Journal: bioRxiv

Article Title: Loss of Flotillin-2 enhances trastuzumab emtansine internalization and cytotoxicity by relieving negative regulation of HER2 internalization in HER2-amplified cancers

doi: 10.64898/2026.05.15.725439

Figure Lengend Snippet: A) HCC1954 cells were fixed, permeabilized, blocked, and probed with PLA probes against HER2 and FLOT2 combined, or either probe alone (negative controls) and DAPI (blue). Green signal indicates interaction between HER2 and FLOT2. Scale bar is 10 µm. B) SKBR3 cells were transfected with siNeg or siFLOT2 for seven days prior to cell lysing and immunoprecipitation with anti-FLOT2, followed by immunoblotting for HER2 or FLOT2. Whole cell lysate was immunoblotted for HER2, FLOT2 or HSC70 (loading control). C) HeLa control or FLOT2 KO cells were lysed and immunoprecipitated with anti-HER2, followed by immunoblotting for HER2 and FLOT2. Whole cell lysate was immunoblotted for HER2, FLOT2 and HSC70 (loading control). D) HEK293T cells were transfected with empty vector, HER2, FLAG-tagged FLOT2 or HER2 and FLAG-tagged FLOT2 for three days, then lysed and immunoprecipitated with anti-FLAG and immunoblotted for HER2 and FLAG. Whole cell lysate was immunoblotted for HER2, FLOT2 and HSC70 (loading control). E) SKBR3 cells were transfected with siNeg or siFLOT2 for five days prior to cell counting or three days prior to lysing and immunoblot to probe for pHER2 (Y1196), HER2, pMAPK (T202/Y204), ERK2, pAKT (S473), AKT, pS6 (S235/236), S6, FLOT2, and HSC70 (loading control). Graphed data represent the average ±SEM of at least three independent experiments, and statistical analysis was performed by Student’s t-test. F) Same as in E, with HCC202 cells transfected for seven days prior to cell counting or three days prior to lysing, and lysates were instead probed for pHER2 (Y1221/1222). G) Same as in E, with HCC1954 cells transfected for seven days prior to cell counting and three days prior to lysing.

Article Snippet: HA-tag Ubiquitin plasmid was kindly provided by Dirk Bohmann. pCMV6 (PS10001) vector control and FLOT2 (RC220884) were purchased from OriGene.

Techniques: Transfection, Immunoprecipitation, Western Blot, Control, Plasmid Preparation, Cell Counting

Journal: bioRxiv

Article Title: Loss of Flotillin-2 enhances trastuzumab emtansine internalization and cytotoxicity by relieving negative regulation of HER2 internalization in HER2-amplified cancers

doi: 10.64898/2026.05.15.725439

Figure Lengend Snippet: A) SKBR3 shFLOT2 cells were treated +/- 500 ng/mL doxycycline for 72 hours, replated, then treated with pHrodo-T-DM1 (1 µg/mL; red) in serum-free RPMI for 7 hours. Cells were stained with CellTracker Blue CMAC dye (1 µM; Blue) in serum-free RPMI for 30 minutes prior to confocal imaging. GFP (green) expression is induced by doxycycline, indicating induction of the shFLOT2 promoter. Intensity of pHrodo signal per cell was quantified and divided by the area of each cell (right). Data represents the average ±SEM of at least three independent experiments, and statistical analysis was performed by Student’s t-test. Scale bar is 40 µm. B) Same as in A, with SKBR3 shControl cells. GFP (green) expression is induced by doxycycline, indicating induction of the shControl promoter. Scale bar is 20 µm. C) SKBR3 cells were transfected with siNeg or siFLOT2 for 48 hours and then treated with DMSO or 1 ng/mL T-DM1 for 24 hours in 1% FBS RPMI. Cells were lysed and immunoblotted for HER2, pAKT (S473), AKT, pS6 (S235/236), S6, FLOT2 and HSC70 (loading control). Band density was normalized to loading control, and then normalized to siNeg DMSO for each individual protein. Data represents the average ±SEM of at least three independent experiments, and statistical analysis was performed by Student’s t-test. D) Same as in C, with HCC202 cells using 10 µg/mL T-DM1.

Article Snippet: HA-tag Ubiquitin plasmid was kindly provided by Dirk Bohmann. pCMV6 (PS10001) vector control and FLOT2 (RC220884) were purchased from OriGene.

Techniques: Staining, Imaging, Expressing, Transfection, Control

Journal: bioRxiv

Article Title: Loss of Flotillin-2 enhances trastuzumab emtansine internalization and cytotoxicity by relieving negative regulation of HER2 internalization in HER2-amplified cancers

doi: 10.64898/2026.05.15.725439

Figure Lengend Snippet: A) SKBR3 shFLOT2 or shControl were pretreated with DMSO control (black) or 500 ng/mL doxycycline (gray) for 48 hours in 1% FBS RPMI, then treated with indicated T-DM1 concentration with continued doxycycline in 1% FBS RPMI for three days prior to cell counting. The table below the graphs indicates the calculated IC50 comparing DMSO + T-DM1 to doxycycline + T-DM1. Data represents the average ±SEM of at least three independent experiments, and statistical analysis was performed by Student’s t-test. B) Same as in A, with HCC202 shFLOT2 or shControl. C) Same as in A, with HCC1954 shFLOT2 or shControl. D) ARK1 (top) or ARK2 (bottom) cells were transfected with siNeg (black) or siFLOT2 (gray) for 48 hours prior to being treated with indicated T-DM1 concentration in 10% FBS RPMI for three days prior to cell viability reading (CellTiter-Glo 2.0). Data represents the average ±SEM of at least three independent experiments, and statistical analysis was performed by Student’s t-test. E) SKBR3, HCC202, HCC1954, BT474, HCC1419, HCC2218, UACC893, ARK1, and ARK2 cells were treated with 0-1000 ng/mL T-DM1 in 1% FBS RPMI for three days prior to cell counting. Average FLOT2 expression by immunoblot (relative to HSC70 loading control) and the calculated IC50 for each cell line was plotted. Protein expression and IC50 were calculated from at least three independent experiments. Line represents linear regression. F) Same as E, with average HER2 expression (relative to HSC70 loading control). G) Overall survival of patients in Caris dataset from T-DXd to last contact with high FLOT2 (orange; 16.22 months) vs low FLOT2 (blue; 18.26 months), p=0.041. H) Overall survival of patients in Caris dataset from T-DM1 to last contact with high FLOT2 (orange; 37.967 months) vs low FLOT2 (blue; 41.29 months), p=0.131.

Article Snippet: HA-tag Ubiquitin plasmid was kindly provided by Dirk Bohmann. pCMV6 (PS10001) vector control and FLOT2 (RC220884) were purchased from OriGene.

Techniques: Control, Concentration Assay, Cell Counting, Transfection, Expressing, Western Blot

Journal: bioRxiv

Article Title: Loss of Flotillin-2 enhances trastuzumab emtansine internalization and cytotoxicity by relieving negative regulation of HER2 internalization in HER2-amplified cancers

doi: 10.64898/2026.05.15.725439

Figure Lengend Snippet: A) SKBR3 shFLOT2 cells were treated +/- 500 ng/mL doxycycline for a total of four days, and transfected with HA-tagged Ubiquitin for a total of three days. Cells were then treated with 10 ng/mL T-DM1 in 1% FBS RPMI for 2 hours, lysed, immunoprecipitated with anti-HA antibody, and immunoblotted for HER2 and HA. Whole cell lysate was immunoblotted for HER2, HA, FLOT2 and HSC70 (loading control). B) SKBR3 (left) or HCC1954 (right) were treated with TAK-243 (1 nM for SKBR3, 100 nM for HCC1954) and T-DM1 (10 ng/mL for SKBR3, 100 ng/mL for HCC1954) in 1% FBS RPMI for three days. Dead cell percentage was calculated using PI stain with the BioTek Cytation. Data represents the average ±SEM of at least three independent experiments, and statistical analysis was performed by Student’s t-test. C) SKBR3 cells were treated with DMSO (control), 1 nM or 10 nM TAK-243 for 24 hours in 1% FBS RPMI, and then treated with pHrodo-T-DM1 (1 µg/mL; red) for 7 hours in serum-free RPMI. Cells were stained with CellTracker Blue CMAC dye (1 µM; Blue) for 30 minutes in serum-free RPMI prior to confocal imaging. Intensity of pHrodo signal per cell was quantified and divided by the area of each cell (right). Data represents the average ±SEM of at least three independent experiments, and statistical analysis was performed by Student’s t-test. Scale bar is 40 µm. D) SKBR3 shFLOT2 were pretreated +/- 500 ng/mL doxycycline in complete RPMI for 48 hours, and then continued doxycycline +/- TAK-243 (1 nM) for 24 hours in 1% FBS RPMI. Cells were then treated with pHrodo-T-DM1 in serum-free RPMI (1 µg/mL; red) for 7 hours. Cells were stained with CellTracker Blue CMAC dye (1 µM; Blue) in serum-free RPMI for 30 minutes prior to confocal imaging. GFP (green) expression is induced by doxycycline, indicating induction of the shFLOT2 promoter. Intensity of pHrodo signal per cell was quantified and divided by the area of each cell (right). Data represents the average ±SEM of at least three independent experiments, and statistical analysis was performed by Student’s t-test. Scale bar is 20 µm.

Article Snippet: HA-tag Ubiquitin plasmid was kindly provided by Dirk Bohmann. pCMV6 (PS10001) vector control and FLOT2 (RC220884) were purchased from OriGene.

Techniques: Transfection, Ubiquitin Proteomics, Immunoprecipitation, Control, Staining, Imaging, Expressing

Journal: bioRxiv

Article Title: Loss of Flotillin-2 enhances trastuzumab emtansine internalization and cytotoxicity by relieving negative regulation of HER2 internalization in HER2-amplified cancers

doi: 10.64898/2026.05.15.725439

Figure Lengend Snippet: A) SKBR3 shFLOT2 cells were transfected with siRNA for Cbl (siCbl) or neg control (siNeg)and +/- 500 ng/mL doxycycline for 4 days total and transfected with HA-tagged ubiquitin for 3 days total. Cells were then treated with 10 ng/mL for 2 hours in 1% FBS RPMI, lysed, immunoprecipitated with anti-HA antibody, and immunoblotted for HER2, HA, and ubiquitin. Whole cell lysate was immunoblotted for HA, Cbl, FLOT2 and HSC70 (loading control). Immunoprecipitation band intensity was quantified and HER2 and ubiquitin and graphed as HER2/ubiquitin (right). siNeg with T-DM1 and doxycycline was normalized to siNeg with DMSO, and siCbl with T-DM1 and doxycycline was normalized to siCbl with DMSO. Data represents the average ±SEM of at least three independent experiments, and statistical analysis was performed by Student’s t-test. B) SKBR3 shFLOT2 cells were transfected with siNeg or siCbl +/- 500 ng/mL doxycycline for 48 hours and re-plated on chambered coverglass, then treated with pHrodo-T-DM1 (1 µg/mL; red) in serum-free RPMI for 7 hours. Cells were stained with CellTracker Blue CMAC dye (1 µM; Blue) in serum-free RPMI for 30 minutes prior to confocal imaging. GFP (green) expression is induced by doxycycline, indicating induction of the shFLOT2 promoter. Intensity of pHrodo signal per cell was quantified and divided by the area of each cell (right). Data represents the average ±SEM of at least three independent experiments, and statistical analysis was performed by one-way ANOVA. Scale bar is 20 µm. C) HCC202, HCC1954 and SKBR3 lysates were immunoblotted for Cbl, Cbl-b and HSC70 (loading control). D) The same SKBR3 shFLOT2 cells that were transfected with siNeg or siCbl and treated +/-doxycycline for 48 hours in B were then re-plated and treated +/- 500 ng/mL doxycycline +/- 100 ng/mL T-DM1 for an additional 72 hours and dead cell percentage was calculated using PI stain with the BioTek Cytation. Cells were lysed and immunoblotted (bottom) for Cbl, FLOT2 and HSC70 (loading control) to confirm knockdown. Data represents the average ±SEM of at least three independent experiments, and statistical analysis was performed by Student’s t-test. Dead cell percentage was normalized to the baseline percentage recorded at time of TDM1 treatment. E) Same as in D, with HCC1954 shFLOT2 cells. SiCbl was co-transfected with si-Cbl-b and cells were treated with 1000 ng/mL T-DM1, and lysates were also immunoblotted for cbl-b.

Article Snippet: HA-tag Ubiquitin plasmid was kindly provided by Dirk Bohmann. pCMV6 (PS10001) vector control and FLOT2 (RC220884) were purchased from OriGene.

Techniques: Transfection, Control, Ubiquitin Proteomics, Immunoprecipitation, Staining, Imaging, Expressing, Knockdown

Journal: bioRxiv

Article Title: Loss of Flotillin-2 enhances trastuzumab emtansine internalization and cytotoxicity by relieving negative regulation of HER2 internalization in HER2-amplified cancers

doi: 10.64898/2026.05.15.725439

Figure Lengend Snippet: A) SKBR3 cells were lysed, lysates treated with DMSO or 250 µM zoledronic acid (ZA) for an hour, and then heated at the indicated temperatures for 3 minutes each. Lysates were then centrifuged, and supernatant was immunoblotted for FLOT2, PHB1, and PHB2 (left). Quantification of bands and calculation of melting temperatures are depicted on the right. Quantification of bands represent the average ±SEM of three independent experiments. B) SKBR3 cells were treated with 0, 1 or 10 µM zoledronic acid for 24 or 48 hours, lysed, immunoprecipitated by anti-HER2, and immunoblotted for HER2, FLOT2, PHB1 or PHB2. Whole cell lysate was immunoblotted for HER2, FLOT2, PHB1, PHB2 and HSC70 (loading control). Quantification of immunoprecipitation bands from three independent experiments for FLOT2 relative to HER2 are depicted on the right, with the average ±SEM. Statistical analysis was performed by Student’s t-test compared to 0 µM zoledronic acid. C) SKBR3 cells were treated with DMSO (control) or 1 µM Zoledronic acid (ZA) for 72 hours then treated with pHrodo-T-DM1 (1 µg/mL; red) in serum-free RPMI for 7 hours. Cells were stained with CellTracker Blue CMAC dye (1 µM; Blue) in serum-free RPMI for 30 minutes prior to confocal imaging. Intensity of pHrodo signal per cell was quantified and divided by the area of each cell (right). Data represents the average ±SEM of at least three independent experiments, and statistical analysis was performed by Student’s t-test. Scale bar is 20 µm. D) SKBR3 cells were treated with the indicated combinations of DMSO (control), 1 ng/mL T-DM1 and 1 µM zoledronic acid (ZA) for 48 hours in 1% FBS RPMI. Dead cell percentage was calculated using PI stain with the BioTek Cytation. Data represents the average ±SEM of three independent experiments, and statistical analysis was performed by Student’s t-test. E) Same as D, with ARK1 cells in 10% FBS RPMI. F) Same as E, with ARK2 cells and 10 µM zoledronic acid (ZA). G) Same as D, with HCC1954 cells with 24 hour treatment.

Article Snippet: HA-tag Ubiquitin plasmid was kindly provided by Dirk Bohmann. pCMV6 (PS10001) vector control and FLOT2 (RC220884) were purchased from OriGene.

Techniques: Immunoprecipitation, Control, Staining, Imaging

Journal: The Journal of Biological Chemistry

Article Title: An N-terminal di-proline motif is essential for fatty acid–dependent degradation of Δ9-desaturase in Drosophila

doi: 10.1074/jbc.M117.801936

Figure Lengend Snippet: Role of the di-proline motif in degradation of Δ9-desaturase. N-terminal amino acid sequences of Δ9-desaturases were compared (D. melanogaster DESAT1 (NP_652731), Mus musculus SCD1 (NP_033153), M. musculus SCD3 (NP_077770), Homo sapiens SCD1 (NP_005054), Danio rerio stearoyl-CoA desaturase (AAO25582), and S. cerevisiae Ole1 (CAA96757)) (A). Schematic illustration of DESAT1, mouse SCD1, and constructed mutants is shown (B). S2 cells expressing mouse SCD1-FLAGC, chimera-FLAGC, chimera (AA)-FLAGC, and SCD1 (di-Pro)-FLAGC were treated with C18:1 (100 μm) for 6 h (C) or DESAT1 inhibitor 37c (1 μm) for 16 h (D), and the amounts of endogenous DESAT1, FLAG-tagged exogenous Δ9-desaturases, and α-tubulin protein were detected with anti-DESAT1 antibody, anti-FLAG antibody, and anti-α-tubulin antibody, respectively. Band intensities were determined by ImageJ software, and levels of SCD1 proteins are shown relative to the amount of SCD1 protein in vehicle-treated cells (C and D). Mean ± S.D. (n = 3). *, p < 0.05; ***, p < 0.001; n.s., not significant.

Article Snippet: Anti-α-tubulin antibody (PM054), anti-FLAG antibody (M2), and anti-ubiquitin antibody (SPC-119) were purchased from MBL, Sigma, and StressMarq Biosciences, respectively.

Techniques: Construct, Expressing, Software

Journal: The Journal of Biological Chemistry

Article Title: An N-terminal di-proline motif is essential for fatty acid–dependent degradation of Δ9-desaturase in Drosophila

doi: 10.1074/jbc.M117.801936

Figure Lengend Snippet: Identification of protease involved in the degradation of DESAT1. S2 cells were treated with calpeptin (50 μm), MG132 (50 μg/ml), or chloroquine (50 μm) for 6 h (A). S2 cells were treated with dsRNA against GFP, CalpA, CalpB, CalpC, or sol for 72 h and then exposed to C18:1 (100 μm) for 6 h (B). S2 cells expressing DESAT1-FLAGC and DESAT1 (P2A/P3A)-FLAGC were treated with calpeptin (50 μm) for 6 h (C). The amounts of endogenous DESAT1, FLAG-tagged exogenously expressed DESAT1, and α-tubulin protein were detected with anti-DESAT1 antibody, anti-FLAG antibody, and anti-α-tubulin antibody, respectively (A–C). Band intensities were determined by ImageJ software, and levels of DESAT1 proteins are shown relative to the amount of DESAT1 protein in vehicle-treated cells (A–C). Mean ± S.D. (n = 3). **, p < 0.01; ***, p < 0.001; n.s., not significant.

Article Snippet: Anti-α-tubulin antibody (PM054), anti-FLAG antibody (M2), and anti-ubiquitin antibody (SPC-119) were purchased from MBL, Sigma, and StressMarq Biosciences, respectively.

Techniques: Expressing, Software