Journal: Acta biomaterialia

Article Title: Bioconjugated liquid-like solid enhances characterization of solid tumor - chimeric antigen receptor T cell interactions.

doi: 10.1016/j.actbio.2023.09.042

Figure Lengend Snippet: Fig. 2. Expression of CD70 on solid tumors and confirmation of target expression and CAR T cell transduction efficiency. (A) Gene expression of CD70 in various solid tumors compared to normal matched tissues was analyzed using data from The University of Alabama at Birmingham Cancer data analysis (UALCAN) and The Cancer Genome Atlas program (TCGA). B-C) Top: confirmation of CD70 (target) expression on cancer cells post transduction – (B) glioblastoma (GBM) and (C) osteosarcoma (OS) models - and Bottom: transduction of CAR construct in (B) C57BL/6 mice - derived T cells (CAR T Kr158B ) and (C) in Balb/c mice - derived T cells (CAR T K7M2 ) as indicated by GFP reporter. On average, (D) the mean CD70 expression was found to be 73% for GBM and 99% for OS models. (E) The mean transduction efficiency was 66% for CAR T Kr158B and 60% for CAR T K7M2 . The names of the cell lines and tumor models, GBM for Kr158B and OS for K7M2, will be used interchangeably in the study. Number of biological replicates (N) is indicated on the graphs.

Article Snippet: Cell lines 293T cell line (ATCC, CRL-3216) was used for lentiviral plasid production encoding for mouse full length CD70 surface antien (GenBank: U78091.1).

Techniques: Expressing, Transduction, Gene Expression, Construct, Derivative Assay

Journal: Acta biomaterialia

Article Title: Bioconjugated liquid-like solid enhances characterization of solid tumor - chimeric antigen receptor T cell interactions.

doi: 10.1016/j.actbio.2023.09.042

Figure Lengend Snippet: Fig. 3. CAR T cell exhibit directed motion towards the target tumor. A) Representative confocal snapshots showed CD70-specific CAR T cells (green) navigating through supported COL1-LLS RhB microgels (red) and infiltrating the target GBM tumor (white), with blue arrows indicating the paths of CAR T cell migration. (B, D) Mean velocity of CAR T cells co-cultured with CD70-positive tumors and the wild-type (WT) control were quantified for GBM (B) and OS (D) tumor models. The number of tracks (n) and biological replicates (N) were indicated on the plots, and statistical significance was determined using an unpaired two-tailed Student’s t-test with p values indicated on the plots. (C, E) Tumor-infiltrating CAR T cells were quantified as a percentage of total CAR T cells on average from 0 to 72 h for GBM (C) and OS (E) tumors, displayed as box plots showing 25th and 75th percentiles, median, and mean, with whiskers representing the minimum to maximum observations. An unpaired two-tailed Student’s t-test was performed for statistical analysis, and p values were indicated on the plots. (F) Top panel: maximum intensity z projection showed snapshots of CAR T – GBM tumor interaction at 0, 24, and 72 h. Middle panel: the segmentation of CAR T with colors indicating individual CAR T cells at each frame. Bottom panel: maximum intensity projection of segmented CAR T cell velocity tracks over time with color-coded velocity gradient, revealing accumulation of CAR T cells inside the tumor. The segmentation employed a deep learning-based method, as discussed in the methods section, and the cells were tracked using a Linear Assignment Problem (LAP) tracker with maximum frame-to-frame linking and allowable track segment gap closing of 150 μm. (G) Evidence of chemotaxis and upregulation in migratory pathways for CAR T cells co-cultured with their target tumors was demonstrated for GBM (top panel) and OS (bottom panel) tumor models. Notably, evidence of immune-mediated cytotoxic function was shown through IFN γ detection. Error bars represent standard deviation.

Article Snippet: Cell lines 293T cell line (ATCC, CRL-3216) was used for lentiviral plasid production encoding for mouse full length CD70 surface antien (GenBank: U78091.1).

Techniques: Migration, Cell Culture, Control, Two Tailed Test, Chemotaxis Assay, Standard Deviation

Journal: Acta biomaterialia

Article Title: Bioconjugated liquid-like solid enhances characterization of solid tumor - chimeric antigen receptor T cell interactions.

doi: 10.1016/j.actbio.2023.09.042

Figure Lengend Snippet: Fig. 4. CAR T expansion, activation, and killing. (A) Representative confocal time-lapse images of the FITC channel (green) illustrating immune activation, expansion, and killing of the target tumors. CAR T cell clustering and rapid expansion were observed in almost all conditions with efficient anti-tumor activity. White arrows indicate CAR T cell clusters. (B, C) The number of CAR T cell clusters rapidly increased within the first 24 h and remained steady for over 72 h in (B) GBM and (C) OS models. (D, E) Cluster size in all co-cultures with target tumors steadily increased over time, but not in WT controls. (F, G) Expansion of CAR T cell clusters revealed an inverse correlation with tumor size. n = 3 for all CD70 pos samples and n = 2 for all WT samples. (H, I) Endpoint flow cytometry data measuring CAR T cell expansion after 96 h of co-culture with cancer cells in the 2D assay for both tumor models. (J, K) A comparison in IFN γ secretion (pg/mL) between 2D and 3D cultures for (J) GBM and (K) OS. Error bars indicate SD and the number of biological replicates is n = 2. (L) Top row: confocal 3D snapshots of GBM tumors after co-cultured with CAR T cells (E:T = 1:4) for 24 h, showing highly tortuous tumor margins in CD70 pos tumors compared to WT counterparts. Bottom row: cross-section view (at z depth: 70 μm) of the sample on the top row revealing infiltrating CAR T cells within the tumor mass. (M) Measurement of tumor tortuosity factors revealed more than 3-fold change for the CD70 pos tumors within the first 48 h of co-culture. The tumor tortuosity factor was calculated as the ratio between the perimeter of the tumor outline and the perimeter of a circle with the same pixel area. Data was obtained from GBM samples ( n = 3) at an initial E:T ratio of 1:2 and from osteosarcoma (K7M2) samples ( n = 3) at an initial E:T ratio of 1:1. Error bars represent standard deviation and biological samples ( n = 3) for each group were performed unless indicated otherwise.

Article Snippet: Cell lines 293T cell line (ATCC, CRL-3216) was used for lentiviral plasid production encoding for mouse full length CD70 surface antien (GenBank: U78091.1).

Techniques: Activation Assay, Activity Assay, Cytometry, Co-Culture Assay, Two-Dimensional Assay, Comparison, Cell Culture, Standard Deviation

Journal: Acta biomaterialia

Article Title: Bioconjugated liquid-like solid enhances characterization of solid tumor - chimeric antigen receptor T cell interactions.

doi: 10.1016/j.actbio.2023.09.042

Figure Lengend Snippet: Fig. 5. Quantification of CAR T–tumor interactions . Top rows: confocal timelapse images at 0, 24, 48, and 72 h showing dynamic immune-cancer interactions and antitumor activities of CAR T cells in (A) GBM and (B) OS tumor models. The bottom row of each group shows a digital reconstruction of the confocal data with distinct segmentation of tumor and immune cell populations. The initial CAR T to tumor cell ratio or effector-to-tar get (E:T) ratio is indicated on the graph. (C, D) Quantification of CAR T–tumor interactions revealing an inverse correlation between tumor size and infiltrating CAR T cells for (C) GBM and D) OS tumors. The percentage of tumor size (left axis) and CAR T cells infiltrating the tumor (right axis) were normalized to the original tumor size at time 0. (E, F) Killing rates over time were calculated as derivatives from C and D, respectively. (G, H) The measured CAR T to cancer cell area ratios (E:T) as a function of time. The E:T ratios dynamically changed over time, while for WT tumors, the ratios remained constant. (I) RNA sequencing data from single CAR T cells isolated from a 3D co-culture with target CD70 pos versus WT tumors. All genes shown in the results have a statistical significance of p ≤0.05. Error bars represent standard deviation and biological samples n = 3 for all CD70 pos samples and n = 2 for all WT samples.

Article Snippet: Cell lines 293T cell line (ATCC, CRL-3216) was used for lentiviral plasid production encoding for mouse full length CD70 surface antien (GenBank: U78091.1).

Techniques: RNA Sequencing, Isolation, Co-Culture Assay, Standard Deviation

Journal: Acta biomaterialia

Article Title: Bioconjugated liquid-like solid enhances characterization of solid tumor - chimeric antigen receptor T cell interactions.

doi: 10.1016/j.actbio.2023.09.042

Figure Lengend Snippet: Fig. 7. Sensitivity of anti-tumor activity to various CAR T: cancer cell (E:T) ratios. (A,B) confocal time-lapse images of CAR T – tumor co-culture in iVITA at different E:T ratios. (C,D) show the digital image reconstruction of confocal data quantifying bulk tumor mass, migrating single cancer cells, immune cells, and immune cell clusters, and killing activity over time. (A,C) GBM CD70 pos tumors and (B,D) OS CD70 pos tumors were co-cultured with their respective CAR T cells at different concentrations corresponding to E:T of 1:4, 1:2, and 4:1 for the GBM model and E:T of 1:4, 1:1, and 4:1 for the OS model. (E–H) CAR T expansion as a function of initial E:T seeding ratios. (E,F) CAR T expansion on average in both models from 0 to 72 h. For each seeding E:T, CAR T expansion at each time point was normalized to the CAR T at time 0, and the average was calculated for all frames. G,H) the number of CAR T clusters counted every 1.5 h for 72 h for each group. The box plots display 25th and 75th percentiles, a line at the median a plus sign at the mean, from the minimum to the maximum observation. (I,J) Quantification of tumor size over time at the initial E:T = 1:4, 1:2, and 4:1 for I) GBM model and at E:T = 1:4, 1:1, and 4:1 for (J) OS model. (K,L) The E:T ratio dynamically changed over time. The CAR T expansion and tumor-killing were presented by the exponential increase of E:T ratios. (M,N) tumor killing rates were calculated as derivatives from (I) and (J), respectively. Error bars represent standard deviation. Statistical analysis was performed using Ordinary One-Way ANOVA. ( n = 3 unless indicated otherwise, ∗∗= p < 0.01, and ∗∗∗∗= p < 0.0 0 01).

Article Snippet: Cell lines 293T cell line (ATCC, CRL-3216) was used for lentiviral plasid production encoding for mouse full length CD70 surface antien (GenBank: U78091.1).

Techniques: Activity Assay, Co-Culture Assay, Cell Culture, Standard Deviation

Journal: Nature Communications

Article Title: ATF4 selectively regulates heat nociception and contributes to kinesin-mediated TRPM3 trafficking

doi: 10.1038/s41467-021-21731-1

Figure Lengend Snippet: a – c TRPM3 expression in the DRG membrane fraction ( a ), cytoplasmic fraction ( b ) and total lysate ( c ) from WT and Atf4 +/− mice. n = 3 mice per group. t 4 = 10.3, P = 0.0005 in a . t 4 = 6.043, P = 0.0038 in b . d TRPM3 surface levels were measured in cultured DRG neurons prepared from WT and Atf4 +/− mice using a surface biotinylation assay. n = 3 cultures per group. t 4 = 6.28, P = 0.0033. e Co-IP showing the ATF4/TRPM3 interaction in DRGs. DRG lysates were immunoprecipitated with an ATF4 (top) or TRPM3 (bottom) antibody and immunoblotted with a TRPM3 or ATF4 antibody as indicated. This experiment was repeated three times. f The SIM images show that the colocalization between ATF4 and TRPM3 in DRG neurons. Scale bar, 5 μm. g The GST pull-down assay with two purified proteins, TRPM3-GST-Flag and ATF4, showed a direct interaction between ATF4 and TRPM3. This experiment was repeated three times. h Interaction between TRPM3 and ATF4 mutants. The ATF4 mutants was transiently co-expressed with TRPM3, and the cell lysates were immunoprecipitated with a His antibody and then immunoblotted with a His or Flag antibody as indicated. This experiment was repeated three times. i – k TRPM3 expression in the DRG membrane fraction ( i ), cytoplasmic fraction ( j ) and total lysate ( k ) after ATF4 overexpression. n = 6 mice per group. F (2,15) = 14.31, P = 0.0007 in i . F (2,15) = 30.15, P < 0.0001 in j . l , n , p Time course of a whole-cell patch-clamp recording showing the effect of CIM0216 (3 μM) and isosakuranetin (Iso; 10 μM) on the TRPM3 current in DRG neurons from WT ( l ), Atf4 +/− ( n ) and rescued ( p ) mice. The open circle indicates the outward current recorded at + 75 mV, and the closed circle indicates the inward current recorded at −75 mV. m , o , q I–V relationship of the TRPM3 current at the time points indicated in I , n and p . r Histogram of the TRPM3 current density (outward and inward, current before CIM0216 was subtracted) of DRG neurons from WT, Atf4 +/− and ATF4 rescued mice after CIM0216 treatment. n = 6 neurons per group. F (2,15) = 8.735, P = 0.0049 in WT vs. Atf4 +/− , P = 0.0097 in Atf4 +/− vs. rescue in +75 mV; F (2,15) = 9.158, P = 0.0038 in WT vs. Atf4 +/− , P = 0.0089 in Atf4 +/− vs. rescue in −75 mV. a – d Two-tailed Independent Student’s t test. i , j , k , r , One-way ANOVA followed by Tukey’s multiple comparisons test, * P < 0.05, ** P < 0.01, n.s. means not significant. The error bars indicate the SEMs.

Article Snippet: The membranes were placed in blocking buffer for 1 h at RT and incubated in primary antibodies against ATF4 (goat, 1:1000, GeneTex, catalogue no.: GTX89973), TRPM3 (rabbit, 1:1000, Bioss, catalogue no.: bs-9046R; rabbit, 1:200, Alomone Labs, catalogue no.: ACC-050), TRPV1 (rabbit, 1:200, Alomone Labs, catalogue no.: ACC-030), TRPA1 (rabbit, 1:1000, ABclonal, catalogue no.: A12544), KIF17 (mouse, 1:100, Santa Cruz Biotechnology, catalogue no.: sc-137040), KIF3A (goat, 1:100, Santa Cruz Biotechnology, catalogue no.: sc-18745), KIF3B (rabbit, 1:100, Santa Cruz Biotechnology, catalogue no.: sc-50456), KIF5A (rabbit, 1:1000, Abcam, catalogue no.: ab5628), KIF5B (rabbit, 1:2000, Abcam, catalogue no.: ab167429), KIFC2 (rabbit, 1:100, Abcam, catalogue no.: ab3476), TfR (mouse, 1:1000, Thermo Fisher Scientific, catalogue no.: 13-6800), CXCR4 (rabbit, 1:1000, Abcam, catalogue no.: ab124824), CXCR7 (rabbit, 1:1000, Abcam, catalogue no.: ab72100), β-tubulin (mouse, 1:2000, Arigo, catalogue no.: ARG62347), Flag (rabbit, 1:1000, Cell Signaling Technology, catalogue no.: 14793) or His (rabbit, 1:1000, Cell Signaling Technology, catalogue no.: 12698) overnight at 4 °C.

Techniques: Expressing, Cell Culture, Surface Biotinylation Assay, Co-Immunoprecipitation Assay, Immunoprecipitation, Pull Down Assay, Purification, Over Expression, Patch Clamp, Two Tailed Test

Journal: Nature Communications

Article Title: ATF4 selectively regulates heat nociception and contributes to kinesin-mediated TRPM3 trafficking

doi: 10.1038/s41467-021-21731-1

Figure Lengend Snippet: a The diagram shows the experimental procedure. The hindpaws of mice were soaked in a 43 °C water bath for 30 s, and the mice were placed at RT for 2, 5, 10, 20 or 40 min. Then, behavioural, co-IP and membrane protein immunoblotting experiments were performed. b , c ATF4/TRPM3 interactions ( b ) and TRPM3 membrane expression ( c ) in mice were evaluated at different time points after heat stimulation. The experiment was repeated four times in b . n = 6 mice per group in c . F (5,18) = 20.17, P = 0.0007 in 2 min, P < 0.0001 in 5 min, P < 0.0001 in 10 min, P = 0.0044 in 20 min in b . F (5,30) = 10.59, P = 0.0277 in 2 min, P = 0.0022 in 5 min, P < 0.0001 in 10 min, P = 0.0027 in 20 min in c . * P < 0.05, ** P < 0.01 versus the RT group. d The diagram shows the experimental procedure. Isolate d DRG neurons were placed in a 43 °C water bath for 30 s and placed at RT for 2, 5, 10, 20 or 40 min. The neurons were then lysed to detect the membrane abundance of TRPM3. e TRPM3 membrane expression in isolated DRG neurons was evaluated at different time points after direct heat stimulation. n = 3. F (5,12) = 12.3, P = 0.0358 in 2 min, P = 0.0100 in 5 min, P < 0.0001 in 10 min, P = 0.0085 in 20 min. * P < 0.05, ** P < 0.01 versus the RT group. f – h Naïve ( f ), ATF4 siRNA-injected ( g ) and Atf4 +/− ( h ) mice were subjected to the Hargreaves test at different time points after heat stimulation. n = 12 mice per group in f , g . n = 6 mice per group in h . F (5,66) = 14.36, P = 0.0004 in 2 min, P < 0.0001 in 5 min, P < 0.0001 in 10 min, P = 0.0057 in 20 min. ** P < 0.01 versus RT group. One-way ANOVA followed by Tukey’s multiple comparisons test. The error bars indicate the SEMs.

Article Snippet: The membranes were placed in blocking buffer for 1 h at RT and incubated in primary antibodies against ATF4 (goat, 1:1000, GeneTex, catalogue no.: GTX89973), TRPM3 (rabbit, 1:1000, Bioss, catalogue no.: bs-9046R; rabbit, 1:200, Alomone Labs, catalogue no.: ACC-050), TRPV1 (rabbit, 1:200, Alomone Labs, catalogue no.: ACC-030), TRPA1 (rabbit, 1:1000, ABclonal, catalogue no.: A12544), KIF17 (mouse, 1:100, Santa Cruz Biotechnology, catalogue no.: sc-137040), KIF3A (goat, 1:100, Santa Cruz Biotechnology, catalogue no.: sc-18745), KIF3B (rabbit, 1:100, Santa Cruz Biotechnology, catalogue no.: sc-50456), KIF5A (rabbit, 1:1000, Abcam, catalogue no.: ab5628), KIF5B (rabbit, 1:2000, Abcam, catalogue no.: ab167429), KIFC2 (rabbit, 1:100, Abcam, catalogue no.: ab3476), TfR (mouse, 1:1000, Thermo Fisher Scientific, catalogue no.: 13-6800), CXCR4 (rabbit, 1:1000, Abcam, catalogue no.: ab124824), CXCR7 (rabbit, 1:1000, Abcam, catalogue no.: ab72100), β-tubulin (mouse, 1:2000, Arigo, catalogue no.: ARG62347), Flag (rabbit, 1:1000, Cell Signaling Technology, catalogue no.: 14793) or His (rabbit, 1:1000, Cell Signaling Technology, catalogue no.: 12698) overnight at 4 °C.

Techniques: Co-Immunoprecipitation Assay, Western Blot, Expressing, Isolation, Injection

Journal: Nature Communications

Article Title: ATF4 selectively regulates heat nociception and contributes to kinesin-mediated TRPM3 trafficking

doi: 10.1038/s41467-021-21731-1

Figure Lengend Snippet: a – f Co-IP showing the ATF4/KIF interaction in the DRG. DRG lysates were immunoprecipitated with an ATF4 antibody and immunoblotted with a KIF17, KIF3A, KIF3B, KIF5A, KIF5B, KIFC2 or ATF4 antibody as indicated. This experiment was repeated three times. g SIM images show that the colocalization between ATF4 and KIF17 in DRG neurons. Scale bar, 5 μm. h The GST pull-down assay with two purified proteins, KIF17-GST-Flag and ATF4, showed a direct interaction between ATF4 and KIF17. This experiment was repeated three times. i Interaction between KIF17 and ATF4 mutants. The ATF4 mutants were transiently co-expressed with KIF17, and the cell lysates were immunoprecipitated with a His antibody and then immunoblotted with a His or Flag antibody as indicated. This experiment was repeated three times. j The interaction level between TRPM3 and KIF17 was examined by co-IP in scrambled and ATF4 siRNA-treated mice DRG lysates. DRG lysates were immunoprecipitated with a TRPM3 antibody and immunoblotted with a KIF17 or TRPM3 antibody as indicated. This experiment was repeated three times. t 4 = 12.88, P = 0.0002. k SIM images showed the colocalization between TRPM3 and KIF17 in DRG neurons from naïve, ATF4 siRNA and ATF4-overexpresing mice. Quantification data showed the colocalization rates of KIF17 with TRPM3 (colocalized yellow spots/total KIF17 positive spots) and those of TRPM3 with KIF17 (colocalized yellow spots/total TRPM3 positive spots) in DRG neurons. n = 3 mice per group. F (2,6) = 27.17, P = 0.0224 in naïve vs. ATF4-siRNA, P = 0.0255 in naïve vs. ATF4-over in KIF17 with TRPM3. F (2,6) = 33.5, P = 0.0069 in naïve vs. ATF4-siRNA, P = 0.0375 in naïve vs. ATF4-over in TRPM3 with KIF17. Scale bar, 10 μm. j Two-tailed Independent Student’s t test. k One-way ANOVA followed by Tukey’s multiple comparisons test, * P < 0.05, ** P < 0.01. The error bars indicate the SEMs.

Article Snippet: The membranes were placed in blocking buffer for 1 h at RT and incubated in primary antibodies against ATF4 (goat, 1:1000, GeneTex, catalogue no.: GTX89973), TRPM3 (rabbit, 1:1000, Bioss, catalogue no.: bs-9046R; rabbit, 1:200, Alomone Labs, catalogue no.: ACC-050), TRPV1 (rabbit, 1:200, Alomone Labs, catalogue no.: ACC-030), TRPA1 (rabbit, 1:1000, ABclonal, catalogue no.: A12544), KIF17 (mouse, 1:100, Santa Cruz Biotechnology, catalogue no.: sc-137040), KIF3A (goat, 1:100, Santa Cruz Biotechnology, catalogue no.: sc-18745), KIF3B (rabbit, 1:100, Santa Cruz Biotechnology, catalogue no.: sc-50456), KIF5A (rabbit, 1:1000, Abcam, catalogue no.: ab5628), KIF5B (rabbit, 1:2000, Abcam, catalogue no.: ab167429), KIFC2 (rabbit, 1:100, Abcam, catalogue no.: ab3476), TfR (mouse, 1:1000, Thermo Fisher Scientific, catalogue no.: 13-6800), CXCR4 (rabbit, 1:1000, Abcam, catalogue no.: ab124824), CXCR7 (rabbit, 1:1000, Abcam, catalogue no.: ab72100), β-tubulin (mouse, 1:2000, Arigo, catalogue no.: ARG62347), Flag (rabbit, 1:1000, Cell Signaling Technology, catalogue no.: 14793) or His (rabbit, 1:1000, Cell Signaling Technology, catalogue no.: 12698) overnight at 4 °C.

Techniques: Co-Immunoprecipitation Assay, Immunoprecipitation, Pull Down Assay, Purification, Two Tailed Test

Journal: Nature Communications

Article Title: ATF4 selectively regulates heat nociception and contributes to kinesin-mediated TRPM3 trafficking

doi: 10.1038/s41467-021-21731-1

Figure Lengend Snippet: a Colocalization of the KIF17, ATF4 and TRPM3 proteins in mouse DRG sections. Scale bar, 200 μm. b Colocalization of KIF17 mRNA , ATF4 mRNA and TRPM3 mRNA in mouse DRG sections. Scale bar, 20 μm. c Colocalization of the KIF17, ATF4 and TRPM3 proteins in DRG neurons was detected by SIM. Scale bar, 5 μm. d , e Changes in the membrane expression of TRPM3 in the DRG after KIF17 knockdown ( d ) or KIF17 overexpression ( e ). n = 6 mice per group. F (2,15) = 34.36, P = 0.0002 in d . F (2,15) = 20.48, P = 0.0005 in e . f The behaviours of KIF17 siRNA- and scrambled siRNA-injected mice were evaluated by the Hargreaves test. n = 6 mice per group. t 10 = 3.485, P = 0.0059. g The behaviours of KIF17-overexpressing and control mice were evaluated by the Hargreaves test. n = 6 mice per group. t 10 = 6.776, P < 0.0001. h ATF4 knockdown suppressed the increased expression of TRPM3 in the membrane induced by KIF17 overexpression. n = 6 mice per group. F (2,15) = 16.19, P = 0.0002 in naïve vs. KIF17-over, P = 0.0014 in KIF17-over vs. KIF17-over + ATF4-siRNA. i KIF17 knockdown inhibited the increase in TRPM3 expression on the cell surface induced by ATF4 overexpression. n = 6 mice per group. F (2,15) = 12.76, P = 0.0025 in naïve vs. ATF4-over, P = 0.0010 in ATF4-over vs. ATF4-over + KIF17-siRNA. ** P < 0.01. d , e , h , i One-way ANOVA followed by Tukey’s multiple comparisons test. f , g Two-tailed Independent Student’s t test. The error bars indicate the SEMs.

Article Snippet: The membranes were placed in blocking buffer for 1 h at RT and incubated in primary antibodies against ATF4 (goat, 1:1000, GeneTex, catalogue no.: GTX89973), TRPM3 (rabbit, 1:1000, Bioss, catalogue no.: bs-9046R; rabbit, 1:200, Alomone Labs, catalogue no.: ACC-050), TRPV1 (rabbit, 1:200, Alomone Labs, catalogue no.: ACC-030), TRPA1 (rabbit, 1:1000, ABclonal, catalogue no.: A12544), KIF17 (mouse, 1:100, Santa Cruz Biotechnology, catalogue no.: sc-137040), KIF3A (goat, 1:100, Santa Cruz Biotechnology, catalogue no.: sc-18745), KIF3B (rabbit, 1:100, Santa Cruz Biotechnology, catalogue no.: sc-50456), KIF5A (rabbit, 1:1000, Abcam, catalogue no.: ab5628), KIF5B (rabbit, 1:2000, Abcam, catalogue no.: ab167429), KIFC2 (rabbit, 1:100, Abcam, catalogue no.: ab3476), TfR (mouse, 1:1000, Thermo Fisher Scientific, catalogue no.: 13-6800), CXCR4 (rabbit, 1:1000, Abcam, catalogue no.: ab124824), CXCR7 (rabbit, 1:1000, Abcam, catalogue no.: ab72100), β-tubulin (mouse, 1:2000, Arigo, catalogue no.: ARG62347), Flag (rabbit, 1:1000, Cell Signaling Technology, catalogue no.: 14793) or His (rabbit, 1:1000, Cell Signaling Technology, catalogue no.: 12698) overnight at 4 °C.

Techniques: Expressing, Over Expression, Injection, Two Tailed Test

Journal: Nature Communications

Article Title: ATF4 selectively regulates heat nociception and contributes to kinesin-mediated TRPM3 trafficking

doi: 10.1038/s41467-021-21731-1

Figure Lengend Snippet: a Cultured DRG neurons were treated with CXCL12 (1 μg/mL) for 120, 240 or 360 min, and then ATF4 expression was measured. n = 6. F (3,20) = 4.642, P = 0.0462 in 120 min, P = 0.0154 in 240 min, P = 0.0128 in 360 min. * P < 0.05 versus 0 min. b CXCL12 (1 μg in 10 μL PBS + 0.5% BSA) was intrathecally injected three times every 3 h, and ATF4 expression was measured in the DRG 2 h after the last injection. n = 6 mice per group. t 10 = 5.151 , P = 0.0004. ** P < 0.01. c ATF4 siRNA signifi c antly relieved CXCL12-induced thermal hyperalgesia. n = 12 mice per group. F (2,33) = 15.68, P < 0.0001 in vehicle vs. CXCL12, P = 0.0101 in CXCL12 vs. CXCL12 + ATF4-siRNA. * P < 0.05, ** P < 0.01. d CXCR4 siRNA but not CXCR7 siRNA abolished the increase in ATF4 expression in the DRG induced by CXCL12 in vivo. n = 6 mice per group. F (3,20) = 54.47, P < 0.0001 in CXCL12, CXCL12 + CXCR4-siRNA and CXCL12 + CXCR7-siRNA. ** P < 0.01 versus the vehicle group, ## P < 0.01 versus the CXCL12 group. e Hypothetical model illustrating that ATF4 interacts with TRPM3 and KIF17 to form a complex to regulate the membrane trafficking of TRPM3 in sensory neurons and thus contributes to thermal sensitivity. a , c , d One-way ANOVA followed by Tukey’s multiple comparisons test. b Two-tailed Independent Student’s t test. The error bars indicate the SEMs.

Article Snippet: The membranes were placed in blocking buffer for 1 h at RT and incubated in primary antibodies against ATF4 (goat, 1:1000, GeneTex, catalogue no.: GTX89973), TRPM3 (rabbit, 1:1000, Bioss, catalogue no.: bs-9046R; rabbit, 1:200, Alomone Labs, catalogue no.: ACC-050), TRPV1 (rabbit, 1:200, Alomone Labs, catalogue no.: ACC-030), TRPA1 (rabbit, 1:1000, ABclonal, catalogue no.: A12544), KIF17 (mouse, 1:100, Santa Cruz Biotechnology, catalogue no.: sc-137040), KIF3A (goat, 1:100, Santa Cruz Biotechnology, catalogue no.: sc-18745), KIF3B (rabbit, 1:100, Santa Cruz Biotechnology, catalogue no.: sc-50456), KIF5A (rabbit, 1:1000, Abcam, catalogue no.: ab5628), KIF5B (rabbit, 1:2000, Abcam, catalogue no.: ab167429), KIFC2 (rabbit, 1:100, Abcam, catalogue no.: ab3476), TfR (mouse, 1:1000, Thermo Fisher Scientific, catalogue no.: 13-6800), CXCR4 (rabbit, 1:1000, Abcam, catalogue no.: ab124824), CXCR7 (rabbit, 1:1000, Abcam, catalogue no.: ab72100), β-tubulin (mouse, 1:2000, Arigo, catalogue no.: ARG62347), Flag (rabbit, 1:1000, Cell Signaling Technology, catalogue no.: 14793) or His (rabbit, 1:1000, Cell Signaling Technology, catalogue no.: 12698) overnight at 4 °C.

Techniques: Cell Culture, Expressing, Injection, In Vivo, Two Tailed Test

Journal: Theranostics

Article Title: Hypofractionated radiotherapy combined with lenalidomide improves systemic antitumor activity in mouse solid tumor models.

doi: 10.7150/thno.88864

Figure Lengend Snippet: Figure 2. The enhanced abscopal effect depends on CD8+ cells, and adding lena to hRT increases tumor-specific CD8+ T cells. A, Scheme for the depletion of CD8+ cells. B, B16-CD133 tumor growth of irradiated primary tumor (left) and non-irradiated secondary tumor (right). C, Survival of mice. D, Gating strategy to identify tumor-specific tetramer+ CD8+ T cells at day 8 after treatment start. E-H, Percentage (E, G), and absolute numbers (F, H) of M8-tetramer+ CD8+ T cells in the primary and secondary tumors (E, F) and TDLNs (G, H) of control mice (n=6, grey), or mice treated with lena (n=7, green), hRT (n=7, blue) or hRT/lena combination (n=7, yellow). Data are presented as mean with SEM and were collected from 3 independent experiments. P values (ns, not significant; * P < 0.05; ** P < 0.01; *** P < 0.001) were determined by unpaired two-tailed Student’s t-test (B), Kaplan–Meier analysis (C), or one-way ANOVA with Tukey’s multiple comparisons test (E-H).

Article Snippet: In the CD8 depletion experiment, 200 μg/mouse of anti-CD8 antibodies (clone 2.43, BioXCell) was injected i.p. 3 days before hRT, on the day of the first hRT, and once weekly thereafter.

Techniques: Irradiation, Control, Two Tailed Test

Journal: Theranostics

Article Title: Hypofractionated radiotherapy combined with lenalidomide improves systemic antitumor activity in mouse solid tumor models.

doi: 10.7150/thno.88864

Figure Lengend Snippet: Figure 3. Lena increases DC numbers in the non-irradiated tumor. A, Numbers of Ly6C+ CD103+ DCs per gram tumor in the B16-OVA model at day 4 after treatment start. To identify cross-presenting DCs, first CD3+, F4/80+, CD49b+, and CD19+ cells were excluded, then the gate was set on the MHC-II+CD11c+ population and further on the CD103+ Ly6C+ subset. B, Percentage of mature CD103+ Ly6C+ CD86+ DCs presenting SIINFEKL peptide by H-2Kb in the B16-OVA model at day 4 after treatment start. Tumor single-cell suspensions derived from B16-OVA tumors were stained with an IgG1 isotype control antibody as a control for H-2Kb/SIINFEKL staining, and TILs from B16-CD133 tumors were used as antigen-negative control. C, Representative flow cytometry plots for H-2Kb/SIINFEKL+ CD86+ cells among CD103+ Ly6C+ DCs. D, Number of total DCs (CD11c+ cells) at day 8 after treatment start in the B16-CD133 model. Data are presented as mean with SEM and were collected from 2 independent experiments. E, F, BMDCs were generated from the BM of healthy mice in the presence of DMSO or 10 µM lena; after 8-9 days, BMDCs were incubated with OVA protein, stimulated with LPS and co-cultured with CFSE-labeled CD8+ OT-I T cells. Numbers (E) and proliferation by CFSE dilution (F) of OT-I cells were determined 4 days later. P values (ns, not significant; * P < 0.05; ** P < 0.01; *** P < 0.001) were determined by one-way ANOVA with Tukey’s multiple comparisons test (A-D) or unpaired two-tailed Student’s t-test (E-F).

Article Snippet: In the CD8 depletion experiment, 200 μg/mouse of anti-CD8 antibodies (clone 2.43, BioXCell) was injected i.p. 3 days before hRT, on the day of the first hRT, and once weekly thereafter.

Techniques: Irradiation, Derivative Assay, Staining, Control, Negative Control, Flow Cytometry, Generated, Incubation, Cell Culture, Labeling, Two Tailed Test

Journal: Theranostics

Article Title: Hypofractionated radiotherapy combined with lenalidomide improves systemic antitumor activity in mouse solid tumor models.

doi: 10.7150/thno.88864

Figure Lengend Snippet: Figure 6. Adding lena to hRT increases the number of stem- and effector-like exhausted TILs. A, Gating strategy employed to identify CD8+ TILs secreting effector molecules after 4 h ex vivo stimulation with M8 peptide and Brefeldin A. B, C, Numbers of CD8+ TILs which secrete GzmB, IFNγ, TNFα, or IL-2 in primary (B), and secondary (C) B16-CD133 tumors at day 8 after treatment start. D, Gating strategy to characterize exhausted subsets of tumor-specific T cells. E, Cell number of stem-like (TCF1+ TIM3- PD1+), transitory (CD101- TCF1- TIM3+ PD1+), and terminally exhausted (CD101+ TCF1- TIM3+ PD1+) M8-tet+ CD8+ T cells in primary and secondary tumor. Data are presented as mean with SEM and were collected from 3 independent experiments. P values (ns, not significant; * P < 0.05; ** P < 0.01; *** P < 0.001) were determined by unpaired two-tailed Student’s t-test.

Article Snippet: In the CD8 depletion experiment, 200 μg/mouse of anti-CD8 antibodies (clone 2.43, BioXCell) was injected i.p. 3 days before hRT, on the day of the first hRT, and once weekly thereafter.

Techniques: Ex Vivo, Two Tailed Test

Journal: PLOS Pathogens

Article Title: Systemic infection by Candida albicans requires FASN-α subunit induced cell wall remodeling to perturb immune response

doi: 10.1371/journal.ppat.1012865

Figure Lengend Snippet: Δ/Δ . (A) Female BALB/c mice were infected with WT, fas2 Δ/Δ, and fas2 Δ/ FAS2 (5 × 10 5 CFUs per mice), the serum levels of the proinflammatory cytokines TNF-α, IL-1β, IL-17a, IL-6, and IFN-γ concentrations at 1- and 8- days postinfection were determined by ELISA (n = 3). (B-E) Peripheral blood of mice was analyzed at 24, 48, 72 h and 6 d after intravenous infection with C. albicans WT, fas2 Δ/Δ, and fas2 Δ/ FAS2 (5 × 10 5 CFUs per mice) using FACS, with cells stained with antibodies to CD11b, CD11c, Ly6C and Ly6G, the absolute numbers of the different populations were statistically analyzed (n = 3). (F) Following intravenously injected with C. albicans WT, fas2 Δ/Δ, and fas2 Δ/ FAS2 (5 × 10 5 CFUs per mice), the kidney homogenates of mice were analyzed at 1- and 8- days postinfection for the levels of TNF-α, IL-6, IL-1β, and IL-17a by ELISA (n = 3). (G) Kidney homogenates of mice were analyzed for immune cell populations at 24, 48, 72 h, and 6 d after intravenous infection with C. albicans WT, fas2 Δ/Δ, and fas2 Δ/ FAS2 (5 × 10 5 CFUs per mice) using FACS stained with antibodies to CD11b, CD11c, Ly6C and Ly6G, the absolute numbers of the different populations were statistically analyzed (n = 3). (H, I) Representative images of kidney sections stained for the neutrophil marker Ly-6G (n = 3) (H) and quantification of kidney area scored as Ly-6G + (I) at 72h postinfection with WT, fas2 Δ/Δ and fas2 Δ/ FAS2 (5 × 10 5 CFUs per mice) (n = 3). Data in (A, B, C, D, E, F, G, and I) are presented as mean ± SD. Statistical significance was assessed using a two-tailed unpaired Student’s t -test for cytokine levels and blood immune cell populations (A, B, C, D, E, F, and I) or two-way ANOVA for kidney immune cell populations (G). Significance is indicated as * P < 0.05, ** P < 0.01, *** P < 0.001, with ns denoting no significant difference.

Article Snippet: In vivo neutrophil depletion was accomplished using an anti-mouse Ly6G (IA8) monoclonal antibody (BioXcell).

Techniques: Infection, Enzyme-linked Immunosorbent Assay, Staining, Injection, Marker, Two Tailed Test

Journal: PLOS Pathogens

Article Title: Systemic infection by Candida albicans requires FASN-α subunit induced cell wall remodeling to perturb immune response

doi: 10.1371/journal.ppat.1012865

Figure Lengend Snippet: Δ/Δ mutant induced protective immunity. (A) UMAP plots showing the distribution of immune cell clusters in WT, fas2 Δ/Δ, and mock-infected mice. The left panel displays overall immune cell clustering by sample group, while the middle and right panels highlight neutrophil and monocyte phagocyte (MP) sub-clusters, respectively. The number of cells analyzed from each group is indicated in the legend. (B, C) Mice treated with Ly6G (IA8) for neutrophil depletion by recurring intraperitoneal injections of 300 µg of anti-mouse Ly6G (IA8) antibody every 2 days, beginning one day before infection. At day 0, mice were intravenously injected with WT and fas2 Δ/Δ (2 × 10 5 CFUs per mice), survival rates (B) (n = 8) and weight loss (C) (n = 8) of mice post infection are shown. (D-G) Fungal burdens in kidneys (D), spleens (E), livers (F), and brains (G) of mice at 1, 24, 48 h post infection with WT and fas2 Δ/Δ (2 × 10 5 CFUs per mice) with PBS drug vehicle (n = 3) or Ly6G (IA8) (n = 3) treatment. (H-J) Fungal burdens in kidneys (H), livers (I), and spleens (J) of mice administered PBS or clodronate liposomes 24 h before and 24 h after intravenous infection with WT and fas2 Δ/Δ (2 × 10 5 CFUs per mice) (n = 3). (K, L) Mice treated with clodronate liposomes for macrophage depletion. Survival curves (K) and weight loss (L) in mice given PBS liposomes or clodronate liposomes 24 h before and 24 h after intravenous infection with WT and fas2 Δ/Δ (2 × 10 5 CFUs per mice) (n = 4). Data from three independent experiments are present as the mean ± SD. Statistical significance was determined using two-way ANOVA for fungal burdens (D, E, F, G, H, I, and J) or log-rank test for survival curves (B, K). Significance is indicated as * P < 0.05, ** P < 0.01, *** P < 0.001, with ns denoting no significant difference.

Article Snippet: In vivo neutrophil depletion was accomplished using an anti-mouse Ly6G (IA8) monoclonal antibody (BioXcell).

Techniques: Mutagenesis, Infection, Injection, Liposomes

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 , Schematic of metastatic LUAD and patient-derived xenograft (PDX) engraftment. b , Box and whisker plots of L1CAM IHC H-score in patient-derived primary tumor and metastasis samples. Primary tumor, n = 15; metastases, n = 54. *** P = 0.0002. c , Box and whisker plots of L1CAM IHC H-score in the primary tumors in ( b ) and in PDXs derived from primary tumor or metastases. Primary tumors, n = 15; primary tumor-derived PDXs, n = 36; metastasis-derived PDXs, n = 70. ns, P = 0.8811; * P = 0.0294 (left), 0.0420 (right). d , Schematic of the oncogenic transformation of AT2 cells into lung adenocarcinoma in the KP GEMM ( left panel ), and box and whisker plots of the percentage of L1CAM + cells in the KP mouse lungs and tumor areas at different time points after lentivirus instillation ( right panel ). Week 0, n = 6; week 14-19, n = 6; week 20-32, n = 15. ns, P = 0.5710; ** P = 0.0032. e , Schematic of the tumor tissue section ( left panel ), and box and whisker plots of the percentage of L1CAM + cells in the tumor center and invasive front ( right panel ). Tumor center, n = 300 cells; invasive front, n = 258 cells. N = 7 image frames. * P = 0.0152. f , L1CAM immunofluorescence (IF) staining of LUAD patient tissue samples at the tumor center (P, papillary) or the invasive front (M, micropapillary). Magnified regions are indicated in red boxes. Scale bar, 20 μm. g , Confocal microscopy image of L1CAM IF staining and Hoechst counterstaining of nuclei in KP tumoroids grown for 5 days. Schematic of tumoroids generated from KP-derived cancer cells ( lower right panel ). Scale bar, 10 μm. h , Percentage of L1CAM + cells in KP primary tumors versus tumoroids. Primary tumor, n = 4 experiments; tumoroids, n = 6 experiments. Mean ± s.e.m. ** P = 0.0095. i , Widefield fluorescence microscopy image of KP tumoroids stained with calcein AM in KP tumor cells sorted by L1CAM expression and grown as tumoroids for 7 days. The magnified region showing a single cell is indicated by a dotted box and that of a tumoroid by a solid box. Scale bar, 400 μm. j , Box and whisker plots of tumoroids formed per 1,000 cells after 7 days in culture. L1CAM − , n = 16; L1CAM + , n = 16. **** P < 0.0001. k , Box and whisker plots of cross-section area per tumoroid in the experiment of panel ( I ). L1CAM - , n = 807; L1CAM + , n = 477. **** P < 0.0001. l , Schematic representation of the generation of L1cam knockout KP LUAD GEMMs. m , H&E staining of KP and KPL1 primary tumors (26 week post-Cre) followed by their histopathological grading using an automated deep neural network. Magnified regions are shown in a red square. Scale bar, 1 mm. n , Fraction of KP and KPL1 tumors (22-26 week post-Cre) based on histopathological grading. Grade 1, green ; Grade 2, orange ; Grade 3, blue ; Grade 4, red . KP, n = 4; KPL1, n = 4. Mean ± S.D. o , H&E staining of KP metastasis in the subcapsular sinus of the lymph node (top panel) or ribcage bone (bottom panel). Invasive front is shown with a dotted yellow line. T, tumor; LN, lymph node. Scale bar, 100 μm. p , Incidence of primary tumor and spontaneous metastases upon viral transduction in KP and KPL1 mice. The organ specificity of spontaneous metastasis is shown as a percentage of the total metastatic tumors. LN, lymph node. KP primary, n = 46; KPL1 primary, n = 30; KP met, n = 42; KPL1 met, n = 16. ns, P = 1; * P = 0.0122. q , Representative images of subcutaneous tumors formed 6 weeks after inoculating 500 KP or KPL1 cells in athymic mice. Scale bar, 2 mm. r , Limiting dilution assay of subcutaneous tumor formation at various doses of KP or KPL1 cells in athymic mice. n = 10 for each condition. s , Fluorescent images of GFP + KP or KPL1 cells seeded in lungs at week 1 after tail vein injection (10 5 cells each) into athymic mice. Scale bar, 10 μm. t , Quantification of KP and KPL1 cells seeded in lungs from the experiment in ( S ). KP, n = 9; KPL1, n = 10. ns, P = 0.3154. u , Representative image of ex vivo lung BLI at week 5 after tail vein injection of single-cell suspension of KP and KPL1 tumoroids (2 x 10 4 cells) into athymic mice. v , Quantification of ex vivo lung BLI signal in the experiment in ( u ). KP, n = 28; KPL1, n = 14. *** P = 0.0006. w , The KM plot showing the overall survival after performing tail vein injections with KP or KPL1 cells. KP, n = 10; KPL1, n = 10. **** P < 0.0001. Statistical significance was assessed using the two-tailed Mann-Whitney test ( b , e , h , j , k , n , t , v ), one-way analysis of variance followed by the Tukey test ( c , d ), two-tailed Fisher’s exact test ( p ) or log-rank (Mantel-Cox) test ( o , w ). Data are shown as a box (median ± 25-75%) and whisker (maximum to minimum values) plot ( b - e , j , k , t , v ).

Article Snippet: For immunofluorescence staining, samples were fixed in 4% PFA for 10 min and permeabilized with 0.5% of Triton X-100 in PBS for another 10 min. After incubating with 10% normal goat serum (Life Technologies Cat# 50062Z) for 1 h at room temperature, the samples were incubated with primary antibodies overnight at 4°C in blocking solution with antibodies against mouse L1CAM (Miltenyi Biotec Cat# 130-115-812, AB_2727206), human L1CAM (Santa Cruz Biotechnology Cat# sc-53386, RRID: AB_628937), Sox2 (Invitrogen Cat# 14-9811-82, RRID: AB_891383), CELSR1 (Millipore Sigma Cat# ABT119, RRID: AB_11215810), c-Jun(pS73) (Cell Signaling Technology Cat# 9164, RRID: AB_330892), GFP (Aves Labs Cat# GFP-1010, RRID: AB_2307313), mouse FZD6 (R&D Systems Cat# AF1526, RRID: AB_354842), human FZD6 (Abcam Cat# AB150545, RRID: AB_3697520), Sox9 (Invitrogen Cat# 14-9765-82, RRID:AB_2573006), Cleaved Caspase-3 (Cell Signaling Technology Cat# 9661, RRID: AB_2341188), anti-TTF1/NKX2-1 (abcam Cat# ab76013, RRID: AB_1310784), or human cytokeratin (Dako Cat# M3515, RRID: AB_2132885).

Techniques: Derivative Assay, Whisker Assay, Transformation Assay, Immunofluorescence, Staining, Confocal Microscopy, Generated, Fluorescence, Microscopy, Expressing, Knock-Out, Transduction, Limiting Dilution Assay, Injection, Ex Vivo, Suspension, 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 , Representative L1CAM IHC staining in primary tumors, pleural fluids, and metastatic samples from LUAD patients and three tissue microarrays (Array 1, green; Array 2, blue; Array 3, red) containing normal human lung tissue and LUAD PDX (Passage 0 or 1). Scale bar, 100 μm. b , Heatmap of L1CAM IHC staining quantification (H-score) in primary tumor, pleural fluid, metastatic samples, and PDXs from LUAD patients. Each column represents an individual sample. Samples are color-coded according to their site of tissue. c , Maximum projection of a PDX tumoroid fluorescently stained with L1CAM. Scale bar, 10 μm. d , Cross-section of a PDX tumoroid stained with L1CAM IF and DAPI (nuclei). Scale bar, 10 μm. e , Close up of a PDX tumoroid stained with L1CAM IF and DAPI. Scale bar, 5 μm.

Article Snippet: For immunofluorescence staining, samples were fixed in 4% PFA for 10 min and permeabilized with 0.5% of Triton X-100 in PBS for another 10 min. After incubating with 10% normal goat serum (Life Technologies Cat# 50062Z) for 1 h at room temperature, the samples were incubated with primary antibodies overnight at 4°C in blocking solution with antibodies against mouse L1CAM (Miltenyi Biotec Cat# 130-115-812, AB_2727206), human L1CAM (Santa Cruz Biotechnology Cat# sc-53386, RRID: AB_628937), Sox2 (Invitrogen Cat# 14-9811-82, RRID: AB_891383), CELSR1 (Millipore Sigma Cat# ABT119, RRID: AB_11215810), c-Jun(pS73) (Cell Signaling Technology Cat# 9164, RRID: AB_330892), GFP (Aves Labs Cat# GFP-1010, RRID: AB_2307313), mouse FZD6 (R&D Systems Cat# AF1526, RRID: AB_354842), human FZD6 (Abcam Cat# AB150545, RRID: AB_3697520), Sox9 (Invitrogen Cat# 14-9765-82, RRID:AB_2573006), Cleaved Caspase-3 (Cell Signaling Technology Cat# 9661, RRID: AB_2341188), anti-TTF1/NKX2-1 (abcam Cat# ab76013, RRID: AB_1310784), or human cytokeratin (Dako Cat# M3515, RRID: AB_2132885).

Techniques: Immunohistochemistry, Staining

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 , L1CAM IHC staining in lung and tumor sections in KP mice at baseline and at different time points after lentivirus instillation to induce adenocarcinoma formation. Yellow, week 0; orange, week 14; red, week 24. Scale bar, 50 μm. b , L1CAM IHC staining of the patient tissue sections including normal tissue and tumor. The invasive tumor front is indicated with a yellow line. Scale bar, 200 μm. c , H&E and L1CAM IHC staining of KP primary tumor sections (26 week post-Cre) showing the indicated histological subtypes. Scale bar, 50 μm. d , Fraction of histological subtypes observed in KP primary tumors. n = 203,870 cells. e , Percentage of L1CAM + cells in KP primary tumors with respect to histological subtypes. Lepidic ( n = 26,871); Papillary ( n = 88,381); Micropapillary (red colored, n = 4,116); Acinar ( n = 3,430); Solid ( n = 81,072). **** P < 0.0001. f , Representative GFP (cancer cells) IF and L1CAM IF images of primary tumor and an autochthonous lymph node metastasis from a KP mouse (35 week post-Cre). Scale bar, 20 μm. g , Quantification of L1CAM IF intensity in the experiment ( f ). LN, lymph node; AU, arbitrary unit. n = 24 image frames; N = 3 mice in each condition. **** P < 0.0001. h , H&E staining of KP tumoroids grown in Matrigel over the course of 7 days. Scale bar, 100 μm. I , Brightfield and GFP fluorescence microscopy images of KP tumoroids at day 7. Scale bar, 10 μm. j , Close up of L1CAM IHC staining of patient samples and KP tumors. Scale bar, 20 μm. Statistical significance was assessed using the two-tailed Mann-Whitney test ( g ) or one-way analysis of variance followed by the Tukey test ( e ). Data are shown as mean ± S.D. ( d , e ).

Article Snippet: For immunofluorescence staining, samples were fixed in 4% PFA for 10 min and permeabilized with 0.5% of Triton X-100 in PBS for another 10 min. After incubating with 10% normal goat serum (Life Technologies Cat# 50062Z) for 1 h at room temperature, the samples were incubated with primary antibodies overnight at 4°C in blocking solution with antibodies against mouse L1CAM (Miltenyi Biotec Cat# 130-115-812, AB_2727206), human L1CAM (Santa Cruz Biotechnology Cat# sc-53386, RRID: AB_628937), Sox2 (Invitrogen Cat# 14-9811-82, RRID: AB_891383), CELSR1 (Millipore Sigma Cat# ABT119, RRID: AB_11215810), c-Jun(pS73) (Cell Signaling Technology Cat# 9164, RRID: AB_330892), GFP (Aves Labs Cat# GFP-1010, RRID: AB_2307313), mouse FZD6 (R&D Systems Cat# AF1526, RRID: AB_354842), human FZD6 (Abcam Cat# AB150545, RRID: AB_3697520), Sox9 (Invitrogen Cat# 14-9765-82, RRID:AB_2573006), Cleaved Caspase-3 (Cell Signaling Technology Cat# 9661, RRID: AB_2341188), anti-TTF1/NKX2-1 (abcam Cat# ab76013, RRID: AB_1310784), or human cytokeratin (Dako Cat# M3515, RRID: AB_2132885).

Techniques: Immunohistochemistry, Staining, Fluorescence, Microscopy, 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 , Representative images of tumor-bearing KP and KPL1 mice analyzed by BLI at week 16 after viral transduction. b , Growth curve of KP and KPL1 primary tumor burden as determined by BLI. KP, n = 37; KPL1, n = 14. ns, P > 0.9999. c , Micro-CT scan images of normal lung and tumor-bearing lungs from KP and KPL1 mice at week 22 after viral transduction. Arrows , tumors; red h , heart. d , Tumor burden comparison between KP and KPL1 mice based on micro-CT scans at week 22. n = 38 KP mice, n = 10 KPL1 mice. ns, P = 0.3192. e , Tumor areas of KP and KPL1 mouse lungs at week 22-30 after viral transduction. n = 4 mice. ns, P = 0.6857. f , Graphical representation of mouse survival prior to reaching the target tumor burden, set at a luciferase bioluminescence flux of 10 7 photons/sec per mouse chest. KP, n = 35; KPL1, n = 14. ns, P = 0.7143. g , Quantification of tumoroids formed per 10,000 cells over 7 days. KP, n = 6; KPL1, n = 6. ns, P = 0.6991. h , Schematic of three different metastasis inoculation routes. i , Representative image of ex vivo lung BLI signals at week 5 after intratracheal delivery of single-cell suspension of KP and KPL1 tumoroids (2 x 10 4 cells) into athymic mice. j , Quantification of ex vivo lung BLI signal in the experiment in panel ( i ). KP, n = 8; KPL1, n = 6. ** P = 0.0027. k , Representative image of ex vivo lung BLI signals at week 3 after intracardiac injection of single-cell suspension of KP and KPL1 tumoroids (5 x 10 4 cells) into athymic mice. l , Quantification of ex vivo lung BLI signals in the experiment in panel ( k ). KP, n = 9; KPL1, n = 5. ** P = 0.0070. m , Representative image of ex vivo kidney BLI signals at week 3 after intracardiac injection of single-cell suspension of KP tumoroids or KPL1 tumoroids (5 x 10 4 cells). n , Quantification of ex vivo kidney BLI signals at week 3 after intracardiac injection of single-cell suspension of KP tumoroids or KPL1 tumoroids. KP, n = 9; KPL1, n = 5. * P = 0.0120. o , Representative image of ex vivo brain BLI signals at week 3 after intracardiac injection of single-cell suspension of KP tumoroids or KPL1 tumoroids (5 x 10 4 cells). p , Quantification of ex vivo brain BLI signals at week 3 after intracardiac injection of single-cell suspension of KP tumoroids or KPL1 tumoroids. KP, n = 9; KPL1, n = 5. * P = 0.0120. q , Representative image of ex vivo liver BLI signals at week 3 after intracardiac injection of single-cell suspension of KP tumoroids or KPL1 tumoroids (5 x 10 4 cells). r , Quantification of ex vivo liver BLI signals at week 3 after intracardiac injection of single-cell suspension of KP tumoroids or KPL1 tumoroids. KP, n = 9; KPL1, n = 5. * P = 0.0120. s , Western immunoblotting analysis of Ru631 PDX cells transduced with two different L1CAM shRNAs. t , H&E staining of lung sections four weeks after injecting Ru631 L1CAM knockdown via tail vein. Scale bar, 50 μm. u , Quantification of Ru631 tumors after tail vein injection. n = 4 mice per condition. * P = 0.0337; ** P = 0.0013. v , Western immunoblotting of L1CAM knockdowns with two different short hairpins in Ru323 PDXs. w , H&E staining of lung sections four weeks after injecting Ru323 with L1CAM knockdown via tail vein. Scale bar, 50 μm. x , Quantification of Ru323 tumors after tail vein injection. n = 5 mice per condition. *P = 0.0123; ** P = 0.0058. Data are shown as a box (median ± 25-75%) and whisker (maximum to minimum values) plot ( d , e , j , l , n , p , r ). Error bar indicates mean ± S.D. ( b ). Statistical significance was assessed using Kolmogorov-Smirnov test ( b ), two-tailed Mann-Whitney test ( d , e , g , j , l , n , p , r ) or one-way analysis of variance followed by the Tukey test ( u , x ).

Article Snippet: For immunofluorescence staining, samples were fixed in 4% PFA for 10 min and permeabilized with 0.5% of Triton X-100 in PBS for another 10 min. After incubating with 10% normal goat serum (Life Technologies Cat# 50062Z) for 1 h at room temperature, the samples were incubated with primary antibodies overnight at 4°C in blocking solution with antibodies against mouse L1CAM (Miltenyi Biotec Cat# 130-115-812, AB_2727206), human L1CAM (Santa Cruz Biotechnology Cat# sc-53386, RRID: AB_628937), Sox2 (Invitrogen Cat# 14-9811-82, RRID: AB_891383), CELSR1 (Millipore Sigma Cat# ABT119, RRID: AB_11215810), c-Jun(pS73) (Cell Signaling Technology Cat# 9164, RRID: AB_330892), GFP (Aves Labs Cat# GFP-1010, RRID: AB_2307313), mouse FZD6 (R&D Systems Cat# AF1526, RRID: AB_354842), human FZD6 (Abcam Cat# AB150545, RRID: AB_3697520), Sox9 (Invitrogen Cat# 14-9765-82, RRID:AB_2573006), Cleaved Caspase-3 (Cell Signaling Technology Cat# 9661, RRID: AB_2341188), anti-TTF1/NKX2-1 (abcam Cat# ab76013, RRID: AB_1310784), or human cytokeratin (Dako Cat# M3515, RRID: AB_2132885).

Techniques: Transduction, Micro-CT, Comparison, Luciferase, Ex Vivo, Suspension, Injection, Western Blot, Staining, Knockdown, 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 , Schematic summary of lung developmental continuum from early to late progenitor stages defined by specific transcription factors. b , Representative 3D maximum intensity projections (MIP) images of SOX2, NKX2-1 and SOX9 IF in the anterior foregut domain in embryonic day E9.5 mouse embryos and the distal lung bud tip in E10.5 mouse embryos. The dashed box demarcates the SOX2/NKX2-1 boundary in E9.5 and NKX2-1/SOX9 boundary in E10.5 as illustrated in the zoomed 2D (Z-Slice) panels on the right. All data were independently validated from replicate samples of at least n = 4 embryos with similar results obtained. c , SOX2, NKX2-1, and SOX9 IF staining in patient-derived primary tumor and metastasis tissue sections. Magnified regions are highlighted in yellow boxes. Scale bar, 50 μm. d , Fraction of cancer cells expressing SOX2, NKX2-1, or SOX9 by IF analysis in patient-derived primary tumor and metastasis tissue sections. Mean ± S.D. n = 6 patient samples each. ns, P = 0.4848 (NKX2-1), 0.3095 (SOX9); ** P = 0.0043. e , SOX2 and SOX9 IHC staining in LUAD patient-derived primary tumor and pleural metastasis samples. Magnified regions are highlighted in red boxes. Scale bar, 100 μm. f , Box and whisker plots of SOX2 and SOX9 IHC H-score in the patient-derived primary tumor and metastasis samples. Primary, n = 15; Metastasis, n = 7. ns, P = 0.1229; ** P = 0.0011. g , Relative median survival of LUAD patients based on the expression of SOX2 , NKX2-1 , and SOX9 in the primary tumor. Gene expression and survival data compiled from GEO, EGA and TCGA. SOX2 (low, n = 706; high, n = 705); NKX2-1 (low, n = 1083; high, n = 1083); SOX9 (low, n = 1083; high, n = 1083). * P = 0.0219; *** P = 0.0003; **** P < 0.0001. h , H&E staining of lung tissue sections harboring metastatic colonies upon tail vein injection of KP tumoroid cells expressing control or Sox2 short hairpins (sh). Magnified regions are indicated in red boxes. Scale bar, 5 mm. i,j , Number per lung ( I ) and percent area ( j ) of metastases in the experiment of panel ( h ). n = 10. **** P < 0.0001. k , UMAP of scRNA-seq data from autochthonous KP tumors collected 32 weeks after viral instillation. n = 4,489 cells. Transcriptionally distinct clusters were computed by Leiden algorithm and numbered. l , Heatmap showing imputed average LUAD developmental marker expression in the clusters from primary tumors ranked left to right according to the average L1cam expression level. m , UMAP of scRNA-seq data from KP tumoroids collected after 7 days. n = 12,962 cells. Transcriptionally distinct clusters were computed by Leiden algorithm and are represented by a different color and a number. n , Heatmap showing imputed average LUAD developmental marker expression in the clusters from 7-day tumoroids ranked left to right according to the average L1cam expression level. Lung developmental progenitor stages represented by the predominant expression of Sox2 ( early developmental stage), Nkx2-1 and Foxa2 ( middle progenitor stage), Hmga2 and Sox9 ( late progenitor stage) are indicated. o , Representative L1CAM and SOX2 IF staining of the invasive front ( dotted yellow line ) within an autochthonous KP tumor (32 week post-Cre) and of KP-derived tumoroids grown for 4 days or 7 days. T , tumor; N , normal tissue. Magnified regions are indicated in red boxes. Scale bar, 50 μm for primary tumor; 20 μm for tumoroids. p , Relative Sox2 mRNA expression of KP tumoroid-derived cells sorted by L1CAM expression. Mean ± S.E.M. n = 3. * P = 0.0258. q , L1CAM and SOX2 IHC staining of representative LUAD PDXs with high or low expression of L1CAM. Scale bar, 50 μm. r , Scatter plot and linear regression ( red line ) of L1CAM versus SOX2 IHC H-score data from LUAD PDXs. Data are log-transformed for visualization. n = 36. s , L1CAM and SOX2 IHC staining in serial sections of patient-derived bone metastasis and liver metastasis tissues. Scale bar, 100 μm. t , Scatter plot and linear regression ( red line ) of L1CAM versus SOX2 IHC H-score data from LUAD patient primary and metastasis samples. Data are log-transformed for visualization. n = 61 samples (primary, 15; metastasis, 46). Spearman correlation was used to calculate the relationship between L1CAM expression and SOX2 expression ( r,t ). Data are shown as a box (median ± 25-75%) and whisker (maximum to minimum values) plot ( f , i,j ). Statistical significance was assessed using the two-tailed Mann-Whitney test ( d , f , g , i , j ) or two-tailed t test after passing the Shapiro-Wilk normality test ( p ).

Article Snippet: For immunofluorescence staining, samples were fixed in 4% PFA for 10 min and permeabilized with 0.5% of Triton X-100 in PBS for another 10 min. After incubating with 10% normal goat serum (Life Technologies Cat# 50062Z) for 1 h at room temperature, the samples were incubated with primary antibodies overnight at 4°C in blocking solution with antibodies against mouse L1CAM (Miltenyi Biotec Cat# 130-115-812, AB_2727206), human L1CAM (Santa Cruz Biotechnology Cat# sc-53386, RRID: AB_628937), Sox2 (Invitrogen Cat# 14-9811-82, RRID: AB_891383), CELSR1 (Millipore Sigma Cat# ABT119, RRID: AB_11215810), c-Jun(pS73) (Cell Signaling Technology Cat# 9164, RRID: AB_330892), GFP (Aves Labs Cat# GFP-1010, RRID: AB_2307313), mouse FZD6 (R&D Systems Cat# AF1526, RRID: AB_354842), human FZD6 (Abcam Cat# AB150545, RRID: AB_3697520), Sox9 (Invitrogen Cat# 14-9765-82, RRID:AB_2573006), Cleaved Caspase-3 (Cell Signaling Technology Cat# 9661, RRID: AB_2341188), anti-TTF1/NKX2-1 (abcam Cat# ab76013, RRID: AB_1310784), or human cytokeratin (Dako Cat# M3515, RRID: AB_2132885).

Techniques: Staining, Derivative Assay, Expressing, Immunohistochemistry, Whisker Assay, Gene Expression, Injection, Control, Marker, Transformation 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 , SOX2 and NKX2-1 IF staining of patient-derived LUAD primary tumor tissue section. The invasive front is marked with a dotted yellow line, and the magnified low-grade and high-grade tumor areas are shown in red boxes. Scale bar, 1 mm. b , SOX2 and NKX2-1 IF staining of patient-derived primary tumor tissue section with different tumor grade areas. Invasive front is shown with a dotted yellow line. Scale bar, 20 μm. c , SOX2 and SOX9 IF staining in KP primary tumor and metastasis tissue sections collected simultaneously from the same mouse (35 week post-Cre). Scale bar, 100 μm. d , Western immunoblotting analysis of SOX2 expression after dox-induced Sox2 knockdown in KP tumoroids. e , f , Box and whisker plots of the imputed transcript levels of Sox2 ( e ) and L1cam ( f ) in the scRNA-seq transcriptional clusters from KP primary tumors. g , Scatter plot of L1cam versus Sox2 expression in the scRNA-seq dataset from KP primary tumors. Data from transcriptional cluster 9 are shown in black. H , I , Box and whisker plots of the imputed transcript levels of Sox2 ( h ) and L1cam ( i ) from 7-day tumoroids. Data from the cluster 12 are shown in red. j , Scatter plot of L1cam versus Sox2 expression in the scRNA-seq dataset from 7-day tumoroids. Data from transcriptional cluster 12 are shown in black. k , Proportion of SOX9 + cells detected by IF in lung colonies after injecting a high number (1 x 10 5 ) of KP or KPL1 cells via the tail vein into athymic mice. KP, n = 10; KPL1, n = 5. ** P = 0.0013. l , Relative expression of SOX9 in two independent KP tumoroid lines upon conditional knockdown of L1CAM. Bar graphs, mean ± S.D. n = 3 in each condition. ns, P = 0.8301; * P = 0.0202, 0.0305; ** P = 0.0094. m , Western immunoblotting analysis of L1CAM levels after L1CAM overexpression in KPL1 tumoroids. Data are shown as a box (median ± 25-75%) and whisker (maximum to minimum values) plot ( e , f , h , i , k ). Spearman correlation was used to calculate the relationship between L1CAM expression and SOX2 expression ( g , j ). Statistical significance was assessed using the two-tailed Mann-Whitney test ( k ) or one-way analysis of variance followed by the Tukey test ( l ).

Article Snippet: For immunofluorescence staining, samples were fixed in 4% PFA for 10 min and permeabilized with 0.5% of Triton X-100 in PBS for another 10 min. After incubating with 10% normal goat serum (Life Technologies Cat# 50062Z) for 1 h at room temperature, the samples were incubated with primary antibodies overnight at 4°C in blocking solution with antibodies against mouse L1CAM (Miltenyi Biotec Cat# 130-115-812, AB_2727206), human L1CAM (Santa Cruz Biotechnology Cat# sc-53386, RRID: AB_628937), Sox2 (Invitrogen Cat# 14-9811-82, RRID: AB_891383), CELSR1 (Millipore Sigma Cat# ABT119, RRID: AB_11215810), c-Jun(pS73) (Cell Signaling Technology Cat# 9164, RRID: AB_330892), GFP (Aves Labs Cat# GFP-1010, RRID: AB_2307313), mouse FZD6 (R&D Systems Cat# AF1526, RRID: AB_354842), human FZD6 (Abcam Cat# AB150545, RRID: AB_3697520), Sox9 (Invitrogen Cat# 14-9765-82, RRID:AB_2573006), Cleaved Caspase-3 (Cell Signaling Technology Cat# 9661, RRID: AB_2341188), anti-TTF1/NKX2-1 (abcam Cat# ab76013, RRID: AB_1310784), or human cytokeratin (Dako Cat# M3515, RRID: AB_2132885).

Techniques: Staining, Derivative Assay, Western Blot, Expressing, Knockdown, Whisker Assay, Over Expression, 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 , SOX2 IF staining of KP and KPL1 primary tumors (32 week post-Cre). Scale bar, 10 μm. b , Percentage of SOX2 + cells in the experiment from panel ( a ). n = 3 mice for each condition. * P = 0.0411. c , d , Relative L1cam mRNA expression and Sox2 mRNA expression in KP and KPL1 tumoroids. KP, n = 4; KPL1, n = 4. * P = 0.0286 ( c , d ). e , IF images of KP and KPL1 tumors in the lung after injecting a high number (1 x 10 5 ) of single cells from KP and KPL1 tumoroids into athymic mice via the tail vein. Magnified regions are highlighted in yellow boxes. Scale bar, 100 μm. f , Proportion of SOX2 + cells detected by IF in lung colonies after injecting a high number (1 x 10 5 ) of KP or KPL1 cells via the tail vein in athymic mice. KP, n = 10; KPL1, n = 5. * P = 0.0400. g , h , Relative expression of L1cam ( g ) and Sox2 ( h ) in two independent KP tumoroid lines upon Dox-dependent conditional knock down of L1cam . n = 4. **** P < 0.0001. i , j , Western immunoblot analysis ( i ) and quantification ( j ) of SOX2 and SOX9 levels in control and L1CAM-overexpressing (OE) KP tumoroids. n = 4. * P = 0.0286. k , l , H&E staining ( k ) and quantification ( l ) of subcutaneous tumors or lung metastases after injection of KPL1 cells with or without L1CAM overexpression into athymic mice, analyzed at 4 weeks after subcutaneous (subQ) injection or 5 weeks after tail vein (TV) injection. Scale bar, 1 mm (SubQ) and 100 μm (TV). n = 5. ns, P > 0.9999; * P = 0.0238. m , GFP (cancer cells) and SOX2 IF staining of control vs L1CAM overexpressing KPL1 cells in lung metastasis after 5 week-post tail vein injection. Magnified regions are highlighted in yellow boxes. Scale bar, 10 μm. n , Fraction of SOX2 + cells in the experiment from panel ( m ). Control, n = 5; L1CAM overexpression, n = 5. ** P = 0.0079. Data are shown as a box (median ± 25-75%) and whisker (maximum to minimum values) plot ( f ). Bar graphs, mean ± S.D. ( c , d , g , h , j ) or ± S.E.M. ( b , l , n ). Statistical significance was assessed using the two-tailed t test after passing the Shapiro-Wilk normality test ( b ), two-tailed Mann-Whitney test ( c , d , f , j , l , n ) or one-way analysis of variance followed by the Tukey test ( g , h ).

Article Snippet: For immunofluorescence staining, samples were fixed in 4% PFA for 10 min and permeabilized with 0.5% of Triton X-100 in PBS for another 10 min. After incubating with 10% normal goat serum (Life Technologies Cat# 50062Z) for 1 h at room temperature, the samples were incubated with primary antibodies overnight at 4°C in blocking solution with antibodies against mouse L1CAM (Miltenyi Biotec Cat# 130-115-812, AB_2727206), human L1CAM (Santa Cruz Biotechnology Cat# sc-53386, RRID: AB_628937), Sox2 (Invitrogen Cat# 14-9811-82, RRID: AB_891383), CELSR1 (Millipore Sigma Cat# ABT119, RRID: AB_11215810), c-Jun(pS73) (Cell Signaling Technology Cat# 9164, RRID: AB_330892), GFP (Aves Labs Cat# GFP-1010, RRID: AB_2307313), mouse FZD6 (R&D Systems Cat# AF1526, RRID: AB_354842), human FZD6 (Abcam Cat# AB150545, RRID: AB_3697520), Sox9 (Invitrogen Cat# 14-9765-82, RRID:AB_2573006), Cleaved Caspase-3 (Cell Signaling Technology Cat# 9661, RRID: AB_2341188), anti-TTF1/NKX2-1 (abcam Cat# ab76013, RRID: AB_1310784), or human cytokeratin (Dako Cat# M3515, RRID: AB_2132885).

Techniques: Staining, Expressing, Knockdown, Western Blot, Control, Injection, 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 , 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: For immunofluorescence staining, samples were fixed in 4% PFA for 10 min and permeabilized with 0.5% of Triton X-100 in PBS for another 10 min. After incubating with 10% normal goat serum (Life Technologies Cat# 50062Z) for 1 h at room temperature, the samples were incubated with primary antibodies overnight at 4°C in blocking solution with antibodies against mouse L1CAM (Miltenyi Biotec Cat# 130-115-812, AB_2727206), human L1CAM (Santa Cruz Biotechnology Cat# sc-53386, RRID: AB_628937), Sox2 (Invitrogen Cat# 14-9811-82, RRID: AB_891383), CELSR1 (Millipore Sigma Cat# ABT119, RRID: AB_11215810), c-Jun(pS73) (Cell Signaling Technology Cat# 9164, RRID: AB_330892), GFP (Aves Labs Cat# GFP-1010, RRID: AB_2307313), mouse FZD6 (R&D Systems Cat# AF1526, RRID: AB_354842), human FZD6 (Abcam Cat# AB150545, RRID: AB_3697520), Sox9 (Invitrogen Cat# 14-9765-82, RRID:AB_2573006), Cleaved Caspase-3 (Cell Signaling Technology Cat# 9661, RRID: AB_2341188), anti-TTF1/NKX2-1 (abcam Cat# ab76013, RRID: AB_1310784), or human cytokeratin (Dako Cat# M3515, RRID: AB_2132885).

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: For immunofluorescence staining, samples were fixed in 4% PFA for 10 min and permeabilized with 0.5% of Triton X-100 in PBS for another 10 min. After incubating with 10% normal goat serum (Life Technologies Cat# 50062Z) for 1 h at room temperature, the samples were incubated with primary antibodies overnight at 4°C in blocking solution with antibodies against mouse L1CAM (Miltenyi Biotec Cat# 130-115-812, AB_2727206), human L1CAM (Santa Cruz Biotechnology Cat# sc-53386, RRID: AB_628937), Sox2 (Invitrogen Cat# 14-9811-82, RRID: AB_891383), CELSR1 (Millipore Sigma Cat# ABT119, RRID: AB_11215810), c-Jun(pS73) (Cell Signaling Technology Cat# 9164, RRID: AB_330892), GFP (Aves Labs Cat# GFP-1010, RRID: AB_2307313), mouse FZD6 (R&D Systems Cat# AF1526, RRID: AB_354842), human FZD6 (Abcam Cat# AB150545, RRID: AB_3697520), Sox9 (Invitrogen Cat# 14-9765-82, RRID:AB_2573006), Cleaved Caspase-3 (Cell Signaling Technology Cat# 9661, RRID: AB_2341188), anti-TTF1/NKX2-1 (abcam Cat# ab76013, RRID: AB_1310784), or human cytokeratin (Dako Cat# M3515, RRID: AB_2132885).

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: 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 , FZD6 IF staining with counterstained nuclei in KP tumoroids upon conditional knockdown of L1cam . Scale bar, 10 μm. b , Relative intensity of FZD6 IF quantified in KP tumoroids upon conditional knockdown of L1cam . Left to right, n = 25, 23, 35, 29, 39, 41, 45, 53 tumoroids. * P = 0.0440, *** P = 0.0010. c , Relative Fzd6 mRNA level in upon conditional knockdown of Fzd6 in KP tumoroids. n = 4 experiments. **** P < 0.0001. d , Relative Sox2 mRNA level upon conditional knockdown of FZD6 in KP tumoroids. n = 4 experiments. ** P = 0.0040. e , IF staining for L1CAM, c-Jun(pS73), and SOX2 in KP tumoroids ( upper panels ) and image segmentation to quantify the signal ( bottom panels ). Scale bar, 10 μm. f , Pie chart showing the percent of cells staining positive for c-Jun(pS73) and SOX2 in KP tumoroids. n = 649 cells. g , IF staining for cytokeratin, L1CAM, and c-Jun(pS73) in a patient-derived LUAD lymph node metastasis. Scale bar, 50 μm. h , Pie chart showing the percent of cells staining positive for c-Jun(pS73) and L1CAM in patient-derived LUAD lymph node metastases. n = 2,166 cells from 12 different lymph nodes. i , c-Jun(pS73) IF staining and counterstained nuclei in KP tumoroids upon conditional knockdown of L1CAM. Scale bar, 20 μm. j , Percentage of cells staining positive for c-Jun(pS73) after conditional knockdown of L1cam in KP tumoroids. Left to right, n = 94, 67, 87, 101, 38, 41, 36, 36 tumoroids. **** P < 0.0001. k , Western immunoblotting analysis of L1CAM, S73 phosphorylated and total c-Jun levels in H23 LUAD cells treated with 10 μM (JNK-IN-8) JNK inhibitor (JNKi) for 2 h. l , Sox2 mRNA relative expression level upon incubation of KP tumoroids with 20 μM JNK inhibitor for 24 h. n = 7 for KP tumoroid #1; 4 for KP tumoroid #2. *** P < 0.0006; * P = 0.0286. m , Western immunoblotting analysis of S73 phosphorylated and total c-Jun levels upon the treatments with anisomycin for 6 h. n , Sox2 mRNA relative expression level upon incubation of KP cells with anisomycin for 6 h. n = 3. ** P = 0.0064 (left), 0.0021 (right). The bar graph indicates mean ± S.E.M. ( b - d , j , l , n ). Statistical significance was assessed using the one-way analysis of variance followed by the Tukey test ( b - d , j , n ) or two-tailed Mann-Whitney test ( l ).

Article Snippet: For immunofluorescence staining, samples were fixed in 4% PFA for 10 min and permeabilized with 0.5% of Triton X-100 in PBS for another 10 min. After incubating with 10% normal goat serum (Life Technologies Cat# 50062Z) for 1 h at room temperature, the samples were incubated with primary antibodies overnight at 4°C in blocking solution with antibodies against mouse L1CAM (Miltenyi Biotec Cat# 130-115-812, AB_2727206), human L1CAM (Santa Cruz Biotechnology Cat# sc-53386, RRID: AB_628937), Sox2 (Invitrogen Cat# 14-9811-82, RRID: AB_891383), CELSR1 (Millipore Sigma Cat# ABT119, RRID: AB_11215810), c-Jun(pS73) (Cell Signaling Technology Cat# 9164, RRID: AB_330892), GFP (Aves Labs Cat# GFP-1010, RRID: AB_2307313), mouse FZD6 (R&D Systems Cat# AF1526, RRID: AB_354842), human FZD6 (Abcam Cat# AB150545, RRID: AB_3697520), Sox9 (Invitrogen Cat# 14-9765-82, RRID:AB_2573006), Cleaved Caspase-3 (Cell Signaling Technology Cat# 9661, RRID: AB_2341188), anti-TTF1/NKX2-1 (abcam Cat# ab76013, RRID: AB_1310784), or human cytokeratin (Dako Cat# M3515, RRID: AB_2132885).

Techniques: Staining, Knockdown, Derivative Assay, Western Blot, Expressing, Incubation, 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 , Venn diagram of transcription factors and chromatin modifiers differentially active in L1CAM + /SOX2 + KP tumoroid cells (scRNA-seq cluster #12, ) based on CHEA and ENCODE databases. b , PLA image of CHD1 and c-Jun in H23 LUAD cells. The PLA signals are shown as red dots overlayed with nuclei staining. Scale bar, 10 μm. c , Quantification of PLA signals per nucleus. Control, n = 63 cells; CHD1/c-Jun, n = 39 cells. **** P < 0.0001. d , Co-immunoprecipitation of CHD1 and c-Jun in KP tumoroids. e , Metaplots of CHD1 only (green), c-Jun only (blue), and CHD1/c-Jun overlap (red) ChIP-seq peak summits relative to peak center of CHD1 (left) or c-Jun (right) in H23 cells. f , Venn diagram showing the overlap between CHD1 and c-Jun genome-wide peaks. g , ChIP-seq analysis of CHD1 and c-Jun binding to the Sox2 locus in H23 cells. Sox2 enhancers ( red ), promoter and gene body ( green ) are indicated. h , ChIP-qPCR analysis of CHD1 binding to the SOX2 promoter in H23 cells with and without JNK inhibitor. n = 3 experiments. *** P = 0.0002. i , Co-immunoprecipitation of CHD1 and the transcriptional elongation factor RTF1 in H23 LUAD cells. j , Western immunoblotting analysis of SOX2 levels upon CHD1 knockout in H23 LUAD cells. k , Western immunoblotting analysis of SOX2 levels upon CHD1 knockdown in KP tumoroids using two different shRNAs. l , H&E staining of lung sections from NSG mice after tail-vein inoculation of H23 cells with CRISPR/Cas9-induced knockouts of CHD1 or L1CAM. Magnified regions are shown ( red boxes ). Scale bar, 100 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 ( j ). n = 6 per experimental condition. ** P = 0.0035, 0.0060, 0.0015 from left to right; * P = 0.0301 in ( m ). ** P = 0.0026, 0.0028, 0.0011 from left to right; *** P = 0.0006 in ( n ). o , Model of L1CAM-dependent PCP activation of c-Jun/CHD1 driven SOX2 expression in LUAD progenitors to generate a metastasis-initiating state. Data are shown as a box (median ± 25-75%) and whisker (maximum to minimum values) plot ( c , m , n ). The bar graph indicates mean ± S.E.M. ( h ). Statistical significance was assessed using a one-way analysis of variance followed by the Tukey test ( h , m , n ) or two-tailed Mann-Whitney test ( c ).

Article Snippet: For immunofluorescence staining, samples were fixed in 4% PFA for 10 min and permeabilized with 0.5% of Triton X-100 in PBS for another 10 min. After incubating with 10% normal goat serum (Life Technologies Cat# 50062Z) for 1 h at room temperature, the samples were incubated with primary antibodies overnight at 4°C in blocking solution with antibodies against mouse L1CAM (Miltenyi Biotec Cat# 130-115-812, AB_2727206), human L1CAM (Santa Cruz Biotechnology Cat# sc-53386, RRID: AB_628937), Sox2 (Invitrogen Cat# 14-9811-82, RRID: AB_891383), CELSR1 (Millipore Sigma Cat# ABT119, RRID: AB_11215810), c-Jun(pS73) (Cell Signaling Technology Cat# 9164, RRID: AB_330892), GFP (Aves Labs Cat# GFP-1010, RRID: AB_2307313), mouse FZD6 (R&D Systems Cat# AF1526, RRID: AB_354842), human FZD6 (Abcam Cat# AB150545, RRID: AB_3697520), Sox9 (Invitrogen Cat# 14-9765-82, RRID:AB_2573006), Cleaved Caspase-3 (Cell Signaling Technology Cat# 9661, RRID: AB_2341188), anti-TTF1/NKX2-1 (abcam Cat# ab76013, RRID: AB_1310784), or human cytokeratin (Dako Cat# M3515, RRID: AB_2132885).

Techniques: Staining, Control, Immunoprecipitation, ChIP-sequencing, Genome Wide, Binding Assay, ChIP-qPCR, Western Blot, Knock-Out, Knockdown, CRISPR, Whisker Assay, Activation Assay, Expressing, Two Tailed Test, MANN-WHITNEY

Journal: bioRxiv

Article Title: Centrosome Migration and Apical Membrane Formation in Polarized Epithelial Cells: Insights from the MDCK Cyst Model

doi: 10.1101/2024.06.17.598507

Figure Lengend Snippet: (A) Single MDCK cells were cultured in Matrigel for 12 h. Immunostaining was performed with the following markers: apical membrane marker glycoprotein 135 (Gp135, green), centrosome marker γ-tubulin (magenta), acetyl-tubulin (white), and DAPI for nuclei (blue). Single confocal sections through the middle of a cyst are shown. The order of polarization is arranged from single cell (1-cell), metaphase (Meta), telophase (Telo), cytokinetic pre-abscission (Pre-Abs), post-cytokinesis (Post-CK), to lumen open (LO). Arrows indicate Gp135 clusters around centrosomes, while arrowheads point to centrosome positions. Scale bar: 5 μm. (B) Illustration showing the use of a fixed-size oval tool to select and measure Gp135 intensity in the chosen region. 1. Cytosol. 2. Cytokinesis (CK) bridge. 3. Centrosome (Cent.). (C–D) Boxplots of Gp135 intensity surrounding centrosomes at different stages normalized to the mean in metaphase (C) and at different cellular regions at the cytokinetic pre-abscission stage normalized to the mean in the cytosol (D). We analyzed >15 cells for each stage or region in three independent experiments. Statistical analyses were performed via one-way ANOVA and Dunn’s multiple comparisons (****p<0.0001, ***p<0.001, **p<0.01, ns: not significant). The midlines and boxes show the mean ± SD, with whiskers indicating minimum and maximum values (a.u., arbitrary units). (E) Illustration showing how the polarity index ( L N-C [nucleus–centrosome distance] divided by r N [average nuclear radius]) and θ N-C (angle between the N-C and N-N axes) are calculated. (F–G) Boxplots depicting centrosome positions at different stages, shown by the polarity index (F) and θ N-C (G). We analyzed >16 cells per measurement in three independent experiments. Statistical analyses used one-way ANOVA and Dunn’s multiple comparisons (*p<0.05, **p<0.01, ****p<0.0001, ns: not significant). Midlines and boxes show the mean ± SD, with whiskers indicating minimum and maximum values. (H) Localization overview summarizing the localization of the centrosome (magenta) and recycling endosome/apical membrane components (green) during cell-division-directed polarization in Matrigel culture. White lines depict intercellular bridge microtubules, and nuclei are shown in blue. Arrows indicate apical membrane component localization around centrosomes and their transport from the centrosome to the AMIS.

Article Snippet: We used rabbit polyclonal antibodies against Gp135 (IF: 1:400, a kind gift from Dr. Jou Tzuu-Shuh, NTU, Taiwan) , Rab11a (IF: 1:400, WB: 1:2000, 20229-1-AP, Proteintech, Rosemont, IL), aPKC (IF: 1:200, 610175, BD Biosciences, Franklin Lakes, NJ), CEP120 (IF: 1:1000, WB: 1:2000, human residues 639–986, described in our previous paper) , p53 (IF: 1:100, 9282, Cell Signaling Technology, Danvers, MA), p21 (WB: 1:2000, 10355-1-AP, Proteintech), CEP164 (IF: 1:400, NBP1-81445, NOVUS, Centennial, CO), PCNT (IF: 1:200, ab4448, Abcam, Cambridge, UK), Par3 (IF: 1:200, WB: 1:1000, 07-330, EMD Millipore, Burlington, MA), and beta-actin (IF: 1:200, A2066, Sigma-Aldrich, St. Louis, MO); rabbit monoclonal antibodies against E-cadherin (IF: 1:200, 24E10, #3195, Cell Signaling Technology); and mouse monoclonal antibodies against γ-tubulin (IF: 1:400, GTU-88, T6557, Sigma-Aldrich), acetylated tubulin (IF: 1:400, T6793, Sigma-Aldrich), Gp135 (IF: 1:10, the culture medium of hybridoma clone 3F2D8, a kind gift from Dr. Jou Tzuu-Shuh, NTU, Taiwan) , polyglutamylated tubulin (IF: 1:100, GT335, AG-20B-0020, Adipogen, San Diego, CA), ODF2 (IF: 1:400, H00004957-M01, NOVUS), α-tubulin (WB: 1:5000, DM1α, T9026, Sigma-Aldrich), CDC42 (WB: 1:1000, 610928, BD Biosciences), and ZO-1 (IF: 1:400, 33-9100, ThermoFisher Scientific, Waltham, MA).

Techniques: Cell Culture, Immunostaining, Membrane, Marker

Journal: bioRxiv

Article Title: Centrosome Migration and Apical Membrane Formation in Polarized Epithelial Cells: Insights from the MDCK Cyst Model

doi: 10.1101/2024.06.17.598507

Figure Lengend Snippet: (A, F) Single MDCK cells were cultured in Matrigel for 12 h with labeled markers: Gp135 (green), γ-tubulin (magenta), and Rab11a (white) in (A), and aPKC (green), γ-tubulin (magenta), and Gp135 (white) in (F). DAPI staining was applied for nuclei (blue). Single confocal sections are shown through the middle of the cells. The centrosome region during telophase (Telo) and the AMIS region in post-cytokinesis (Post-CK) cells, as shown in the yellow box, are enlarged. Yellow arrows indicate Rab11a and aPKC around centrosomes or at the AMIS. White arrows indicate Gp135. Arrowheads point to the centrosome position. LO: lumen open. Scale bar: 5 μm. (B, D) Illustration showing L O-C (origin to centrosome distance) and L IC (inter-centrosomal distance) for cell doublets. The center point between the two nuclei is taken as the origin (O). (C, E) Boxplots depicting centrosome positions at different stages, shown by L O-C (C) and L IC (E). We analyzed >15 cells per measurement in three independent experiments. Statistical analyses used one-way ANOVA and Dunn’s multiple comparisons (*p<0.05, **p<0.01, ****p<0.0001). Midlines and boxes show the mean ± SD, with whiskers indicating minimum and maximum values.

Article Snippet: We used rabbit polyclonal antibodies against Gp135 (IF: 1:400, a kind gift from Dr. Jou Tzuu-Shuh, NTU, Taiwan) , Rab11a (IF: 1:400, WB: 1:2000, 20229-1-AP, Proteintech, Rosemont, IL), aPKC (IF: 1:200, 610175, BD Biosciences, Franklin Lakes, NJ), CEP120 (IF: 1:1000, WB: 1:2000, human residues 639–986, described in our previous paper) , p53 (IF: 1:100, 9282, Cell Signaling Technology, Danvers, MA), p21 (WB: 1:2000, 10355-1-AP, Proteintech), CEP164 (IF: 1:400, NBP1-81445, NOVUS, Centennial, CO), PCNT (IF: 1:200, ab4448, Abcam, Cambridge, UK), Par3 (IF: 1:200, WB: 1:1000, 07-330, EMD Millipore, Burlington, MA), and beta-actin (IF: 1:200, A2066, Sigma-Aldrich, St. Louis, MO); rabbit monoclonal antibodies against E-cadherin (IF: 1:200, 24E10, #3195, Cell Signaling Technology); and mouse monoclonal antibodies against γ-tubulin (IF: 1:400, GTU-88, T6557, Sigma-Aldrich), acetylated tubulin (IF: 1:400, T6793, Sigma-Aldrich), Gp135 (IF: 1:10, the culture medium of hybridoma clone 3F2D8, a kind gift from Dr. Jou Tzuu-Shuh, NTU, Taiwan) , polyglutamylated tubulin (IF: 1:100, GT335, AG-20B-0020, Adipogen, San Diego, CA), ODF2 (IF: 1:400, H00004957-M01, NOVUS), α-tubulin (WB: 1:5000, DM1α, T9026, Sigma-Aldrich), CDC42 (WB: 1:1000, 610928, BD Biosciences), and ZO-1 (IF: 1:400, 33-9100, ThermoFisher Scientific, Waltham, MA).

Techniques: Cell Culture, Labeling, Staining

Journal: bioRxiv

Article Title: Centrosome Migration and Apical Membrane Formation in Polarized Epithelial Cells: Insights from the MDCK Cyst Model

doi: 10.1101/2024.06.17.598507

Figure Lengend Snippet: (A) Time-lapse snapshots of MDCK cells expressing EGFP-Gp135 (green) and PACT-mKO1 (magenta, centrosome marker) in Matrigel. Nuclei were labeled with SiR-DNA (blue) before live imaging. Z-projection images of a dividing cell are shown. Time stamps show hours and minutes, with 0:00 set at the first frame of anaphase onset. Scale bar: 10 μm. (B–D) Change in polarity index, θ N-C , and L O-C over time. Each data point represents the average at a given time (>10 cells in Matrigel culture from three independent experiments). The lines show the means, and the shaded regions indicate SD values. (E) Fluorescent profiles of EGFP-Gp135 along the line connecting the two nuclei in a cell doublet. White arrows indicate the central value used to indicate the level of Gp135 on the AMIS (a.u., arbitrary units). Time stamps show hours and minutes, with 0:00 set to the first frame after anaphase onset. Scale bar: 10 μm. (F) Change in the central value in the EGFP-Gp135 fluorescent profile over time. The value of each time point was normalized to the value at 0:00. Each data point represents the average fluorescent intensity at a given time (10 cells in Matrigel culture from three independent experiments). The line shows the mean, and the shaded region indicates SD values (a.u., arbitrary units).

Article Snippet: We used rabbit polyclonal antibodies against Gp135 (IF: 1:400, a kind gift from Dr. Jou Tzuu-Shuh, NTU, Taiwan) , Rab11a (IF: 1:400, WB: 1:2000, 20229-1-AP, Proteintech, Rosemont, IL), aPKC (IF: 1:200, 610175, BD Biosciences, Franklin Lakes, NJ), CEP120 (IF: 1:1000, WB: 1:2000, human residues 639–986, described in our previous paper) , p53 (IF: 1:100, 9282, Cell Signaling Technology, Danvers, MA), p21 (WB: 1:2000, 10355-1-AP, Proteintech), CEP164 (IF: 1:400, NBP1-81445, NOVUS, Centennial, CO), PCNT (IF: 1:200, ab4448, Abcam, Cambridge, UK), Par3 (IF: 1:200, WB: 1:1000, 07-330, EMD Millipore, Burlington, MA), and beta-actin (IF: 1:200, A2066, Sigma-Aldrich, St. Louis, MO); rabbit monoclonal antibodies against E-cadherin (IF: 1:200, 24E10, #3195, Cell Signaling Technology); and mouse monoclonal antibodies against γ-tubulin (IF: 1:400, GTU-88, T6557, Sigma-Aldrich), acetylated tubulin (IF: 1:400, T6793, Sigma-Aldrich), Gp135 (IF: 1:10, the culture medium of hybridoma clone 3F2D8, a kind gift from Dr. Jou Tzuu-Shuh, NTU, Taiwan) , polyglutamylated tubulin (IF: 1:100, GT335, AG-20B-0020, Adipogen, San Diego, CA), ODF2 (IF: 1:400, H00004957-M01, NOVUS), α-tubulin (WB: 1:5000, DM1α, T9026, Sigma-Aldrich), CDC42 (WB: 1:1000, 610928, BD Biosciences), and ZO-1 (IF: 1:400, 33-9100, ThermoFisher Scientific, Waltham, MA).

Techniques: Expressing, Marker, Labeling, Imaging

Journal: bioRxiv

Article Title: Centrosome Migration and Apical Membrane Formation in Polarized Epithelial Cells: Insights from the MDCK Cyst Model

doi: 10.1101/2024.06.17.598507

Figure Lengend Snippet: (A) Time-lapse snapshots of MDCK cells expressing EGFP-Gp135 (green) and PACT-mKO1 (magenta, centrosome marker) on a cover glass. Nuclei were labeled with SiR-DNA (blue) before live imaging. Z-projection images of a dividing cell are shown. Time stamps show hours and minutes, with 0:00 set at the first frame of anaphase onset. Scale bar: 10 μm. (B–C) Change in θ N-C and L O-C over time. Each data point represents the average at a given time (6 cells on cover glass from three independent experiments). The lines show the means, and the shaded regions indicate SD values.

Article Snippet: We used rabbit polyclonal antibodies against Gp135 (IF: 1:400, a kind gift from Dr. Jou Tzuu-Shuh, NTU, Taiwan) , Rab11a (IF: 1:400, WB: 1:2000, 20229-1-AP, Proteintech, Rosemont, IL), aPKC (IF: 1:200, 610175, BD Biosciences, Franklin Lakes, NJ), CEP120 (IF: 1:1000, WB: 1:2000, human residues 639–986, described in our previous paper) , p53 (IF: 1:100, 9282, Cell Signaling Technology, Danvers, MA), p21 (WB: 1:2000, 10355-1-AP, Proteintech), CEP164 (IF: 1:400, NBP1-81445, NOVUS, Centennial, CO), PCNT (IF: 1:200, ab4448, Abcam, Cambridge, UK), Par3 (IF: 1:200, WB: 1:1000, 07-330, EMD Millipore, Burlington, MA), and beta-actin (IF: 1:200, A2066, Sigma-Aldrich, St. Louis, MO); rabbit monoclonal antibodies against E-cadherin (IF: 1:200, 24E10, #3195, Cell Signaling Technology); and mouse monoclonal antibodies against γ-tubulin (IF: 1:400, GTU-88, T6557, Sigma-Aldrich), acetylated tubulin (IF: 1:400, T6793, Sigma-Aldrich), Gp135 (IF: 1:10, the culture medium of hybridoma clone 3F2D8, a kind gift from Dr. Jou Tzuu-Shuh, NTU, Taiwan) , polyglutamylated tubulin (IF: 1:100, GT335, AG-20B-0020, Adipogen, San Diego, CA), ODF2 (IF: 1:400, H00004957-M01, NOVUS), α-tubulin (WB: 1:5000, DM1α, T9026, Sigma-Aldrich), CDC42 (WB: 1:1000, 610928, BD Biosciences), and ZO-1 (IF: 1:400, 33-9100, ThermoFisher Scientific, Waltham, MA).

Techniques: Expressing, Marker, Labeling, Imaging

Journal: bioRxiv

Article Title: Centrosome Migration and Apical Membrane Formation in Polarized Epithelial Cells: Insights from the MDCK Cyst Model

doi: 10.1101/2024.06.17.598507

Figure Lengend Snippet: (A) Time-lapse snapshots of p53-KO and centrinone (CN)-treated cells expressing EGFP-Gp135 (green) and PACT-mKO1 (magenta, centrosome marker) in Matrigel. Nuclei were labeled with SiR-DNA (blue) before live imaging. Z-projection images of dividing cells are shown. Time stamps show hours and minutes, with 0:00 set at the first frame of anaphase onset. Scale bar: 10 μm. (B–C) Change in θ N-C and L O-C over time. Each data point represents the average at a given time (>3 p53-KO cells in Matrigel culture from three independent experiments). The lines show the means, and the shaded regions indicate SD values. (D) Change in the central value of the EGFP-Gp135 fluorescent profile over time. The value of each time point was normalized to the value at 0:00. Each data point represents the average fluorescent intensity at a given time (n = 3 [p53-KO; olive] and 3 [p53-KO + CN; gray]). The lines show the means, and the shaded regions indicate SD values (a.u., arbitrary units). (E) Single MDCK WT and p53-KO cells, with or without CN treatment, after 72 h of Matrigel culture. Single confocal sections through the middle of cysts are shown with immunofluorescent signals of indicated markers: apical membrane Gp135 (green), centrosome marker γ-tubulin (magenta), and DAPI for nuclei (blue). “L” denotes the lumen. WT cells treated with CN remain at the single-cell stage. The insets display enlarged images of the region in the yellow box. Arrowheads indicate the presence of centrosomes. Scale bar: 10 μm. The bottom panels represent a larger view of the 3D culture. Scale bar: 50 μm. (F) Quantification of the proportion of MDCK cysts with a single central lumen after being cultured for different durations (24, 48, 72 h). The lumen structure was identified by Gp135 staining. n = 92 (24 h), 231 (48 h), 78 (72 h) cysts (MDCK WT); n = 22 (24 h), 133 (48 h), 59 (72 h) cysts (WT + CN); n = 154 (24 h), 170 (48 h), 204 (72 h) cysts (p53-KO); n = 105 (24 h), 128 (48 h), 149 (72 h) cysts (p53-KO + CN) analyzed for each measurement in three independent experiments. Statistical analyses used two-way ANOVA and Tukey multiple comparisons (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, ns: not significant). Values represent the mean ± SD. (G) Boxplot of Gp135 intensity on the apical membrane normalized to the mean in WT cells. n = 25 (MDCK WT), 26 (p53-KO), 25 (p53-KO + CN) cysts were analyzed in three independent experiments. Statistical analyses were performed via one-way ANOVA and Dunn’s multiple comparisons (ns: not significant, ****p<0.0001). The midlines and boxes show the mean ± SD, with whiskers indicating minimum and maximum values (a.u., arbitrary units).

Article Snippet: We used rabbit polyclonal antibodies against Gp135 (IF: 1:400, a kind gift from Dr. Jou Tzuu-Shuh, NTU, Taiwan) , Rab11a (IF: 1:400, WB: 1:2000, 20229-1-AP, Proteintech, Rosemont, IL), aPKC (IF: 1:200, 610175, BD Biosciences, Franklin Lakes, NJ), CEP120 (IF: 1:1000, WB: 1:2000, human residues 639–986, described in our previous paper) , p53 (IF: 1:100, 9282, Cell Signaling Technology, Danvers, MA), p21 (WB: 1:2000, 10355-1-AP, Proteintech), CEP164 (IF: 1:400, NBP1-81445, NOVUS, Centennial, CO), PCNT (IF: 1:200, ab4448, Abcam, Cambridge, UK), Par3 (IF: 1:200, WB: 1:1000, 07-330, EMD Millipore, Burlington, MA), and beta-actin (IF: 1:200, A2066, Sigma-Aldrich, St. Louis, MO); rabbit monoclonal antibodies against E-cadherin (IF: 1:200, 24E10, #3195, Cell Signaling Technology); and mouse monoclonal antibodies against γ-tubulin (IF: 1:400, GTU-88, T6557, Sigma-Aldrich), acetylated tubulin (IF: 1:400, T6793, Sigma-Aldrich), Gp135 (IF: 1:10, the culture medium of hybridoma clone 3F2D8, a kind gift from Dr. Jou Tzuu-Shuh, NTU, Taiwan) , polyglutamylated tubulin (IF: 1:100, GT335, AG-20B-0020, Adipogen, San Diego, CA), ODF2 (IF: 1:400, H00004957-M01, NOVUS), α-tubulin (WB: 1:5000, DM1α, T9026, Sigma-Aldrich), CDC42 (WB: 1:1000, 610928, BD Biosciences), and ZO-1 (IF: 1:400, 33-9100, ThermoFisher Scientific, Waltham, MA).

Techniques: Expressing, Marker, Labeling, Imaging, Membrane, Cell Culture, Staining

Journal: bioRxiv

Article Title: Centrosome Migration and Apical Membrane Formation in Polarized Epithelial Cells: Insights from the MDCK Cyst Model

doi: 10.1101/2024.06.17.598507

Figure Lengend Snippet: (A) Single MDCK WT and p53-KO cells, with or without centrinone (CN) treatment, after 8–12h of Matrigel culture. Single confocal sections through the middle of cysts are shown with immunofluorescent signals of indicated markers: Gp135 (green), γ-tubulin (magenta), acetyl-tubulin (white), and DAPI for nuclei (blue). Acentrosomal cells fail to cluster Gp135-positive vesicles and affect lumen formation in the two-cell stage (Pre-Abs, pre-abscission; LO, lumen open). Scale bar: 5 μm. (B) Boxplot of Gp135 intensity at the AMIS normalized to the mean in WT cells. n = 22 (MDCK WT), 26 (p53-KO), 28 (p53-KO + CN) post-CK cells were analyzed in three independent experiments. Statistical analyses were performed via one-way ANOVA and Dunn’s multiple comparisons (ns: not significant, ***p<0.001, ****p<0.0001). The midlines and boxes show the mean ± SD, with whiskers indicating minimum and maximum values (a.u., arbitrary units). (C) Proliferation curves of MDCK WT and p53-KO cells, with or without CN treatment. The cells with CN treatment underwent a 3-day pre-treatment before examination. Values are presented as the mean ± SD. (D) Proportion of lumen opening in MDCK cell doublets after 24 h of Matrigel culture based on Gp135 staining. n = 17 (MDCK WT), 30 (p53-KO), and 45 (p53-KO + CN) cell doublets were analyzed in three independent experiments. Statistical analyses used two-way ANOVA and Tukey multiple comparisons (ns: not significant, ****p<0.0001). Values represent the mean ± SD.

Article Snippet: We used rabbit polyclonal antibodies against Gp135 (IF: 1:400, a kind gift from Dr. Jou Tzuu-Shuh, NTU, Taiwan) , Rab11a (IF: 1:400, WB: 1:2000, 20229-1-AP, Proteintech, Rosemont, IL), aPKC (IF: 1:200, 610175, BD Biosciences, Franklin Lakes, NJ), CEP120 (IF: 1:1000, WB: 1:2000, human residues 639–986, described in our previous paper) , p53 (IF: 1:100, 9282, Cell Signaling Technology, Danvers, MA), p21 (WB: 1:2000, 10355-1-AP, Proteintech), CEP164 (IF: 1:400, NBP1-81445, NOVUS, Centennial, CO), PCNT (IF: 1:200, ab4448, Abcam, Cambridge, UK), Par3 (IF: 1:200, WB: 1:1000, 07-330, EMD Millipore, Burlington, MA), and beta-actin (IF: 1:200, A2066, Sigma-Aldrich, St. Louis, MO); rabbit monoclonal antibodies against E-cadherin (IF: 1:200, 24E10, #3195, Cell Signaling Technology); and mouse monoclonal antibodies against γ-tubulin (IF: 1:400, GTU-88, T6557, Sigma-Aldrich), acetylated tubulin (IF: 1:400, T6793, Sigma-Aldrich), Gp135 (IF: 1:10, the culture medium of hybridoma clone 3F2D8, a kind gift from Dr. Jou Tzuu-Shuh, NTU, Taiwan) , polyglutamylated tubulin (IF: 1:100, GT335, AG-20B-0020, Adipogen, San Diego, CA), ODF2 (IF: 1:400, H00004957-M01, NOVUS), α-tubulin (WB: 1:5000, DM1α, T9026, Sigma-Aldrich), CDC42 (WB: 1:1000, 610928, BD Biosciences), and ZO-1 (IF: 1:400, 33-9100, ThermoFisher Scientific, Waltham, MA).

Techniques: Staining

Journal: bioRxiv

Article Title: Centrosome Migration and Apical Membrane Formation in Polarized Epithelial Cells: Insights from the MDCK Cyst Model

doi: 10.1101/2024.06.17.598507

Figure Lengend Snippet: (A) Illustration depicting individual centrosomal structures (DA, SDA, and PCM proteins) and the state of microtubules on the centrosome in cells with knockout (KO) of CEP164 , ODF2 , p53/CEP120 , or PCNT genes. (B, F) Single MDCK cells of different genotypes were cultured in Matrigel for 12 h. Images show post-cytokinesis (CK) cells (B) or cells during pre-abscission (pre-Abs, F) with labeled markers: Gp135 (green), γ-tubulin (magenta), acetyl-tubulin (white), and DAPI for nuclei (blue). Images shown are single confocal sections through the middle of cells and side-view x-z cross-sections. Scale bar: 5 μm. (C–D, G–H) Boxplots of centrosome positions, represented by θ N-C and L O-C , for post-CK cells (C– D, n = 16 [WT], 10 [CEP164-KO], 15 [ODF2-KO], 20 [CEP120-KO], 14 [PCNT-KO]) and pre-Abs cells (G–H, n = 36 [WT], 14 [CEP164-KO], 16 [ODF2-KO], 12 [CEP120-KO], 14 [PCNT-KO]). Cells were analyzed in three independent experiments. Statistical analyses used one-way ANOVA and Dunn’s multiple comparisons (ns: not significant). Midlines and boxes show the mean ± SD, with whiskers indicating minimum and maximum values. (E) Boxplot of Gp135 intensity at the AMIS normalized to the mean in WT cells. n = 25 (WT), 10 (CEP164-KO), 12 (ODF2-KO), 10 (CEP120-KO), and 10 (PCNT-KO) cell doublets were analyzed in three independent experiments. Statistical analyses were performed via one-way ANOVA and Dunn’s multiple comparisons (ns: not significant). The midlines and boxes show the mean ± SD, with whiskers indicating minimum and maximum values (a.u., arbitrary units).

Article Snippet: We used rabbit polyclonal antibodies against Gp135 (IF: 1:400, a kind gift from Dr. Jou Tzuu-Shuh, NTU, Taiwan) , Rab11a (IF: 1:400, WB: 1:2000, 20229-1-AP, Proteintech, Rosemont, IL), aPKC (IF: 1:200, 610175, BD Biosciences, Franklin Lakes, NJ), CEP120 (IF: 1:1000, WB: 1:2000, human residues 639–986, described in our previous paper) , p53 (IF: 1:100, 9282, Cell Signaling Technology, Danvers, MA), p21 (WB: 1:2000, 10355-1-AP, Proteintech), CEP164 (IF: 1:400, NBP1-81445, NOVUS, Centennial, CO), PCNT (IF: 1:200, ab4448, Abcam, Cambridge, UK), Par3 (IF: 1:200, WB: 1:1000, 07-330, EMD Millipore, Burlington, MA), and beta-actin (IF: 1:200, A2066, Sigma-Aldrich, St. Louis, MO); rabbit monoclonal antibodies against E-cadherin (IF: 1:200, 24E10, #3195, Cell Signaling Technology); and mouse monoclonal antibodies against γ-tubulin (IF: 1:400, GTU-88, T6557, Sigma-Aldrich), acetylated tubulin (IF: 1:400, T6793, Sigma-Aldrich), Gp135 (IF: 1:10, the culture medium of hybridoma clone 3F2D8, a kind gift from Dr. Jou Tzuu-Shuh, NTU, Taiwan) , polyglutamylated tubulin (IF: 1:100, GT335, AG-20B-0020, Adipogen, San Diego, CA), ODF2 (IF: 1:400, H00004957-M01, NOVUS), α-tubulin (WB: 1:5000, DM1α, T9026, Sigma-Aldrich), CDC42 (WB: 1:1000, 610928, BD Biosciences), and ZO-1 (IF: 1:400, 33-9100, ThermoFisher Scientific, Waltham, MA).

Techniques: Knock-Out, Cell Culture, Labeling

Journal: bioRxiv

Article Title: Centrosome Migration and Apical Membrane Formation in Polarized Epithelial Cells: Insights from the MDCK Cyst Model

doi: 10.1101/2024.06.17.598507

Figure Lengend Snippet: (A) Single MDCK cells, stably expressing pLKO-GFP-shRNA to knock down specified genes, cultured in Matrigel for 12 h. Images show post-cytokinesis cells with labeled markers: γ-tubulin (magenta), Gp135 (white), pLKO-GFP (green), and DAPI for nuclei (blue). Z-projection images between two centrosomes and the side-view x-z cross-section (bottom) are shown. “Ctrl” indicates the expression of scrambled-sequence shRNA. Arrowheads point to mislocalized centrosomes. Scale bar: 5 μm. (B–D) Boxplots of centrosome positions, represented by θ N-C , L O-C (B–C, n = 22 [sh-Ctrl], 20 [sh-Rab11a], 20 [sh-Sec15a], 20 [sh-MyoVb], 44 [sh-Par3], 20 [sh-Crb3], 20 [sh-Cdc42]), and L IC (D, n = 10 [sh-Ctrl], 10 [sh-Rab11a], 10 [sh-Sec15a], 10 [sh-MyoVb], 16 [sh-Par3], 10 [sh-Crb3], 10 [sh-Cdc42]), in post-cytokinesis cells. Cells were analyzed in three independent experiments. Statistical analyses used one-way ANOVA and Dunn’s multiple comparisons (ns: not significant, ***p<0.001, **p<0.01, *p<0.05). Midlines and boxes show the mean ± SD, with whiskers indicating minimum and maximum values. (E) Single WT and Rab11a-KO MDCK cells were cultured in Matrigel for 12 h with labeled markers: Par3 (green), γ-tubulin (magenta), acetyl-tubulin (white), and DAPI for nuclei (blue). Single confocal sections of pre-abscission cells are shown. The bridge region is enlarged from the yellow box. Scale bar: 5 μm. (F) Single confocal sections of p53-KO cells, with or without centrinone (CN) treatment, cultured in Matrigel for 12 h with labeled markers: Par3 (green), γ-tubulin (magenta), acetyl-tubulin (white), and DAPI for nuclei (blue). The bridge region shown in the yellow box is enlarged. The bottom panels display Z-projected images to demonstrate the complete depletion of centrosomes. Scale bar: 5 μm.

Article Snippet: We used rabbit polyclonal antibodies against Gp135 (IF: 1:400, a kind gift from Dr. Jou Tzuu-Shuh, NTU, Taiwan) , Rab11a (IF: 1:400, WB: 1:2000, 20229-1-AP, Proteintech, Rosemont, IL), aPKC (IF: 1:200, 610175, BD Biosciences, Franklin Lakes, NJ), CEP120 (IF: 1:1000, WB: 1:2000, human residues 639–986, described in our previous paper) , p53 (IF: 1:100, 9282, Cell Signaling Technology, Danvers, MA), p21 (WB: 1:2000, 10355-1-AP, Proteintech), CEP164 (IF: 1:400, NBP1-81445, NOVUS, Centennial, CO), PCNT (IF: 1:200, ab4448, Abcam, Cambridge, UK), Par3 (IF: 1:200, WB: 1:1000, 07-330, EMD Millipore, Burlington, MA), and beta-actin (IF: 1:200, A2066, Sigma-Aldrich, St. Louis, MO); rabbit monoclonal antibodies against E-cadherin (IF: 1:200, 24E10, #3195, Cell Signaling Technology); and mouse monoclonal antibodies against γ-tubulin (IF: 1:400, GTU-88, T6557, Sigma-Aldrich), acetylated tubulin (IF: 1:400, T6793, Sigma-Aldrich), Gp135 (IF: 1:10, the culture medium of hybridoma clone 3F2D8, a kind gift from Dr. Jou Tzuu-Shuh, NTU, Taiwan) , polyglutamylated tubulin (IF: 1:100, GT335, AG-20B-0020, Adipogen, San Diego, CA), ODF2 (IF: 1:400, H00004957-M01, NOVUS), α-tubulin (WB: 1:5000, DM1α, T9026, Sigma-Aldrich), CDC42 (WB: 1:1000, 610928, BD Biosciences), and ZO-1 (IF: 1:400, 33-9100, ThermoFisher Scientific, Waltham, MA).

Techniques: Stable Transfection, Expressing, shRNA, Knockdown, Cell Culture, Labeling, Sequencing

Journal: bioRxiv

Article Title: Centrosome Migration and Apical Membrane Formation in Polarized Epithelial Cells: Insights from the MDCK Cyst Model

doi: 10.1101/2024.06.17.598507

Figure Lengend Snippet: Centrosome position relative to the cytokinesis site in polarized epithelial sheets (A) Experimental design: Aphidicolin (Aphi)-treated cells (synchronized at the S phase) were seeded into Matrigel and observed within 8 h to exclude the possibility of cell doublet formation through cell division. (B) Bright-field images of MDCK cells cultured in low-attachment microwells. The images are merged with the DAPI (cyan) signal to show cell division occurring over time. (C–D) Polarized MDCK cyst and epithelial sheet with labeled markers: the centrosome markers PACT-mKO1 and γ-tubulin, acetyl-tubulin, F-actin, and DAPI. Insets show enlargements of the image in the boxed area. “L” indicates the lumen in (C). The z-projection of the top view and the X-Z cross-section of the side view (bottom) are shown in (D). Telo, telophase; Pre-Abs, pre-abscission. Arrowheads point to the centrosome position. Scale bar: 10 μm. (E) Time-lapse snapshots of MDCK cells expressing EGFP-Gp135 and PACT-mKO1 cultured on a Transwell insert to form polarized epithelial sheets. The microtubule probe SiR-tubulin (white) was applied before live cell imaging. The X-Y top view and the X-Z cross-section of the side view (bottom) are shown. Time stamps show hours and minutes, with 0:00 set to the first frame after anaphase onset. Arrowheads point to the centrosomes. The yellow arrow indicates the abscission of the cytokinesis bridge. Scale bar: 10 μm. A schematic depicts the centrosomes moving to the apical membrane before bridge abscission. (F) Change in distance from the centrosome to the apical membrane, as measured in each frame (data from three independent experiments, n = 5 cells). The graph presents the mean ± SD.

Article Snippet: We used rabbit polyclonal antibodies against Gp135 (IF: 1:400, a kind gift from Dr. Jou Tzuu-Shuh, NTU, Taiwan) , Rab11a (IF: 1:400, WB: 1:2000, 20229-1-AP, Proteintech, Rosemont, IL), aPKC (IF: 1:200, 610175, BD Biosciences, Franklin Lakes, NJ), CEP120 (IF: 1:1000, WB: 1:2000, human residues 639–986, described in our previous paper) , p53 (IF: 1:100, 9282, Cell Signaling Technology, Danvers, MA), p21 (WB: 1:2000, 10355-1-AP, Proteintech), CEP164 (IF: 1:400, NBP1-81445, NOVUS, Centennial, CO), PCNT (IF: 1:200, ab4448, Abcam, Cambridge, UK), Par3 (IF: 1:200, WB: 1:1000, 07-330, EMD Millipore, Burlington, MA), and beta-actin (IF: 1:200, A2066, Sigma-Aldrich, St. Louis, MO); rabbit monoclonal antibodies against E-cadherin (IF: 1:200, 24E10, #3195, Cell Signaling Technology); and mouse monoclonal antibodies against γ-tubulin (IF: 1:400, GTU-88, T6557, Sigma-Aldrich), acetylated tubulin (IF: 1:400, T6793, Sigma-Aldrich), Gp135 (IF: 1:10, the culture medium of hybridoma clone 3F2D8, a kind gift from Dr. Jou Tzuu-Shuh, NTU, Taiwan) , polyglutamylated tubulin (IF: 1:100, GT335, AG-20B-0020, Adipogen, San Diego, CA), ODF2 (IF: 1:400, H00004957-M01, NOVUS), α-tubulin (WB: 1:5000, DM1α, T9026, Sigma-Aldrich), CDC42 (WB: 1:1000, 610928, BD Biosciences), and ZO-1 (IF: 1:400, 33-9100, ThermoFisher Scientific, Waltham, MA).

Techniques: Cell Culture, Labeling, Expressing, Live Cell Imaging, Membrane

Journal: bioRxiv

Article Title: Centrosome Migration and Apical Membrane Formation in Polarized Epithelial Cells: Insights from the MDCK Cyst Model

doi: 10.1101/2024.06.17.598507

Figure Lengend Snippet: (A) Time-lapse snapshots of MDCK cells expressing EGFP-Gp135 (green) and PACT-mKO1 (magenta, centrosome marker) in Matrigel. Nuclei were labeled with SiR-DNA (cyan) before live imaging. Z-projection images of an aggregated cell doublet are shown. Arrows indicate Gp135 on the outer surface of the cell aggregate. Arrowheads point to mislocalized centrosomes that did not move over 3 h. Time stamps show hours and minutes, with 0:00 set at the beginning of live cell imaging. Scale bar: 10 μm. (B, E) MDCK cells, with or without aphidicolin (Aphi) synchronization, were cultured in Matrigel for 6h with labeled markers: Rab11a (B, green), Par3 (E, green), γ-tubulin (magenta), and DAPI for nuclei (blue). Single confocal sections are shown. Two types of cell doublets, formed by cell division (post cytokinesis [CK]) or aggregation, were compared. Arrows indicate Gp135 clusters around centrosomes. Arrowheads point to mislocalized centrosomes. Scale bar: 5 μm. (C–D) Boxplots of centrosome positions, represented by θ N-C (C) and L IC (D), in cell doublets (n = 15 [unsynchronized control], 20 [Aphi-synchronized cells]). Cells were analyzed in three independent experiments. Statistical analyses used an unpaired two-tailed Mann–Whitney U test (*p<0.05, ****p<0.0001). Midlines and boxes show the mean ± SD, with whiskers indicating minimum and maximum values. (F) MDCK cells were cultured in low-attachment microwells for 24h with labeled markers: Gp135 (green), γ-tubulin (magenta), Par3 (white), and DAPI for nuclei (blue). A single confocal section through the middle of a cell doublet is shown. Yellow arrows indicate the Par3signal at the edge of cell-cell contacts. The 3D reconstruction reveals Par3 forming a ring around the edge between cell doublets. Scale bar: 10 μm. (G) Time-lapse snapshots of MDCK cells expressing EGFP-Gp135 (green) and PACT-mKO1 (magenta, centrosome marker) in low-attachment microwells. Nuclei were labeled with SiR-DNA (cyan) before live imaging. Z-projection images of a dividing cell are shown. White and yellow arrows indicate Gp135 on the outer surface of the cell doublet and around the centrosome, respectively. Arrowheads point to the centrosome position. Time stamps show hours and minutes, with 0:00 set at the first frame of anaphase onset. Scale bar: 10 μm. (H–J) Change in polarity index (H), θ N-C (I), and L O-C (J) over time. Each data point represents the average at a given time (three cells in low-attachment microwells from three independent experiments). The lines show the means, and the shaded regions indicate SD values.

Article Snippet: We used rabbit polyclonal antibodies against Gp135 (IF: 1:400, a kind gift from Dr. Jou Tzuu-Shuh, NTU, Taiwan) , Rab11a (IF: 1:400, WB: 1:2000, 20229-1-AP, Proteintech, Rosemont, IL), aPKC (IF: 1:200, 610175, BD Biosciences, Franklin Lakes, NJ), CEP120 (IF: 1:1000, WB: 1:2000, human residues 639–986, described in our previous paper) , p53 (IF: 1:100, 9282, Cell Signaling Technology, Danvers, MA), p21 (WB: 1:2000, 10355-1-AP, Proteintech), CEP164 (IF: 1:400, NBP1-81445, NOVUS, Centennial, CO), PCNT (IF: 1:200, ab4448, Abcam, Cambridge, UK), Par3 (IF: 1:200, WB: 1:1000, 07-330, EMD Millipore, Burlington, MA), and beta-actin (IF: 1:200, A2066, Sigma-Aldrich, St. Louis, MO); rabbit monoclonal antibodies against E-cadherin (IF: 1:200, 24E10, #3195, Cell Signaling Technology); and mouse monoclonal antibodies against γ-tubulin (IF: 1:400, GTU-88, T6557, Sigma-Aldrich), acetylated tubulin (IF: 1:400, T6793, Sigma-Aldrich), Gp135 (IF: 1:10, the culture medium of hybridoma clone 3F2D8, a kind gift from Dr. Jou Tzuu-Shuh, NTU, Taiwan) , polyglutamylated tubulin (IF: 1:100, GT335, AG-20B-0020, Adipogen, San Diego, CA), ODF2 (IF: 1:400, H00004957-M01, NOVUS), α-tubulin (WB: 1:5000, DM1α, T9026, Sigma-Aldrich), CDC42 (WB: 1:1000, 610928, BD Biosciences), and ZO-1 (IF: 1:400, 33-9100, ThermoFisher Scientific, Waltham, MA).

Techniques: Expressing, Marker, Labeling, Imaging, Live Cell Imaging, Cell Culture, Control, Two Tailed Test, MANN-WHITNEY

Journal: bioRxiv

Article Title: Centrosome Migration and Apical Membrane Formation in Polarized Epithelial Cells: Insights from the MDCK Cyst Model

doi: 10.1101/2024.06.17.598507

Figure Lengend Snippet: Summary of centrosome migration, apical membrane component trafficking, and Par3 recruitment in different experiments and culture conditions (A) In conventional Matrigel culture, centrosomes move directionally toward AMIS following cell division (magenta arrow). Apical membrane components like Gp135, Crb3, and Cdc42 follow the centrosomes (green arrow). Par3 first emerges at the cytokinesis site, which regulates centrosome positioning and polarized vesicle trafficking during polarization. (B) Loss of centrosomes diminishes the effectiveness of apical membrane component trafficking (smaller green arrow), yet Par3 still localizes to the cytokinetic bridge. Additionally, centrosome loss mainly impacts the initial stages of epithelial polarization rather than the later stages of lumen formation. (C) Loss of Par3 results in randomized centrosome positioning, obstructs polarized trafficking, and traps apical membrane components around mislocalized centrosomes. (D) Two-cell aggregates without cell division cannot effectively guide centrosome migration and polarized vesicle trafficking. Despite Par3 being recruited to the cell-cell interface, apical membrane components remain trapped around mislocalized centrosomes. (E) MDCK cells suspended in the ECM-free condition. Centrosomes migrate to the center of cell doublets during cytokinesis (magenta arrow), while Apical membrane components transport in the opposite direction (green arrow). After mitosis, Par3 exhibits a pattern distinct from that observed in Matrigel culture.

Article Snippet: We used rabbit polyclonal antibodies against Gp135 (IF: 1:400, a kind gift from Dr. Jou Tzuu-Shuh, NTU, Taiwan) , Rab11a (IF: 1:400, WB: 1:2000, 20229-1-AP, Proteintech, Rosemont, IL), aPKC (IF: 1:200, 610175, BD Biosciences, Franklin Lakes, NJ), CEP120 (IF: 1:1000, WB: 1:2000, human residues 639–986, described in our previous paper) , p53 (IF: 1:100, 9282, Cell Signaling Technology, Danvers, MA), p21 (WB: 1:2000, 10355-1-AP, Proteintech), CEP164 (IF: 1:400, NBP1-81445, NOVUS, Centennial, CO), PCNT (IF: 1:200, ab4448, Abcam, Cambridge, UK), Par3 (IF: 1:200, WB: 1:1000, 07-330, EMD Millipore, Burlington, MA), and beta-actin (IF: 1:200, A2066, Sigma-Aldrich, St. Louis, MO); rabbit monoclonal antibodies against E-cadherin (IF: 1:200, 24E10, #3195, Cell Signaling Technology); and mouse monoclonal antibodies against γ-tubulin (IF: 1:400, GTU-88, T6557, Sigma-Aldrich), acetylated tubulin (IF: 1:400, T6793, Sigma-Aldrich), Gp135 (IF: 1:10, the culture medium of hybridoma clone 3F2D8, a kind gift from Dr. Jou Tzuu-Shuh, NTU, Taiwan) , polyglutamylated tubulin (IF: 1:100, GT335, AG-20B-0020, Adipogen, San Diego, CA), ODF2 (IF: 1:400, H00004957-M01, NOVUS), α-tubulin (WB: 1:5000, DM1α, T9026, Sigma-Aldrich), CDC42 (WB: 1:1000, 610928, BD Biosciences), and ZO-1 (IF: 1:400, 33-9100, ThermoFisher Scientific, Waltham, MA).

Techniques: Migration, Membrane

Journal: bioRxiv

Article Title: FGF receptor modulates planar cell polarity in the neuroectoderm via Vangl2 tyrosine phosphorylation

doi: 10.1101/2025.05.28.656647

Figure Lengend Snippet: (A) Experimental scheme. Thirty-two-cell Xenopus embryos were co-injected with 5 nl of control (Co) or FGFR1 morpholino (MO), 15 ng each, along with GFP RNA (90 pg) as a lineage tracer into one dorsal animal blastomere. Embryos were collected at stage 15 (st.15) and co-immunostained for Vangl2 (red) and GFP (green). The anteroposterior (AP) axis is indicated. Representative en face neural plate images are shown in (B) and (C), white box areas are magnified on the right to show merged (green+red) and single (red) channel images. (B) Control neuroectoderm with anteriorly polarized Vangl2 (arrows). (C) FGFR1MO-injected neuroectoderm lacking anterior Vangl2 accumulation (asterisks). (D) Frequencies of GFP-positive cells with anteriorly enriched Vangl2 in the CoMO and FGFR1MO injected neuroectoderm. Numbers of scored cells (n) are indicated on the top of each bar. Scoring was performed on 50-90 cells per embryo, three embryos per group. Data represent four independent experiments; *** p<0.001, two-tailed unpaired Student’s t test. Scale bar, 30 µm.

Article Snippet: Vangl2 pulldowns were immunoblotted with mouse Vangl2-specific antibody (C8, Santa Cruz), pY pulldowns were immunoblotted with rat anti-Vangl2 monoclonal antibody (MABN750, Millipore Sigma) and rabbit Cyclin B1 antibody (H-433, Santa Cruz) that served as negative control.

Techniques: Injection, Control, Two Tailed Test

Journal: bioRxiv

Article Title: FGF receptor modulates planar cell polarity in the neuroectoderm via Vangl2 tyrosine phosphorylation

doi: 10.1101/2025.05.28.656647

Figure Lengend Snippet: Representative image of neural plate, stage 15 of an embryo injected with FGFR1 morpholino (MO) and GFP RNA, and co-immunostained for Vangl2 and GFP as described in legend and Methods. A cell was scored as polarized if the fluorescence intensity at an anteroposterior (A-P) junction was at least twofold higher compared to the fluorescence intensity at the mediolateral (M-L) junction. Examples of a planar-polarized cell (arrow) and a non-polarized cell depleted of FGFR1 (asterisk) are shown. Merged channels (Vangl2 (red) +GFP(green)) are shown. A-P and M-L junctions are indicated by dashed boxes. Anteroposterior (A-P) axis is marked. Scale bar: 50 µm.

Article Snippet: Vangl2 pulldowns were immunoblotted with mouse Vangl2-specific antibody (C8, Santa Cruz), pY pulldowns were immunoblotted with rat anti-Vangl2 monoclonal antibody (MABN750, Millipore Sigma) and rabbit Cyclin B1 antibody (H-433, Santa Cruz) that served as negative control.

Techniques: Injection, Fluorescence

Journal: bioRxiv

Article Title: FGF receptor modulates planar cell polarity in the neuroectoderm via Vangl2 tyrosine phosphorylation

doi: 10.1101/2025.05.28.656647

Figure Lengend Snippet: (A) Experimental scheme. Thirty-two-cell stage Xenopus embryos were injected with RNAs encoding GFP (200 pg) and dominant-interfering FGFR1 construct (XFD, 400 pg) into one dorsal animal blastomere. Embryos were collected at stage 15 and co-immunostained for Vangl2 (red) and GFP (green). The dashed boxed area is magnified in (B-C’). The anteroposterior (A-P) axis is indicated. (B-B’) Representative images of neural plate cells expressing GFP tracer with anteriorly enriched Vangl2 (arrows). (C-C’) Vangl2 is not restricted to anterior cell boundaries in cells expressing XFD (asterisks). (D) Quantification of the mean ± s.d. frequencies of cells with anteriorly enriched Vangl2. Numbers of scored cells (n) are indicated on top of each bar. Three embryos were scored for each group (40-100 cells per embryo). Data represent three independent experiments. Statistical significance was assessed using a two-tailed Student’s t -test (***, p<0.001). Scale bar: 30 µm.

Article Snippet: Vangl2 pulldowns were immunoblotted with mouse Vangl2-specific antibody (C8, Santa Cruz), pY pulldowns were immunoblotted with rat anti-Vangl2 monoclonal antibody (MABN750, Millipore Sigma) and rabbit Cyclin B1 antibody (H-433, Santa Cruz) that served as negative control.

Techniques: Injection, Construct, Expressing, Two Tailed Test

Journal: bioRxiv

Article Title: FGF receptor modulates planar cell polarity in the neuroectoderm via Vangl2 tyrosine phosphorylation

doi: 10.1101/2025.05.28.656647

Figure Lengend Snippet: Thirty-two-cell stage Xenopus embryos were injected into one dorsal animal blastomere with RNAs encoding GFP-Pk3 (150 pg), HA-Vangl2 (20 pg), and myristoylated BFP (BFP, 80 pg), with or without the dominant-interfering FGFR1 construct (XFD, 400 pg), and fixed at stage 14 (st.14). GFP-Pk3 (Pk3, green) and BFP (blue) fluorescence channels are shown. GFP-Pk3 (A, B) and merged GFP-Pk3+BFP fluorescence (A’, B’) is shown. Anteroposterior (A-P) axis is indicated. Dorsal view of representative images of neural plate cells expressing GFP-Pk3 without (A-A’, arrows) and with XFD (B-B’, asterisks). Cell membranes are marked by BFP (A’, B’). Scale bar: 30 µm. (C, D) GFP-Pk3 fluorescence around cell boundaries in control (C) and XFD-expressing (D) mosaic neuroepithelial cells was quantified as described in legend and Methods and plotted as a function of angle. In control cells, GFP-Pk3 fluorescence is enriched at the anterior side (90°), whereas it is evenly distributed in cells co-expressing XFD. Cell numbers (n) are shown.

Article Snippet: Vangl2 pulldowns were immunoblotted with mouse Vangl2-specific antibody (C8, Santa Cruz), pY pulldowns were immunoblotted with rat anti-Vangl2 monoclonal antibody (MABN750, Millipore Sigma) and rabbit Cyclin B1 antibody (H-433, Santa Cruz) that served as negative control.

Techniques: Injection, Construct, Fluorescence, Expressing, Control

Journal: bioRxiv

Article Title: FGF receptor modulates planar cell polarity in the neuroectoderm via Vangl2 tyrosine phosphorylation

doi: 10.1101/2025.05.28.656647

Figure Lengend Snippet: (A-C) FGFR signaling inhibition decreases Vangl2 tyrosine phosphorylation in Xenopus embryos. (A) experimental scheme for (B-C). Ectoderm explants were dissected from stage 9 embryos and used for pull down with Vangl2 antibody. (B) Explants were dissected from embryos injected with HA-Vangl2 RNA (40 pg), with or without dominant-interfering FGFR1 (XFD) and cultured until stage 12. (C) Reduction of endogenous Vangl2 tyrosine phosphorylation in ectoderm explants (stage 16) depleted of FGFR1 with FGFR1 MO (20 ng). (D-F) Vangl2 tyrosine phosphorylation depends on FGFR signaling activity in mouse embryonic stem (ES) cells. (D) Experimental scheme. (E, F) Lysates from wild-type and FGFR1/2 double knockout mouse ES cells were precipitated with anti-Vangl2 (E) or anti-phosphotyrosine (pY) (F) antibodies and immunoblotted as indicated. Cyclin B1 is a negative control (F).

Article Snippet: Vangl2 pulldowns were immunoblotted with mouse Vangl2-specific antibody (C8, Santa Cruz), pY pulldowns were immunoblotted with rat anti-Vangl2 monoclonal antibody (MABN750, Millipore Sigma) and rabbit Cyclin B1 antibody (H-433, Santa Cruz) that served as negative control.

Techniques: Inhibition, Phospho-proteomics, Injection, Cell Culture, Activity Assay, Double Knockout, Negative Control

Journal: bioRxiv

Article Title: FGF receptor modulates planar cell polarity in the neuroectoderm via Vangl2 tyrosine phosphorylation

doi: 10.1101/2025.05.28.656647

Figure Lengend Snippet: Xenopus embryos were injected with RNAs for HA-Vangl2 (40 pg) with or without XFD-3xFLAG (150 pg) and cultured until stage 12. Vangl2 tyrosine phosphorylation was analyzed by HA-trap pulldown followed by immunoblotting (IB) with anti-pY, anti-HA, or anti-FLAG antibodies as indicated.

Article Snippet: Vangl2 pulldowns were immunoblotted with mouse Vangl2-specific antibody (C8, Santa Cruz), pY pulldowns were immunoblotted with rat anti-Vangl2 monoclonal antibody (MABN750, Millipore Sigma) and rabbit Cyclin B1 antibody (H-433, Santa Cruz) that served as negative control.

Techniques: Injection, Cell Culture, Phospho-proteomics, Western Blot

Journal: bioRxiv

Article Title: FGF receptor modulates planar cell polarity in the neuroectoderm via Vangl2 tyrosine phosphorylation

doi: 10.1101/2025.05.28.656647

Figure Lengend Snippet: Left: Experimental scheme. Ectoderm explants were dissected from Xenopus embryos injected with HA-Vangl2 RNA (40 pg) at stage 9 and cultured in the presence of FGF2 (25–100 ng/ml), with or without SU5402 (100 µM), until stage 12. Vangl2 phosphorylation was analyzed in anti-Vangl2 immunoprecipitates immunoblotted with anti-phosphotyrosine (pY) and anti-HA antibodies. Intensity ratios of pY to HA signals for control and FGF2-treated explants are shown.

Article Snippet: Vangl2 pulldowns were immunoblotted with mouse Vangl2-specific antibody (C8, Santa Cruz), pY pulldowns were immunoblotted with rat anti-Vangl2 monoclonal antibody (MABN750, Millipore Sigma) and rabbit Cyclin B1 antibody (H-433, Santa Cruz) that served as negative control.

Techniques: Injection, Cell Culture, Phospho-proteomics, Control

Journal: bioRxiv

Article Title: FGF receptor modulates planar cell polarity in the neuroectoderm via Vangl2 tyrosine phosphorylation

doi: 10.1101/2025.05.28.656647

Figure Lengend Snippet: Four- to eight-cell Xenopus embryos were injected with RNAs for GFP-Vangl2, PTK7-GFP and FGFR1-FLAG (100 pg each) (A) or RNAs for HA-Vangl2 (50 pg) and either FGFR1-FLAG (40 pg) or Frizzled3-FLAG (400 pg) (B), lysed at stage 12 and subjected to immunoprecipitation (IP) with anti-FLAG to assess FGFR1-Vangl2 binding (A) or anti-Vangl2 to assess Vangl2 tyrosine phosphorylation (B). PTK7-GFP is a negative control in (A). Frizzled3-dependent Vangl2 band shift in (B) reflects S/T phosphorylation . An irrelevant portion of the membrane was removed. (C) FGF8 induces Vangl2 phosphorylation in Xenopus embryos. Embryos were injected with HA-Vangl2 with or without FGF8 plasmid DNAs (50 pg) each, lysed at stage 14 and precipitated with HA-trap followed by immunoblot with anti-pY antibodies.

Article Snippet: Vangl2 pulldowns were immunoblotted with mouse Vangl2-specific antibody (C8, Santa Cruz), pY pulldowns were immunoblotted with rat anti-Vangl2 monoclonal antibody (MABN750, Millipore Sigma) and rabbit Cyclin B1 antibody (H-433, Santa Cruz) that served as negative control.

Techniques: Injection, Immunoprecipitation, Binding Assay, Phospho-proteomics, Negative Control, Electrophoretic Mobility Shift Assay, Membrane, Plasmid Preparation, Western Blot

Journal: bioRxiv

Article Title: FGF receptor modulates planar cell polarity in the neuroectoderm via Vangl2 tyrosine phosphorylation

doi: 10.1101/2025.05.28.656647

Figure Lengend Snippet: Formation of the complex between Vangl2 and FGFR1 was assessed in C17.2 neural progenitor cells (A) and HEK293T (B) cells. Cell lysates were precipitated with 1 µg of anti-Vangl2 antibody or control rabbit IgG. The immunoprecipitates and input lysates were probed with FGFR1 and Vangl2 antibodies. The band corresponding to the upper FGFR1 band in C17.2 and HEK293T cell lysates is detected in Vangl2 pulldowns from both cell lines (A, B; arrows). Molecular weight markers are indicated.

Article Snippet: Vangl2 pulldowns were immunoblotted with mouse Vangl2-specific antibody (C8, Santa Cruz), pY pulldowns were immunoblotted with rat anti-Vangl2 monoclonal antibody (MABN750, Millipore Sigma) and rabbit Cyclin B1 antibody (H-433, Santa Cruz) that served as negative control.

Techniques: Control, Molecular Weight

Journal: bioRxiv

Article Title: FGF receptor modulates planar cell polarity in the neuroectoderm via Vangl2 tyrosine phosphorylation

doi: 10.1101/2025.05.28.656647

Figure Lengend Snippet: (A) Xenopus embryos were injected with RNAs for Xenopus HA-Vangl2 (40 pg) with or without mouse FGFR1 and FGFR2 (40 pg each), or (B) DNA constructs encoding HA-Vangl2 (25 pg) with or without human FGFR1, FGFR1 Y372>C, or FGFR4 (50 pg each), (see Methods), and cultured until stage 12 (A) or stage 14 (B). Vangl2 tyrosine phosphorylation (Vangl2-pY) was analyzed by pull-downs with rabbit anti-HA antibody (A) or HA-trap beads (B), immunoblotted with anti-pY, anti-HA, or anti-FLAG antibodies as indicated. Expression of wild-type and mutated human FGFR constructs (B) was assessed with anti-phospho-Y653-FGFR1 antibody. Asterisk marks non-specific band in lysates (A, B).

Article Snippet: Vangl2 pulldowns were immunoblotted with mouse Vangl2-specific antibody (C8, Santa Cruz), pY pulldowns were immunoblotted with rat anti-Vangl2 monoclonal antibody (MABN750, Millipore Sigma) and rabbit Cyclin B1 antibody (H-433, Santa Cruz) that served as negative control.

Techniques: Injection, Construct, Cell Culture, Phospho-proteomics, Expressing

Journal: bioRxiv

Article Title: FGF receptor modulates planar cell polarity in the neuroectoderm via Vangl2 tyrosine phosphorylation

doi: 10.1101/2025.05.28.656647

Figure Lengend Snippet: Four- to eight-cell Xenopus embryos were injected into four animal blastomeres with GFP-Vangl2 RNA (40 pg), with or without FGFR1-FLAG RNA (10 pg or 20 pg). Ectoderm explants were dissected at stage 9, cultured until stage 12, lysed, and GFP-Vangl2 was pulled down using GFP-trap beads. Ectoderm explants were cultured with or without FGF2 (100 ng/mL) or SU5402 (100 µM). FGFR1 dose-dependently induced Vangl2 tyrosine phosphorylation. FGF2 strongly activated ERK (pERK) and weakly induced Vangl2 phosphorylation. Neither Vangl2 nor ERK phosphorylation was detected in SU5402-treated explants. Data represent three independent experiments.

Article Snippet: Vangl2 pulldowns were immunoblotted with mouse Vangl2-specific antibody (C8, Santa Cruz), pY pulldowns were immunoblotted with rat anti-Vangl2 monoclonal antibody (MABN750, Millipore Sigma) and rabbit Cyclin B1 antibody (H-433, Santa Cruz) that served as negative control.

Techniques: Injection, Cell Culture, Phospho-proteomics

Journal: bioRxiv

Article Title: FGF receptor modulates planar cell polarity in the neuroectoderm via Vangl2 tyrosine phosphorylation

doi: 10.1101/2025.05.28.656647

Figure Lengend Snippet: Four-to-eight cell embryos were injected into four animal blastomeres with GFP-Vangl2 RNA, FGFR1-FLAG or FGFR1 FCPG -FLAG RNA (40 pg each), and were cultured until stage 12. GFP-Vangl2 was immunoprecipitated (IP) from embryo lysates using GFP-trap beads followed by immunoblotting (IB) with anti-pY, anti-GFP, anti-FLAG, anti-pERK1/2 and anti-ERK1/2 antibodies. FGFR1 FCPG containing mutations in the binding sites of known signaling mediators induced Vangl2 tyrosine phosphorylation but did not upregulate ERK1/2 activity.

Article Snippet: Vangl2 pulldowns were immunoblotted with mouse Vangl2-specific antibody (C8, Santa Cruz), pY pulldowns were immunoblotted with rat anti-Vangl2 monoclonal antibody (MABN750, Millipore Sigma) and rabbit Cyclin B1 antibody (H-433, Santa Cruz) that served as negative control.

Techniques: Injection, Cell Culture, Immunoprecipitation, Western Blot, Binding Assay, Phospho-proteomics, Activity Assay

Journal: bioRxiv

Article Title: FGF receptor modulates planar cell polarity in the neuroectoderm via Vangl2 tyrosine phosphorylation

doi: 10.1101/2025.05.28.656647

Figure Lengend Snippet: (A) Mapping major phospho-tyrosine (pY) sites in Vangl2. Embryos were injected with 40 pg of RNA encoding FGFR1 and the indicated HA-Vangl2 constructs. Vangl2 phosphorylation was analyzed in anti-HA pull-downs from stage 13 embryo lysates. Immunoblotting (IB) was performed using anti-pY, anti-HA or anti-FLAG antibodies, as indicated. One representative set of duplicate samples is shown. The graph below shows average pY/HA intensity ratios for the duplicates. (B) Alignment of N-terminal amino acid sequences of Vangl2 from several chordate species and Drosophila Van Gogh. The N-terminal tyrosine cluster (in red) is conserved in vertebrates but not in Drosophila . (C-E) Planar polarization of Vangl2 tyrosine phosphosite mutants in the neuroectoderm. (C) Experimental scheme. Two dorsal blastomeres of 16-cell embryos were coinjected with myrBFP RNA (80 pg) and RNAs encoding GFP-Pk3 (150 pg) HA-tagged Vangl2 (in D), Y7,10,12>F (Y>F, in E) or Y7,10,12>E (Y>E, in F) (20 pg each). Embryos were fixed at stages 14-15, and GFP and BFP fluorescence were imaged. Anterior enrichment of GFP-Pk3 is indicated by arrows (D-E’), while a non-polarized cell is marked by an asterisk (F-F’). The anteroposterior (AP) axis is indicated. (G) Quantification of GFP fluorescence for the GFP-Pk3-Vangl2 complexes in mosaically expressing cells is shown as a graph. Mean fluorescence is plotted along the cell circumference as a function of circular angle from 0 to 360 degrees relative to the AP axis, with fluorescence intensity shown in green (HA-Vangl2), pink (HA-Vangl2Y>F), and brown (HA-Vangl2Y>E) lines. The number of scored cells is indicated. The data are representative of four independent experiments.

Article Snippet: Vangl2 pulldowns were immunoblotted with mouse Vangl2-specific antibody (C8, Santa Cruz), pY pulldowns were immunoblotted with rat anti-Vangl2 monoclonal antibody (MABN750, Millipore Sigma) and rabbit Cyclin B1 antibody (H-433, Santa Cruz) that served as negative control.

Techniques: Injection, Construct, Phospho-proteomics, Western Blot, Fluorescence, Expressing

Journal: bioRxiv

Article Title: FGF receptor modulates planar cell polarity in the neuroectoderm via Vangl2 tyrosine phosphorylation

doi: 10.1101/2025.05.28.656647

Figure Lengend Snippet: Embryos were injected with 40 pg of RNAs encoding FGFR1, FGFR1D623A and various HA-Vangl2 constructs as indicated. Vangl2 phosphorylation was analyzed in HA-trap immunoprecipitates from stage 12 embryo lysates. Immunoblotting (IB) was done with anti-pY, anti-HA or anti-FLAG antibodies as indicated.

Article Snippet: Vangl2 pulldowns were immunoblotted with mouse Vangl2-specific antibody (C8, Santa Cruz), pY pulldowns were immunoblotted with rat anti-Vangl2 monoclonal antibody (MABN750, Millipore Sigma) and rabbit Cyclin B1 antibody (H-433, Santa Cruz) that served as negative control.

Techniques: Injection, Construct, Phospho-proteomics, Western Blot

Journal: bioRxiv

Article Title: FGF receptor modulates planar cell polarity in the neuroectoderm via Vangl2 tyrosine phosphorylation

doi: 10.1101/2025.05.28.656647

Figure Lengend Snippet: Four- to eight-cell Xenopus embryos were injected with RNAs encoding HA-Vangl2, Y7Y10Y12>F (Y>F), or Y7Y10Y12>E (Y>E) constructs (40 pg each). Vangl2 proteins were pulled down from stage 13 lysates using anti-HA antibody and probed with anti-pS14S17, anti-pT78S79S82, and anti-HA antibodies. Band intensity ratios (pS14S17/HA and pT78S79S82/HA) are shown. Molecular weights are indicated. Data represent two independent experiments.

Article Snippet: Vangl2 pulldowns were immunoblotted with mouse Vangl2-specific antibody (C8, Santa Cruz), pY pulldowns were immunoblotted with rat anti-Vangl2 monoclonal antibody (MABN750, Millipore Sigma) and rabbit Cyclin B1 antibody (H-433, Santa Cruz) that served as negative control.

Techniques: Injection, Construct

Journal: bioRxiv

Article Title: FGF receptor modulates planar cell polarity in the neuroectoderm via Vangl2 tyrosine phosphorylation

doi: 10.1101/2025.05.28.656647

Figure Lengend Snippet: (A) Schematic for the proximity biotinylation assay to assess the interaction between Vangl2 and Prickle3 ( Pk3). Vangl2 is biotinylated (asterisks) when in proximity to the Pk3 fused to the large N-terminal fragment of a bacterial biotin ligase (BLN). (B) Animal blastomeres of four-to-eight-cell stage embryos were co-injected with 100 pg of FLAG-BLN-Pk3 RNA and 40 pg of HA-Vangl2, HA-Vangl2 Y7,10,12>F (Y>F), or HA-Vangl2 Y7,10,12>E (Y>E) RNAs. At stages 8-9, 20 nl of biotin (0.8 mM) was injected into the blastocoel. Embryos were collected at stage 13, and Vangl2 constructs were pulled down using anti-HA antibody. Biotinylation of Vangl2 and protein expression levels were assessed in independent duplicate samples using anti-biotin, anti-HA, and anti-FLAG antibodies. Data represent three independent experiments.

Article Snippet: Vangl2 pulldowns were immunoblotted with mouse Vangl2-specific antibody (C8, Santa Cruz), pY pulldowns were immunoblotted with rat anti-Vangl2 monoclonal antibody (MABN750, Millipore Sigma) and rabbit Cyclin B1 antibody (H-433, Santa Cruz) that served as negative control.

Techniques: Cell Surface Biotinylation Assay, Injection, Construct, Expressing

Journal: bioRxiv

Article Title: FGF receptor modulates planar cell polarity in the neuroectoderm via Vangl2 tyrosine phosphorylation

doi: 10.1101/2025.05.28.656647

Figure Lengend Snippet: (A, B) Vangl2-PTK7 binding is enhanced by non-phosphorylated Vangl2 and reduced by phosphorylated Vangl2. Four-to-eight-cell Xenopus embryos were injected with RNAs encoding HA-Vangl2, HA-Vangl2 Y7Y10Y12>F (Y>F) or HA-Vangl2 Y7Y10Y12>E (Y>E) (40 pg each) as indicated, with or without PTK7-GFP RNA, 100 pg, into four animal blastomeres. Embryos were collected at stage 12 and PTK7-GFP (A) or HA-Vangl2 (B) were immunoprecipitated using GFP-trap or anti-HA antibody, respectively, for Vangl2 analysis (A) or PTK7 analysis (B). Protein levels in pull-downs and lysates were analyzed with anti-GFP and anti-HA antibodies. Irrelevant part of the membrane was removed (A). Data are representative of three experiments.

Article Snippet: Vangl2 pulldowns were immunoblotted with mouse Vangl2-specific antibody (C8, Santa Cruz), pY pulldowns were immunoblotted with rat anti-Vangl2 monoclonal antibody (MABN750, Millipore Sigma) and rabbit Cyclin B1 antibody (H-433, Santa Cruz) that served as negative control.

Techniques: Binding Assay, Injection, Immunoprecipitation, Membrane

Journal: bioRxiv

Article Title: FGF receptor modulates planar cell polarity in the neuroectoderm via Vangl2 tyrosine phosphorylation

doi: 10.1101/2025.05.28.656647

Figure Lengend Snippet: Neural folding defects were assessed in embryos injected with RNAs encoding PTK7 and different forms of Vangl2 (200 pg each) into two dorsal animal blastomeres (inset in A, red arrows). Dorsal views of representative embryos exhibit no (A), mild (B) or severe (C) disruption of neural tube closure. Co-injection of PTK7 and Y>F Vangl2 RNAs causes stronger neural tube defects (NTDs) compared to the co-injection of the wild type or Y>E Vangl2 RNAs. The degree of neural tube closure was scored at stage 17 by the distance between the opposing neural folds near the brain-spinal cord border (arrowheads), anterior is to the top. Dashed line marks the midline. (D) Quantification showing frequencies of normal neural folds (A), as compared to mild (B) and severe (C) defects. The number of scored embryos is shown in the graph. The significance was determined by full 2×3 Fisher’s exact test that was calculated in Prism 10 software; **** p<0.0001, * p<0.05. Data represent two independent experiments. (E) Model. Graded FGF/FGFR1 activity triggers Vangl2 tyrosine phosphorylation at posterior cell edges, inhibiting the formation of Vangl2-Pk3 and Vangl2-PTK7 complexes, leading to reduced posterior accumulation of Vangl2. PTK7 is proposed to act as a feedback regulator and FGFR1 antagonist, promoting Vangl2 dephosphorylation through phosphatase recruitment.

Article Snippet: Vangl2 pulldowns were immunoblotted with mouse Vangl2-specific antibody (C8, Santa Cruz), pY pulldowns were immunoblotted with rat anti-Vangl2 monoclonal antibody (MABN750, Millipore Sigma) and rabbit Cyclin B1 antibody (H-433, Santa Cruz) that served as negative control.

Techniques: Injection, Disruption, Software, Activity Assay, Phospho-proteomics, De-Phosphorylation Assay

Journal: eBioMedicine

Article Title: Disulfiram metabolite Cu(DDC) 2 enhances radionuclide uptake in vivo revealing insights into tumoural ablation resistance

doi: 10.1016/j.ebiom.2026.106165

Figure Lengend Snippet: Copper-bound metabolite of disulfiram augments NIS activity and radioiodide u ptake. (A) Overview of rational design and reformulation drug strategies used in study. (B) RAI uptake of copper gluconate [Cu(II)]-treated 8505C–NIS and TPC-1-NIS cells alone or in combination with disulfiram (DSF) versus untreated (UT). (C) Schematic illustrating metabolic conversion of DSF to copper diethyldithiocarbamate [Cu(DDC) 2 ]. (D) RAI uptake of Cu(DDC) 2 -treated 8505C–NIS and TPC-1-NIS cells. (E) Same as (D) but using parental 8505C and TPC-1 cells. (F) Western blot analysis of NIS expression in Cu(DDC) 2 -treated 8505C–NIS and TPC-1-NIS cells; HE, higher exposure. (G and H) Confocal imaging of 8505C–NIS-HA (G) and TPC-1-NIS-HA (H) cells treated with Cu(DDC) 2 or vehicle (DMSO). Confocal images represent HA expression (green), NIS expression (red), and a merged image (yellow). Arrows (white) indicate regions of greater NIS plasma membrane localisation; HA, haemagglutinin. Scale bar: 20 μm. (I) Western blot analysis of NIS protein levels at the PM relative to Na+/K + ATPase following the cell-surface biotinylation assay (CSBA) in 8505C–NIS cells after Cu(DDC) 2 treatment. Control: Biotin tag omitted ( upper ), total protein before biotin separation ( lower ). (J) RAI uptake of Cu(DDC) 2 -treated human primary thyrocytes. Data presented as mean ± SEM (n = 3); one-way ANOVA, Dunnett's or Tukey's post hoc test (ns, not significant; ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001); unpaired two-tailed t-test ( # P < 0.05).

Article Snippet: Western blotting and cell surface biotinylation assays (CSBA) were performed as described previously., , Blots were probed with specific antibodies against Na,K-ATPase (1:1000; Cell Signalling Technology), NIS (1:1000; Proteintech), NPL4 (1:500; Cell Signalling Technology), VCP (1:1000; Cell Signalling Technology) and β-actin (1:10000; Sigma–Aldrich).

Techniques: Activity Assay, Western Blot, Expressing, Imaging, Clinical Proteomics, Membrane, Cell Surface Biotinylation Assay, Control, Two Tailed Test

Journal: eBioMedicine

Article Title: Disulfiram metabolite Cu(DDC) 2 enhances radionuclide uptake in vivo revealing insights into tumoural ablation resistance

doi: 10.1016/j.ebiom.2026.106165

Figure Lengend Snippet: Dual agonist effect of Cu(DDC) 2 enhances NIS activity . (A – C) RAI uptake in parental 8505C cells (A), parental SW1736 cells (B) and human primary thyrocytes (C) following PAX8-siRNA depletion and Cu(DDC) 2 treatment. CON: scrambled control siRNA. (D) Relative NIS mRNA in human primary thyrocytes following PAX8-siRNA depletion and Cu(DDC) 2 treatment. (E) Relative PAX8 mRNA in human primary thyrocytes following PAX8-siRNA depletion. (F) RAI uptake in 8505C–NIS and TPC-1-NIS cells following NPL4-or VCP-siRNA depletion and Cu(DDC) 2 treatment. (G) Western blot analysis of NIS, NPL4 and VCP in 8505C–NIS and TPC-1-NIS cells after NPL4-or VCP-siRNA depletion. (H) Same as (F) but in human primary thyrocytes. (I) NanoBiT evaluation of protein: protein interaction between NIS and VCP in living HeLa cells treated with CB5339 or Cu(DDC) 2 versus controls. (J) Schematic illustrating NanoBRET assay to monitor proximity of NIS with plasma membrane protein KRAS, as well as subcellular markers RAB5 (early endosome) and RAB11 (recycling endosome). Created with BioRender.com . Modified from Read ML et al. Clinical Cancer Research, 2024. (K and L) NanoBRET evaluation of NIS localisation at the PM (K, KRAS) or in ER-golgi (L, RAB1) in live HeLa and HEK293 cells treated with Cu(DDC) 2 . Data presented as mean ± SEM (n ≥ 3), one-way ANOVA, Dunnett's or Tukey's post hoc test (ns, not significant, ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001), unpaired two-tailed t-test ( # P < 0.05, ## P < 0.01, ### P < 0.001).

Article Snippet: Western blotting and cell surface biotinylation assays (CSBA) were performed as described previously., , Blots were probed with specific antibodies against Na,K-ATPase (1:1000; Cell Signalling Technology), NIS (1:1000; Proteintech), NPL4 (1:500; Cell Signalling Technology), VCP (1:1000; Cell Signalling Technology) and β-actin (1:10000; Sigma–Aldrich).

Techniques: Activity Assay, Control, Western Blot, Clinical Proteomics, Membrane, Modification, Two Tailed Test

Journal: eBioMedicine

Article Title: Disulfiram metabolite Cu(DDC) 2 enhances radionuclide uptake in vivo revealing insights into tumoural ablation resistance

doi: 10.1016/j.ebiom.2026.106165

Figure Lengend Snippet: Copper-bound metabolite stimulates NIS activity to enhance radionuclide uptake in vivo . (A) Schematic illustrating albumin nanoencapsulation of Cu(DDC) 2 . Lower : RAI uptake in 8505C–NIS cells treated with Cu(DDC) 2 in DMSO or albumin nanoencapsulated (ALB). (B) Overview of in vivo study to investigate the effect of Cu(DDC) 2 given by IP (step 1) or IV routes (step 2) on thyroidal NIS function in WT BALB/c mice. (C and D) 99m TcO4 - uptake (C, n = 6–11) and relative NIS mRNA (D) in thyroid glands from Cu(DDC) 2 -ALB treated WT BALB/c mice given by IP route. Total animals used = 18). (E and F) Same as (C and D) but Cu(DDC) 2 -ALB given by IV route at indicated doses (n = 5–7). Total animals used = 21. ( G ) Pearson correlation analysis between thyroidal 99m TcO4 - uptake (FC, log 2 ) and relative NIS mRNA (FC, log 2 ) in Cu(DDC) 2 -ALB treated WT BALB/c mice as outlined (B). 95% CI (upper/lower) are shown. (H and I) Relative PAX8 and NKX2-1 mRNA in thyroids from Cu(DDC) 2 -ALB treated WT BALB/c mice given by IP (H) or IV (I) routes. (J and K) Same as (H and I) but relative TPO and TG mRNA. (L) Schematic illustrating the dual impact of Cu(DDC) 2 on NIS function to enhance RAI uptake by inducing NIS mRNA and inhibiting VCP activity. Inset —promoter/enhancer regions of TPO , TG and NIS genes with relative positions of NKX2-1 and PAX8 binding sites. (M) Representative H&E stained images of thyroid tissue from Tg-rtTA/tetO-BRAF V600E mice fed with DOX ( upper ) versus normal ( lower ) chow for 7 days. Scale bars, 100 μM. (N) 99m TcO4 - uptake in thyroid tissue in DOX chow fed Tg-rtTA/tetO-BRAF V600E mice treated with Cu(DDC) 2 -ALB given by IV route (n = 3–6). Total animals used = 9. Data presented as mean ± SEM, unpaired two-tailed t-test (ns, not significant, ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001). Images created with BioRender.com .

Article Snippet: Western blotting and cell surface biotinylation assays (CSBA) were performed as described previously., , Blots were probed with specific antibodies against Na,K-ATPase (1:1000; Cell Signalling Technology), NIS (1:1000; Proteintech), NPL4 (1:500; Cell Signalling Technology), VCP (1:1000; Cell Signalling Technology) and β-actin (1:10000; Sigma–Aldrich).

Techniques: Activity Assay, In Vivo, Binding Assay, Staining, Two Tailed Test

Journal: eBioMedicine

Article Title: Disulfiram metabolite Cu(DDC) 2 enhances radionuclide uptake in vivo revealing insights into tumoural ablation resistance

doi: 10.1016/j.ebiom.2026.106165

Figure Lengend Snippet: Transcription factor and VCP/proteostasis genes predict recurrence risk in RAI-treated PTC . (A) Volcano plot comparing log 2 FC with q -value (-log base 10) for the THCA PTC cohort and 337 transcription factor (TF) genes. (B) Volcano plot comparing log 2 FC with q -value (-log base 10) for the GSE33630 dataset and 323 TF genes. (C) Venn diagram illustrating TF genes common to both GSE33630 and THCA PTC datasets. (D) Volcano plot illustrating log 2 FC compared to q -value (-log base 10) for disease-free survival (DFS) in the BRAF-like, RAI-treated PTC cohort and 337 TF gene panel. (E) Mean number of dysregulated TF genes stratified into high-risk group (bars; left y-axis) and recurrence rate (white crosses; right y-axis) in patient clusters 1 to 4 (n = 20–49). (F) Representative Kaplan–Meier analysis of DFS for BRAF-like, RAI treated PTC stratified into patient clusters 1 to 4, log-rank test. (G) Box and whisker plot showing NIS expression (log 2 ) in BRAF-like, RAI-treated PTC stratified into patient clusters 1 to 4, Kruskal–Wallis test followed by Dunn's post hoc test (ns, not significant, ∗∗∗ P < 0.001). (H) LASSO regression analysis used to construct a 22 TF gene riskscore classifier. LASSO coefficient plot (loglambda). Y-axis: coefficient value; x-axis (lower): log(λ) value, and x-axis (upper): gene number. (I – K) ROC analysis (I) and Kaplan–Meier curve of the 22 TF gene riskscore classifier in BRAF-like, RAI-treated PTC (J) or RAI-treated PTC (K). (L) Kaplan–Meier analysis of DFS in BRAF-like, RAI-treated PTC ( left ) or RAI-treated PTC ( right ) stratified with the dual TF + VCP riskscore classifier. (M) Uni- ( left ) and multivariate analysis ( right ) of RAI-treated PTC (n = 211) stratified with the dual TF + VCP classifier, adjusting for the covariates age, sex, disease stage and ATA risk group in the multivariate model. (N) Same as (M) but with the entire TCGA THCA cohort (n = 399).

Article Snippet: Western blotting and cell surface biotinylation assays (CSBA) were performed as described previously., , Blots were probed with specific antibodies against Na,K-ATPase (1:1000; Cell Signalling Technology), NIS (1:1000; Proteintech), NPL4 (1:500; Cell Signalling Technology), VCP (1:1000; Cell Signalling Technology) and β-actin (1:10000; Sigma–Aldrich).

Techniques: Whisker Assay, Expressing, Construct

Journal: bioRxiv

Article Title: BRI2-mediated regulation of TREM2 processing in microglia and its potential implications for Alzheimer’s disease and related dementias

doi: 10.1101/2023.06.14.544924

Figure Lengend Snippet: UMAP visualization of mouse hippocampal cell clusters (a, left panel) and human DFC cell clusters (b, left panel), classified by cell type based on DEG identified by Seurat v4. (b) Violin plots represent the log-normalized expression of Itm2b and Trem2 across cell populations in mouse hippocampal cell clusters (a, right panel) and human DFC cell clusters (b, right panel). (c) Itm2b and Trem2 mRNA expression in mouse microglia and non-microglia cells analyzed by quantitative RT-PCR.

Article Snippet: The probes Mm01310552_mH (exon junction 1–2) and Mm04209424_g1 (exon junction 3-4) were used to detect mouse Itm2b and Trem2 .

Techniques: Expressing, Quantitative RT-PCR

Journal: bioRxiv

Article Title: BRI2-mediated regulation of TREM2 processing in microglia and its potential implications for Alzheimer’s disease and related dementias

doi: 10.1101/2023.06.14.544924

Figure Lengend Snippet: (a) UMAPs of re-clustered microglia in Object 1. (b) UMAPs split by individual samples. (c) Gene expression heatmap showing the top 5 enriched genes for each microglia cluster. The number of cells per cluster is denoted above the cluster. Itm2b is one of the top genes downregulated in cluster 3 because 89% of the cells in this cluster are from Itm2b-KO mice. (d) Proportional contribution of each genotype to each cluster. Cluster 3 was highly represented in Itm2b-KO mice, with 89% of microglia in this cluster originating from these mice. Conversely, Cluster 7 was preponderant in WT controls. However, ∼93% of the cells assigned to cluster 7 derived from one WT control animal (the male WT control, as depicted in UMAP plot b). Therefore, the observed expansion of cluster 7 is attributed to animal-specific factors rather than genotype-specific factors.

Article Snippet: The probes Mm01310552_mH (exon junction 1–2) and Mm04209424_g1 (exon junction 3-4) were used to detect mouse Itm2b and Trem2 .

Techniques: Gene Expression, Derivative Assay, Control

Journal: bioRxiv

Article Title: BRI2-mediated regulation of TREM2 processing in microglia and its potential implications for Alzheimer’s disease and related dementias

doi: 10.1101/2023.06.14.544924

Figure Lengend Snippet: (a) Schematic representation of ELISA 1 and ELISA 2. Both ELISAs use the same Biotinylated-αTrem2 capture antibody (in black). ELISA 1 uses αTrem2-CT (red) + Sulfo-αRabbit (blue) detection antibodies. ELISA 2 uses αTrem2-NT (orange) + Sulfo-αRat (green) detection antibodies. Trem2 can be detected by both ELISAs, sTrem2 can be detected only by ELISA 2: neither ELISA can detect Trem2-CTF. (b) Quantification of Trem2 and sTrem2 in the P100 and S100 brain fractions of ∼245 days old control ( w/w , 7 females and 12 males), Itm2b-KO (6 females and 7 males) and Trem2-KO (6 females and 7 males) mice. Data were analyzed by ordinary one-way ANOVA followed by post-hoc Tukey’s multiple comparisons test when ANOVA showed significant differences. ELISA1, Trem2 in P100 fraction F (2, 42) = 60.10, P<0.0001; post-hoc Tukey’s multiple comparisons test: w/w vs. Itm2b- KO P=0.6266, not significant. ELISA 2 sTrem2 F (2, 42) = 82.69, P<0.0001; post-hoc Tukey’s multiple comparisons test: w/w vs. Itm2b-KO P<0.0001. ELISA 2 Trem2+sTrem2 in S100 F (2, 6) = 21.06, P<0.0001; post-hoc Tukey’s multiple comparisons test: w/w vs. Itm2b- KO P=0.0703, not significant. All w/w vs. Trem2-KO and Itm2b-KO vs. Trem2-KO comparisons have P<0.0001. (c) Western blot analysis of P100 fractions from a representative w/w, Trem2-KO and Itm2b-KO P100 sample with αTrem2-CT and an αBri2 antibody. (d) Detection and quantification of Trem2-CTF in the P100 fraction by Western blot analysis and with Image Lab software. GAPDH was used as a loading control. Data were analyzed by two–way ANOVA followed by post-hoc Sidak’s multiple comparisons test when ANOVA showed significant differences. Trem2-CTF: sex factor, F (1, 28) = 1.584, P=0.2186; genotype factor, F (1, 28) = 33.25, P<0.0001; sex/genotype interaction, F (1, 28) = 2.682, P=0.1127; post-hoc Sidak’s multiple comparisons test: females, w/w vs. Itm2b-KO P<0.0001; males, w/w vs. Itm2b-KO P=0.0072. GAPDH did not show any significant differences. (e) ELISA measurements of endogenous Aβ40 and Aβ42 in brain homogenates of w/w, Trem2-KO and Itm2b-KO animals. Data were analyzed by ordinary one-way ANOVA followed by post-hoc Tukey’s multiple comparisons test when ANOVA showed significant differences. Aβ40: F (2, 36) =36.34, P<0.0001; post-hoc Tukey’s multiple comparisons test: w/w vs. Itm2b-KO, P=0.0001; w/w vs. Trem2-KO, P=0.0001; Itm2b-KO vs. Trem2-KO, P<0.0001. Aβ42: F (2, 36) =26.40, P<0.0001; post-hoc Tukey’s multiple comparisons test: w/w vs. Itm2b-KO, P=0.0003; w/w vs. Trem2-KO, P=0.0021; Itm2b-KO vs. Trem2-KO, P<0.0001. All data are shown as means +/-SEM: P=0.0005. *=P<0.05, **=P<0.01, ***=P<0.001, ****=P<0.0001.

Article Snippet: The probes Mm01310552_mH (exon junction 1–2) and Mm04209424_g1 (exon junction 3-4) were used to detect mouse Itm2b and Trem2 .

Techniques: Enzyme-linked Immunosorbent Assay, Control, Western Blot, Software

Journal: bioRxiv

Article Title: BRI2-mediated regulation of TREM2 processing in microglia and its potential implications for Alzheimer’s disease and related dementias

doi: 10.1101/2023.06.14.544924

Figure Lengend Snippet: (a) CD11b staining and FACS analysis of brain cells isolated from Cx3cr1 CreER/wt an d Cx3cr1 wt/wt animals. (b) FACS analysis of sorted EYFP + (microglia) and EYFP - (non-microglia) brain cell populations from Itm2b f/f :Cx3cr1 CreER/wt animals. (c) Schematic representation of the PCR test used to identify the Itm2b f and Itm2b KO alleles. (d) PCR analysis of genomic DNA isolated from EYFP + and EYFP - cells sorted from Itm2b f/f :Cx3cr1 CreER/wt brains. (e) ELISA 2 was used to measure sTrem2 levels in Itm2b f/f :Cx3cr1 CreER/wt (females=15, males=12) and Itm2b f/f :Cx3cr1 wt/wt (females=10, males=11) littermates. Data were analyzed by two-tailed unpaired t test: P=0.0004. All data are shown as means +/-SEM: ***=P<0.001.

Article Snippet: The probes Mm01310552_mH (exon junction 1–2) and Mm04209424_g1 (exon junction 3-4) were used to detect mouse Itm2b and Trem2 .

Techniques: Staining, Isolation, Enzyme-linked Immunosorbent Assay, Two Tailed Test

Journal: International Journal of Molecular Sciences

Article Title: A Novel Mouse Model of TGFβ2-Induced Ocular Hypertension Using Lentiviral Gene Delivery

doi: 10.3390/ijms23136883

Figure Lengend Snippet: LV_TGFβ2 transduction induced extracellular matrix (ECM) and cytoskeletal alterations in primary human TM cells. Primary human TM cells were treated with 5 MOI viral load of LV_Null (gray circles) and LV_TGFβ2 (black squares) vectors and incubated for 11 days ( n = 2 responder strain, representative images shown). ( A ) Active TGFβ2 levels were determined in conditioned media of the transduced cells. The total protein loading was determined via Coomassie staining of the PVDF membrane. ( B ) TGFβ2, ECM markers (fibronectin (FN) and collagen-I (Col-I)) (scale 75 μm), and ( C ) cytoskeletal changes (αSMA and F-actin) were also determined via immunostaining (scale 25 μm). LV_TGFβ2-induced expression of all the markers with morphological changes in the cells that included the formation of cross-linked actin networks (CLANs; marked within white circles) and F-actin stress fibers as depicted via phalloidin staining. ( D ) Quantitative analysis of FN Col-I, αSMA, and phalloidin staining from at least 6 different non-overlapping regions of the treated wells. Paired (one-tailed) student t -test, data represented as mean ± SEM, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Fibronectin (FN) , Ab2413 , Abcam , 1:400.

Techniques: Transduction, Incubation, Staining, Immunostaining, Expressing, One-tailed Test

Journal: International Journal of Molecular Sciences

Article Title: A Novel Mouse Model of TGFβ2-Induced Ocular Hypertension Using Lentiviral Gene Delivery

doi: 10.3390/ijms23136883

Figure Lengend Snippet: Primary antibodies used for immunostaining or Western blot.

Article Snippet: Fibronectin (FN) , Ab2413 , Abcam , 1:400.

Techniques: Immunostaining, Western Blot

Journal: Nature cancer

Article Title: L1CAM defines the regenerative origin of metastasis-initiating cells in colorectal cancer

doi: 10.1038/s43018-019-0006-x

Figure Lengend Snippet: a, L1CAM expression is increased in liver metastases (Met) as compared to matched primary tumors (Pri). Immunohistochemistry (IHC) for L1CAM is shown in matched normal colon, primary CRC tumor and liver metastasis sections from a representative patient. Arrows indicate L1CAM staining at the invasion front of the primary tumor. Detail of the boxed region is shown in Extended Data Fig. 1a. b, The percentage of L1CAM-expressing tumor cells in each section. In box plots, boxes show the 25th–75th percentile with the median, and whiskers show the minimum-maximum; n = 18 paired patient samples; two-sided Wilcoxon matched-pairs signed-rank test. c, Percentage of L1CAM-expressing cells in matched pretreatment (pre-treat.) biopsies and post-treatment (post-treat.) surgically resected residual disease in patients with locally advanced rectal cancer. In box plots, boxes show the 25th–75th percentile with the median, and whiskers show the minimum-maximum; n = 31 patients; two-sided Wilcoxon matched-pairs signed-rank test. d, Representative sections of paired pretreatment core biopsies and matched surgical resection specimens obtained after chemoradiation, from two patients with rectal adenocarcinoma, showing L1CAM expression in peripheral areas of residual adenocarcinoma after treatment. e, L1CAM immunohistochemistry in a human CRC liver metastasis resected after neoadjuvant chemotherapy, showing dense stromal infiltration and L1CAM-expressing residual tumor cell clusters. Representative of 18 samples analyzed. f, Tumor L1CAM expression is associated with greater organoid generation capacity. Median L1CAM expression is shown for freshly resected and dissociated patient CRC liver metastases measured by flow cytometry before plating of 10,000 cells in 40 μl of Matrigel using organoid medium. Organoid generation ability was assessed 14 d after plating. In box plots, boxes show the 25th–75th percentile with the median, and whiskers show the minimum-maximum; n = 14 paired patient tumor samples; two-sided Mann-Whitney U test. g, Number of organoids (mean ± s.e.m.) grown from 10,000 L1CAMhigh or L1CAMlow cells flow-sorted from freshly resected patient CRC primary tumors (P, left) or liver metastases (Li, right), counted 14 d after surgical resection and flow sorting. From left to right, n = 3, 3, 3, 3, 3, 3, 3, 3, 3, 4, 3, 4, 4 and 11 replicates per group from each of seven patients; two-tailed Student’s t tests. h, Subcutaneous tumor volumes measured 35 d after transplantation of mice with 50,000 organoid-derived flow-sorted L1CAMhigh or L1CAMlow cells (mean ± s.e.m.); n = 5 mice per group; two-tailed Mann-Whitney U test. i, Biaxial density plot showing relative expression of L1CAM and LGR5 in 9,974 cells from four independent patient-derived metastatic CRC organoids subjected to scRNA-seq. Five clusters identified according to relative L1CAM and LGR5 expression are overlaid as colors on the density plot. j, Dual LGR5 mRNA FISH and L1CAM immunofluorescence (IF) on a patient primary CRC tissue section (top, low magnification; bottom, high magnification), showing discrete expression levels of LGR5 and L1CAM in different cell clusters, including double-positive cells. Representative field of eight tumor sections from four patients analyzed.

Article Snippet: Solid-phase L1CAM ligand binding assays used recombinant human L1CAM (human Fc tag, R&D Systems; His tag, Thermo Fisher Scientific), UltraPure BSA (Thermo Fisher Scientific), purified mouse laminin-111 (Sigma-Aldrich), purified mouse collagen IV (Cultrex, R&D Systems), purified human collagen V (Sigma-Aldrich), recombinant human tenascin C (R&D Systems) and recombinant human laminin-411, laminin-421, laminin-511 and laminin-521 (Biolamina).

Techniques: Expressing, Immunohistochemistry, Staining, Flow Cytometry, MANN-WHITNEY, Two Tailed Test, Transplantation Assay, Derivative Assay, Immunofluorescence

Journal: Nature cancer

Article Title: L1CAM defines the regenerative origin of metastasis-initiating cells in colorectal cancer

doi: 10.1038/s43018-019-0006-x

Figure Lengend Snippet: a, L1CAM inhibition rescues the increase in organoid generation secondary to REST inhibition. MSK107Li organoids stably expressing the indicated shRNAs were grown in the presence or absence of doxycycline for 7 d before measuring cell viability (luminescence relative to day 0; in box plots, boxes show the 25th–75th percentile with the median, and whiskers show the minimum-maximum; n = 6 organoid cultures per group; two-sided Mann-Whitney U tests). b, Relative mRNA levels of L1CAM and REST on day 0, normalized to GAPDH, in organoid-derived cells transduced with lentiviruses directing expression of the indicated shRNAs in the presence or absence of doxycycline. Data are shown as the mean ± s.e.m.; n = 4 organoid cultures per group; two-sided Student’s t tests. c, Left: schematic diagram showing how loss of epithelial integrity induces L1CAM expression during wound healing and tumor invasion, ultimately driving metastatic relapse. Right schematic diagram showing that loss of membrane E-cadherin in cells detached from their epithelial niche downregulates and displaces REST from the L1CAM enhancer, thus enabling L1CAM expression.

Article Snippet: Solid-phase L1CAM ligand binding assays used recombinant human L1CAM (human Fc tag, R&D Systems; His tag, Thermo Fisher Scientific), UltraPure BSA (Thermo Fisher Scientific), purified mouse laminin-111 (Sigma-Aldrich), purified mouse collagen IV (Cultrex, R&D Systems), purified human collagen V (Sigma-Aldrich), recombinant human tenascin C (R&D Systems) and recombinant human laminin-411, laminin-421, laminin-511 and laminin-521 (Biolamina).

Techniques: Inhibition, Stable Transfection, Expressing, MANN-WHITNEY, Derivative Assay, Transduction, Membrane

Journal: Nature cancer

Article Title: L1CAM defines the regenerative origin of metastasis-initiating cells in colorectal cancer

doi: 10.1038/s43018-019-0006-x

Figure Lengend Snippet: a, Immunohistochemistry for L1CAM (top) and Ki67 (bottom) in serial sections of a representative human CRC primary tumor invasion front showing an inverse relationship between L1CAM and Ki67 expression. Representative of 16 tumors analyzed. b, Immunohistochemistry for L1CAM (top) and Ki67 (bottom) in serial sections of a representative post-treatment human CRC liver metastasis demonstrating L1CAMhighKi67low cells in organized epithelial structures and L1CAMhighKi67high cells in disrupted epithelia. Representative of 16 tumors analyzed. c, Percentage of L1CAMhigh and L1CAMlow cells that are also Ki67high in regions of intact versus disrupted glandular epithelial architecture within CRCs. In box plots, boxes show the 25th–75th percentile with the median, and whiskers show the minimum-maximum; from left to right, n = 64, 54, 28 and 34 independent fields from 16 patient tumors; two-sided Mann-Whitney U tests. d, Immunohistochemistry for L1CAM (top) and Ki67 (bottom) in serial sections of MSK107Li matched surgically resected patient CRC liver metastasis, metastasis-derived organoids and organoid-derived subcutaneous xenograft. Dashed red lines indicate the tumor-stromal boundary. Box plots indicate the percentage of Ki67+ cells among L1CAMhigh cells in the indicated sections. In box plots, boxes show the 25th–75th percentile with the median, and whiskers show the minimum-maximum; from left to right, n = 7, 9 and 9 independent fields; two-sided Mann-Whitney U tests. e,f, L1CAM is induced during normal epithelial organoid formation. Relative L1CAM mRNA levels (mean ± s.e.m.) are shown for human (e) and mouse (f) cells freshly isolated from intact colons or collected after 14 d of growth in organoid conditions. Data were normalized to GAPDH mRNA levels. n = 4 crypts or organoid cultures from each of three patients or mice; two-sided Student’s t tests. g, Relative LGR5 mRNA levels (mean ± s.e.m.) in human cells freshly isolated from intact colons or collected after 14 d of growth in organoid conditions. Data were normalized to GAPDH mRNA levels. n = 4 crypts or organoid cultures from each of three patients; two-sided Student’s t tests. h, L1CAM is induced during epithelial regeneration after colitis. C57BL/6J mice were given 3.5% DSS in their drinking water for 5 d, inducing maximal colitis by day 7, and were then maintained on water without DSS for 12 d. Mice were killed at each of the indicated time points, and their colons were collected, sectioned and either stained with Kreyberg-Jareg stain (blue, mucin; pink, collagen) or subjected to immunohistochemistry for L1CAM. Representative images of three independent experiments are shown. i, High-magnification view showing detail of L1CAM immunohistochemical staining throughout the length of the intestinal crypt, representative of three mice each from three independent experiments.

Article Snippet: Solid-phase L1CAM ligand binding assays used recombinant human L1CAM (human Fc tag, R&D Systems; His tag, Thermo Fisher Scientific), UltraPure BSA (Thermo Fisher Scientific), purified mouse laminin-111 (Sigma-Aldrich), purified mouse collagen IV (Cultrex, R&D Systems), purified human collagen V (Sigma-Aldrich), recombinant human tenascin C (R&D Systems) and recombinant human laminin-411, laminin-421, laminin-511 and laminin-521 (Biolamina).

Techniques: Immunohistochemistry, Expressing, MANN-WHITNEY, Derivative Assay, Isolation, Staining, Immunohistochemical staining

Journal: Nature cancer

Article Title: L1CAM defines the regenerative origin of metastasis-initiating cells in colorectal cancer

doi: 10.1038/s43018-019-0006-x

Figure Lengend Snippet: a, L1CAM immunohistochemistry in a representative colon section from an L1CAMΔIEC mouse maintained on water with DSS, showing L1CAM staining restricted to submucosal neurons and no L1CAM expression in the epithelial cells of the crypt. Representative of three independent mice. b–f, L1CAM deficiency impairs epithelial healing following DSS-induced colitis. L1CAMfl/y and L1CAMΔIEC mice were given 3.5% DSS in their drinking water for 5 d, followed by 12 d of water without DSS before being killed. b, Kaplan-Meier survival curves. n = 26 mice per group from three independent experiments; two-sided Mantel-Cox test. c, Disease activity index (composite of weight loss, diarrhea and rectal bleeding) measured at the time of maximal colitis on day 7. In box plots, boxes show the 25th–75th percentile with the median, and whiskers show the minimum-maximum; n = 26 mice per group; two-sided Mann-Whitney U test. d, Histological score (composite of inflammation, mucosal denudation and crypt dysmorphia) on day 14. L1CAMfl/y, n = 24; L1CAMΔIEC, n = 12. In box plots, boxes show the 25th–75th percentile with the median, and whiskers show the minimum-maximum; two-sided Mann-Whitney U test. e, Representative histological sections with Kreyberg-Jareg staining (blue, mucin; pink, collagen) and immunohistochemical staining for L1CAM showing denudation of mucin-producing crypts in L1CAMΔIEC mice on day 14. Panels on the right show higher magnification of less damaged areas of the colon exhibiting loss of L1CAM immunostaining in crypts of L1CAMΔIEC mice. Representative of three independent experiments. f–l, L1CAM deficiency in the progeny of LGR5-expressing cells impairs epithelial healing after DSS-induced colitis. f, Immunohistochemistry for GFP in a representative colon section from an L1CAMΔLGR5 mouse killed after 5 d of daily tamoxifen treatment. n = 3 mice. g, WTΔLGR5 and L1CAMΔLGR5 mice were treated as in f and killed, and their colon crypts were isolated and seeded for organoid generation. Representative flow cytometry assessment is shown of L1CAM expression 24 h after seeding. n = 2 mice per genotype. h, Schematic of experimental design. Mice were treated with tamoxifen for 5 d to induce Lgr5-GFP-IRES-creERT2 expression and subsequently treated with 3% DSS for 5 d, with maximal colitis by day 7. They were then maintained on water without DSS for a further 12 d before being killed. i, Representative histological sections with Kreyberg-Jareg staining (blue, mucin; pink, collagen) and immunohistochemical staining for L1CAM showing denudation of mucin-producing crypts in the distal colon in L1CAMΔLGR5 mice on day 14, while crypts are restored in L1CAMfl/y and WTΔLGR5 mice. The panels on the right show higher magnification of less damaged areas of the colon exhibiting loss of L1CAM immunostaining in crypts in L1CAMΔLGR5 mice in comparison to L1CAMfl/y and WTΔLGR5 mice. Representative of 20 evaluable mice from two independent experiments. j, Kaplan-Meier plot showing cumulative survival of L1CAMfl/y, WTΔLGR5 and L1CAMΔLGR5 mice. n = 33 mice from two independent experiments; two-sided Mantel-Cox tests. k, Disease activity index (composite of weight loss, diarrhea and rectal bleeding) measured at the time of maximal colitis on day 7. In box plots, boxes show the 25th–75th percentile with the median, and whiskers show the minimum-maximum; n = 33 mice from two independent experiments; two-sided Mann-Whitney U tests. l, Histological scores (composite of inflammation, mucosal denudation and crypt dysmorphia) on day 14. In box plots, boxes show the 25th–75th percentile with the median, and whiskers show the minimum-maximum; n = 20 evaluable mice from two independent experiments; two-sided Mann-Whitney U tests.

Article Snippet: Solid-phase L1CAM ligand binding assays used recombinant human L1CAM (human Fc tag, R&D Systems; His tag, Thermo Fisher Scientific), UltraPure BSA (Thermo Fisher Scientific), purified mouse laminin-111 (Sigma-Aldrich), purified mouse collagen IV (Cultrex, R&D Systems), purified human collagen V (Sigma-Aldrich), recombinant human tenascin C (R&D Systems) and recombinant human laminin-411, laminin-421, laminin-511 and laminin-521 (Biolamina).

Techniques: Immunohistochemistry, Staining, Expressing, Activity Assay, MANN-WHITNEY, Immunohistochemical staining, Immunostaining, Isolation, Flow Cytometry, Comparison

Journal: Nature cancer

Article Title: L1CAM defines the regenerative origin of metastasis-initiating cells in colorectal cancer

doi: 10.1038/s43018-019-0006-x

Figure Lengend Snippet: a, L1CAM is required for organoid regeneration. CRC107Li organoid-derived cells were transduced with lentivirus directing the expression of either Cas9 alone or Cas9 with sgRNAs targeting L1CAM and allowed to grow under antibiotic selection for 14 d, when they were flow-sorted and seeded at a concentration of 2,000 cells per 40 μl of Matrigel in independent wells of a 96-well plate. The number of organoids (mean ± s.e.m.) established from each population 14 d after sorting and seeding is shown. From left to right, n = 10, 13 and 11 organoid cultures per group; two-tailed Mann-Whitney U test. b,c, L1CAM knockdown inhibits regrowth of multiple patient-derived organoids. Organoids derived from four patients with metastatic CRC were transduced with lentiviruses directing the expression of doxycycline (Dox)-inducible shRNA targeting L1CAM, expanded and, where indicated, treated with doxycycline for 48 h before dissociation and seeding at a concentration of 2,000 cells per 40 μl of Matrigel. Knockdown efficiencies of two independent L1CAM-targeting shRNAs in four patient-derived organoids (b) and relative cell viability on day 14 as compared to day 0 (mean ± s.e.m.) after plating of organoid-derived single cells (c) are shown. n = 6 organoid cultures per group; two-sided Student’s t tests. d, L1CAM is required for subcutaneous tumor growth in vivo. MSK107Li organoid-derived cells (50,000) expressing a doxycycline-inducible shRNA targeting L1CAM were injected subcutaneously into each flank of immunodeficient NSG mice. Where indicated, organoids were treated with doxycycline 2 d before transplantation and mice were maintained on a doxycycline diet for the duration of the experiment. Tumor volume (mean ± s.e.m.) was measured with calipers at the indicated time points after subcutaneous inoculation. n = 10 tumors from five mice per group; two-tailed Mann-Whitney U test. e, Representative image and quantification of tumor bioluminescence measured 35 d after inoculation. In box plots, boxes show the 25th–75th percentile with the median, and whiskers show the minimum-maximum; n = 10 tumors from five mice per group; two-sided Mann-Whitney U test. f, Day 21 steady-state MSK107Li and MSK121Li organoids were incubated in medium containing 50 μM irinotecan, and L1CAM expression was measured in residual DAPI− cells 7 d later. Top- flow cytometry plots showing distribution of the data. Bottom: bars showing median fluorescence intensity of L1CAM expression in each population. From left to right, n = 6,512, 130, 8,542 and 49 cells per group, representative of three independent experiments. g, Single cells derived from CRC107Li organoids transduced with lentivirus directing expression of the indicated shRNAs were seeded at a concentration of 2,000 cells per 40 μl grown as organoids for 21 d and then treated with doxycycline and/or irinotecan (irino) as indicated. The viability assay shows the luminescence (mean ± s.e.m.) of each population relative to the luminescence at the time that drug treatment was started (day 0); n = 5 organoid cultures per group; two-sided Mann-Whitney U test. h, Solid-phase binding assay showing dose-response curves of recombinant human L1CAM-Fc binding to plates coated with equimolar concentrations of the indicated proteins. After washing, bound L1CAM-Fc was detected with horseradish peroxidase (HRP)-conjugated anti-human IgG, HRP substrate was added and OD450 was measured. Data are shown as the mean ± s.e.m; n = 5 wells per time point, representative of three independent experiments; two-tailed Mann-Whitney U test. i, L1CAM mediates the interaction of dissociated CRC cells with laminin isoforms. Single cells derived from MSK121Li organoids (3,000) cultured in the presence or absence of doxycycline to knock down L1CAM were seeded in wells coated with 30 nM of the indicated proteins. After 1 h of adhesion and extensive washing, the percentage of adherent cells (mean ± s.e.m.) was measured as the relative luminescence of each well immediately after plating. n = 10 organoid cultures per condition; two-tailed Mann-Whitney U tests.

Article Snippet: Solid-phase L1CAM ligand binding assays used recombinant human L1CAM (human Fc tag, R&D Systems; His tag, Thermo Fisher Scientific), UltraPure BSA (Thermo Fisher Scientific), purified mouse laminin-111 (Sigma-Aldrich), purified mouse collagen IV (Cultrex, R&D Systems), purified human collagen V (Sigma-Aldrich), recombinant human tenascin C (R&D Systems) and recombinant human laminin-411, laminin-421, laminin-511 and laminin-521 (Biolamina).

Techniques: Derivative Assay, Transduction, Expressing, Selection, Concentration Assay, Two Tailed Test, MANN-WHITNEY, shRNA, In Vivo, Injection, Transplantation Assay, Incubation, Flow Cytometry, Fluorescence, Viability Assay, Binding Assay, Recombinant, Cell Culture

Journal: Nature cancer

Article Title: L1CAM defines the regenerative origin of metastasis-initiating cells in colorectal cancer

doi: 10.1038/s43018-019-0006-x

Figure Lengend Snippet: a, L1CAM expression is dynamically regulated during organoid growth. L1CAM immunohistochemistry of CRC organoids of varying size shows progressive restriction of L1CAM expression to cells at the periphery and an overall decrease in L1CAM expression with increasing organoid size. Representative of six organoid lines analyzed. b, L1CAM is induced by dissociation of normal, primary tumor and metastatic human organoids. Relative L1CAM mRNA levels, normalized to GAPDH mRNA levels (mean ± s.e.m.), were measured in intact organoids versus organoid-derived single-cell suspensions plated in Matrigel and assayed 24 h after dissociation. n = 4 replicates per group; two-sided Student’s t tests. c, Time course of regenerating organoids derived from L1CAMhigh (left) and L1CAMlow (right) cells flow-sorted from day 21 MSK107Li (top) and MSK121Li (bottom) organoids. Histograms show the distribution of L1CAM expression (measured by APC fluorescence) in each population at the indicated time points after flow sorting. Representative of three independent experiments. d,e, Dynamic induction of the L1CAMhigh phenotype by a subset of pre-existing L1CAMlow cells. CRC107Li organoids were labeled with lentivirally expressed tdTomato or GFP, and flow-sorted tdTomato+GFP−L1CAMhigh and tdTomato−GFP+L1CAMlow cells were mixed in equal proportions and allowed to regrow as organoids in the presence or absence of irinotecan. Flow plots of the distribution of tdTomato- and GFP-expressing cells (d) and the relative proportions of these cells in the population (e) are shown from monitoring by flow cytometry at the indicated time points after mixing; from left to right, n = 17, 107, 68, 251, 484 and 348 DAPI− single cells representative of three independent experiments with two organoid lines; two-sided chi-squared tests. f,g, Flow cytometry contour plots showing the distribution of L1CAM expression at the indicated time points in the presence or absence of chemotherapy (f) and median fluorescence intensity (MFI) of L1CAM expression (measured by APC) in cells derived from tdTomato+GFP−L1CAMhigh and tdTomato−GFP+L1CAMlow precursors on each day (g), representative of three independent experiments with two organoid lines.

Article Snippet: Solid-phase L1CAM ligand binding assays used recombinant human L1CAM (human Fc tag, R&D Systems; His tag, Thermo Fisher Scientific), UltraPure BSA (Thermo Fisher Scientific), purified mouse laminin-111 (Sigma-Aldrich), purified mouse collagen IV (Cultrex, R&D Systems), purified human collagen V (Sigma-Aldrich), recombinant human tenascin C (R&D Systems) and recombinant human laminin-411, laminin-421, laminin-511 and laminin-521 (Biolamina).

Techniques: Expressing, Immunohistochemistry, Derivative Assay, Fluorescence, Labeling, Flow Cytometry

Journal: Nature cancer

Article Title: L1CAM defines the regenerative origin of metastasis-initiating cells in colorectal cancer

doi: 10.1038/s43018-019-0006-x

Figure Lengend Snippet: a,b, L1CAM is not required for intestinal adenoma formation. Male APCΔIEC and L1CAM/APCΔIEC mice were killed at 3 months of age, and their colons were collected, sectioned and examined for adenoma formation. a, Number of adenomas per mouse intestine. In box plots, boxes show the 25th–75th percentile with the median and whiskers show the minimum-maximum; n = 5 mice per group; two-sided Mann-Whitney U test. b, Mean adenoma diameter per mouse. In box plots, boxes show the 25th–75th percentile with the median, and whiskers show the minimum-maximum; n = 5 mice per group; two-sided Mann-Whitney U test. c–e, L1CAM inhibition impairs orthotopic rectal tumor engraftment. NSG mice were given 3% DSS in their water for 5 d and then maintained on water without DSS for 2 d before intraluminal transplantation with 2 × 105 cells from dissociated MSK107Li organoids expressing doxycycline-inducible shRNA targeting L1CAM or control shRNA. Where indicated, organoids were treated in vitro with doxycycline starting 2 d before transplantation, and mice were maintained on a doxycycline diet. Mice were killed 90 d after transplantation, and their colons were collected and examined for tumor engraftment. c, Percentage of mice with an engrafted orthotopic tumor. From left to right, the stacked bar graphs show n = 7, 7, 10, 13, 9 and 7 mice per group from three independent experiments; two-sided chi-squared tests. d,e, Tumor diameter per engrafted mouse (in box plots, boxes show the 25th–75th percentile with the median, and whiskers show the minimum-maximum; from left to right, n = 6, 5, 6, 4, 5 and 1 mice from three independent experiments; two-sided Mann-Whitney U tests) (d) and representative H&E-stained sections (e). Arrows indicate tumour diameter. f,g, L1CAM inhibition impairs metastatic colonization of the liver. Cells (5 × 104) were derived from dissociated MSK107Li organoids with doxycycline-inducible expression of shRNA targeting L1CAM. Where indicated, organoids were treated with doxycycline starting 2 d before transplantation and mice were maintained on a doxycycline diet. Representative H&E-stained sections of liver metastases at the experimental endpoint (arrows indicate tumour diameter) (f) and quantification of ex vivo liver bioluminescence signal measured 60 d after transplantation (g) are shown. In box plots, boxes show the 25th–75th percentile with the median, and whiskers show the minimum-maximum; from left to right, n = 9, 10, 8, 9, 5 and 5 mice per group; two-sided Mann-Whitney U tests. h–l, L1CAM inhibition impairs local tumor expansion and metastasis from orthotopic cecal xenografts. h, Schematic of the experiment: cells (4 × 105) derived from MSK121Li organoids transduced with lentivirus directing the expression of tdTomato-luciferase and shRNA targeting L1CAM or control shRNA were injected into the cecal submucosa. Mice were monitored until cecal tumors were evident by ex vivo bioluminescence imaging 3 weeks after injection, randomized on the basis of bioluminescence signal and maintained on or off a doxycycline diet for 7 weeks before being killed. i–l, Quantification of whole-mouse bioluminescence signal (i) and ex vivo bioluminescence signal in the cecum (j), liver (k) and lung (l), normalized to bioluminescence at the time of randomization. In box plots, boxes show the 25th–75th percentile with the median, and whiskers show the minimum-maximum; from left to right, n = 5, 6, 12 and 11 mice per group; two-sided Mann-Whitney U tests. m–o, Combination of L1CAM inhibition with chemotherapy impairs tumor growth to a greater extent than chemotherapy alone. m, Schematic of the experiment. Cells (2 × 105) derived from MSK107Li organoids transduced with lentivirus directing the expression of tdTomato-luciferase and shRNA targeting L1CAM or control shRNA were injected subcutaneously; mice were randomized on the basis of bioluminescence intensity 5 weeks after injection and maintained on a doxycycline diet and/or treated weekly with irinotecan as indicated for 4 weeks before being killed. n,o, Ex vivo tumor volume (n) and mass (o), normalized to tumor bioluminescence at the time of randomization. From left to right, n = 10, 10, 8, 7, 10, 10, 10 and 9 tumors per group; mean ± s.e.m.; two-sided Mann-Whitney U tests.

Article Snippet: Solid-phase L1CAM ligand binding assays used recombinant human L1CAM (human Fc tag, R&D Systems; His tag, Thermo Fisher Scientific), UltraPure BSA (Thermo Fisher Scientific), purified mouse laminin-111 (Sigma-Aldrich), purified mouse collagen IV (Cultrex, R&D Systems), purified human collagen V (Sigma-Aldrich), recombinant human tenascin C (R&D Systems) and recombinant human laminin-411, laminin-421, laminin-511 and laminin-521 (Biolamina).

Techniques: MANN-WHITNEY, Inhibition, Transplantation Assay, Expressing, shRNA, In Vitro, Staining, Derivative Assay, Ex Vivo, Transduction, Luciferase, Injection, Imaging

Journal: Nature cancer

Article Title: L1CAM defines the regenerative origin of metastasis-initiating cells in colorectal cancer

doi: 10.1038/s43018-019-0006-x

Figure Lengend Snippet: a, REST ChIP-seq analysis, showing diminution of the REST peak at the L1CAM intronic enhancer in dissociated organoid-derived cells, collected 16 h after dissociation, in comparison to intact organoids. Input control is also shown. LCA10 (gray) is not expressed in CRC organoids. Two independent organoid cultures from two patient-derived organoid lines were analyzed per condition. b, Relative mRNA levels (mean ± s.e.m.) of REST and L1CAM in intact MSK107Li organoids (day 0), cells collected 24 h after dissociation and plating as single cells (day 1), and cells collected at the indicated time points during organoid regeneration. Organoids were transduced with lentivirus constitutively expressing shRNA targeting REST or control shRNA. Gene expression was normalized to GAPDH mRNA levels. Day 1 shControl versus shREST.1: P < 0.0001 (REST), P < 0.0001 (L1CAM); day 1 shControl versus shREST.2: P = 0.007 (REST), P < 0.0001 (L1CAM); n = 4 organoid cultures per sample per time point; two-sided Student’s t tests. c, Relative expression of CDH1, REST and L1CAM (mean ± s.e.m.) in intact MSK107Li organoids transduced with lentivirus constitutively expressing shRNA targeting CDH1 or control shRNA. n = 4 organoid cultures per group; two-sided Student’s t tests. d, ChIP-PCR using antibodies against REST or isotype-control immunoglobulin in intact MSK107Li organoids transduced with lentivirus constitutively expressing shRNA targeting CDH1 or control shRNA. Fold enrichment (mean ± s.e.m.) is shown relative to the corresponding 2% input. PCR primers were selected to amplify immunoprecipitated DNA at the indicated positions relative to the L1CAM transcriptional start site. P values correspond to the comparison between shControl anti-REST and shCDH1 anti-REST; n = 3 organoid cultures per condition; two-sided Student’s t tests. e, Induction of L1CAM expression by E-cadherin knockdown can be rescued by REST but not by dominant-negative REST (dnREST). Relative mRNA levels of L1CAM, CDH1 and REST are shown in MSK107Li organoids stably expressing shRNA targeting CDH1 or control shRNA as well as cDNA expressing REST or dnREST. Gene expression was normalized to the mRNA levels of GAPDH. Data are shown as the mean ± s.e.m.; n = 4 organoid cultures per group; two-sided Student’s t tests.

Article Snippet: Solid-phase L1CAM ligand binding assays used recombinant human L1CAM (human Fc tag, R&D Systems; His tag, Thermo Fisher Scientific), UltraPure BSA (Thermo Fisher Scientific), purified mouse laminin-111 (Sigma-Aldrich), purified mouse collagen IV (Cultrex, R&D Systems), purified human collagen V (Sigma-Aldrich), recombinant human tenascin C (R&D Systems) and recombinant human laminin-411, laminin-421, laminin-511 and laminin-521 (Biolamina).

Techniques: ChIP-sequencing, Derivative Assay, Comparison, Transduction, Expressing, shRNA, Immunoprecipitation, Dominant Negative Mutation, Stable Transfection

Journal: Nature immunology

Article Title: Control of nutrient uptake by IRF4 orchestrates innate immune memory

doi: 10.1038/s41590-023-01620-z

Figure Lengend Snippet: a, Kaplan–Meier survival curves from Rag2−/− Il2rg−/− mice transferred with WT Ly49H+ NK, Irf4−/−Ly49H+ NK or no NK cells (n as indicated). b, Percentage of Ly49H+ NK cells from adoptive co-transfer of WT (CD45.1) and Irf4−/− (CD45.2) NK cells into Ly49H-deficient mice from the blood throughout the course of MCMV infection (left) and percent chimerism of WT versus Irf4−/− transferred Ly49H+ NK cells (right) (n = 4 biological replicates). c, Representative flow plots gated on transferred WT (CD45.1) and Irf4−/− (CD45.2) Ly49H+ NK cells on day 7 in different tissues (left) and the percent chimerism of WT and Irf4−/− NK cells within transferred Ly49H+ NK cells between different tissues (n = 4 biological replicates per group). d, Representative histogram of CTV dilution on day 0 and day 4 PI gated on transferred Ly49H+ NK cells (left). Dashed gray and orange lines represent WT and Irf4−/− NK cells on day 0, respectively; solid gray and orange lines represent WT and Irf4−/− NK cells on day 4 PI, respectively. Percentage of transferred NK cells that have divided at least once (n = 5 biological replicates per group) (right). e, Representative histogram of BIM expression on day 5 PI from splenic WT or Irf4−/−Ly49H+ NK cells (left) and BIM gMFI (right) (n = 4 biological replicates). gMFI, geometric mean fluorescence intensity. f, Percentage of FLICA+ (marking activated caspase) WT or Irf4−/−Ly49H+ NK cells taken from day 5 PI WT:Irf4−/− mBMC splenocytes upon ex vivo treatment with ABT199 (BCL2 inhibitor) (n = 3 biological replicates in each condition). Data are represented as mean ± s.e.m. and are representative of, or pooled from, at least two independent experiments. For statistical testing, a log-rank test was used for a and a two-tailed paired t-test was used for b–f and adjusted for multiple comparisons for b and c.

Article Snippet: Plated splenocytes were then treated with the indicated concentrations of BCL2 inhibitor, ABT199 (also known as venetoclax) (MedChemExpress, cat. no. HY-15531) in the presence of CaspGLOW Fluorescein Active Caspase Staining kit (Thermo Fisher, cat. no. 88–7003-42) per manufacturer’s protocol for 90 min at 37 °C.

Techniques: Infection, Expressing, Fluorescence, Ex Vivo, Two Tailed Test