE complex, while all other factors remain unchanged.

Article Snippet: The antibodies listed below were purchased from commercial sources: anti Lamin A/C (Cell Signaling Technology CST #4777 and Diatheva #ANT0072), FITC-conjugated anti Lamin A/C (CST #8617), anti LAP2α (BETHYL, A304-839A-M), anti phospho S22 Lamin A/C (CST #13448), anti phospho S404 Lamin A/C was kindly provided by Prof. Marmiroli , anti pRB (CST #9309, BETHYL #A302-433A-T), anti E2F1 (Santa Cruz Biotechnology #sc-251, BETHYL #A300-766A-M).

Techniques: Binding Assay