Journal: OncoImmunology

Article Title: An epitope-specific novel anti-EMMPRIN polyclonal antibody inhibits tumor progression

doi: 10.1080/2162402x.2015.1078056

Figure Lengend Snippet: Figure 1: EMMPRIN structure and peptide design, and specificity of 161-Ab: (A) Partial

Article Snippet: One strip was probed with the 1:1,000 diluted commercial rat anti-mouse EMMPRIN (MAB772, R&D systems) and then with the 1:5,000 diluted HRP-conjugated goat anti-rat IgG (112-035- D ow nl oa de d by [ U ni ve rs ity o f R eg in a] a t 0 6: 58 2 4 Fe br ua ry 2 01 6 062, Jackson) and served as positive control (P.C).

Techniques:

Journal: Stem cell research

Article Title: Establishment of a human embryonic stem cell line with homozygous TP53 R248W mutant by TALEN mediated gene editing

doi: 10.1016/j.scr.2018.04.013

Figure Lengend Snippet: Reagents details.

Article Snippet: Pluripotency Markers , Mouse anti-TRA-1-85 Alexa Fluor 555-conjugated , 1:600 , R and D Systems Cat# FAB3195A RRID:AB_663789.

Techniques: Immunocytochemistry, Western Blot, Mutagenesis

Journal: bioRxiv

Article Title: MCT4 and CD147 co-localize with MMP14 in invadopodia and autolysosomes and collectively stimulate breast cancer cell invasion by increasing extracellular matrix degradation

doi: 10.1101/2023.09.03.556100

Figure Lengend Snippet: MDA-MB-231 cells were transfected with siRNAs targeting MCT4 ( a, b ), CD147 ( c, d ) or scrambled siRNA control (mock), or with MCT4, CD147 or MCT4 and CD147 plasmids or empty pcDNA3.1 (+) vector as negative control ( e-h ). 48 h (KD) or 24 h (overexpression) post-transfection, cells were lysed and subjected to Western blot or qPCR analysis as indicated. (a-d) Representative Western blots and corresponding quantification of MCT4 and CD147 protein levels. DCTN1 was used as loading control. MCT4 and HG-CD147 band intensities were normalized to their respective loading controls and control (mock). n=4. (e-f) Representative Western blots and corresponding quantification of MCT4 and CD147. DCTN1 was used as loading control. MCT4 and CD147 band intensities were normalized to loading controls and control (vector). n=10 (MCT4), 8 (CD147). (g-h) Relative mRNA levels (qPCR) of MCT4 and CD147. Data was normalized to housekeeping genes β-actin and TBP and to vector control. n=4. Statistical analysis: Two-tailed paired Student’s t -test (b, g), one-way ANOVA with Dunnett’s post-test (d, f, h). *, **, ***, ****: p < 0.05, 0.01, 0.001, 0.0001.

Article Snippet: Primary antibodies against GAPDH (#2118), LC3A/B (#4108), and Golgin-97 (#97537) were purchased from Cell Signaling Technology; against CD147/EMMPRIN (#MAB972) and CD147 (#AF972) from R&D Systems; against MCT4 (#376140) and LAMP-1 (#20011) from Santa Cruz; against MMP14 (#271840 and #192782), p62 (#56416), and p-cortactin (Y421) (#47768) from Abcam; against DCTN1/p150 (#610473), from BD Transduction Laboratories; PDI (#MA3-019) and Rhodamine-conjugated Phalloidin (#R415) were from Invitrogen; against TOM20 (#11802-1-AP) from Proteintech; and against Src (#OP07) from Calbiochem.

Techniques: Transfection, Control, Plasmid Preparation, Negative Control, Over Expression, Western Blot, Two Tailed Test

Journal: bioRxiv

Article Title: MCT4 and CD147 co-localize with MMP14 in invadopodia and autolysosomes and collectively stimulate breast cancer cell invasion by increasing extracellular matrix degradation

doi: 10.1101/2023.09.03.556100

Figure Lengend Snippet: (a) Representative overview images (left) and zooms (right) of MDA-MB-231 cells transfected with empty vector, MCT4, CD147 or MCT4 and CD147 plasmids. Cells were grown for 24 h, fixed and subjected to IFM analysis with primary antibodies targeting MCT4 (magenta) and CD147 (green). Nuclei were stained with DAPI (blue). White arrowheads show co-localization of MCT4 and CD147 in the plasma membrane and white lines represent the area analyzed by line scan analysis in (b). Scale bars: 20 µm. n=3. (b) Representative line scan analysis plots showing intensities (MCT4 magenta, CD147 green) along a selected line (white lines in (a)). (c-d) Relative membrane localization of MCT4 and CD147, determined by line scan analysis. Color intensities were normalized to cell count and to their respective control (vector). n=3. (e-i) Representative flow cytometric dot plots (FSC vs. FL2 (PE anti-human CD147). n=3 (j) Quantification of CD147 overexpression population. Statistical analysis was performed on log-transformed data. n=3. (k) Relative change in pH i of MDA-MB-231 cells transfected with MCT4, CD147, or both plasmids, or with empty pcDNA3.1 (+) vector as negative control, measured as the change in SNARF-AM ratio after addition of 20 mM Na + -lactate. Representative experiment showing the mean 660/580 nm ratio for 50 s after adding 20 mM lactate. The data was normalized to vector t 0 . (l) Lactate concentration (mM) in culture medium from MDA-MB-231 cells cultured for 48 h after transfection with MCT4, CD147 or MCT4 and CD147 plasmids or empty pcDNA3.1 (+) vector as negative control. n=6. Statistics: (c), (d), (j), (l): one-way ANOVA followed by Dunnett’s multiple comparisons post-test.

Article Snippet: Primary antibodies against GAPDH (#2118), LC3A/B (#4108), and Golgin-97 (#97537) were purchased from Cell Signaling Technology; against CD147/EMMPRIN (#MAB972) and CD147 (#AF972) from R&D Systems; against MCT4 (#376140) and LAMP-1 (#20011) from Santa Cruz; against MMP14 (#271840 and #192782), p62 (#56416), and p-cortactin (Y421) (#47768) from Abcam; against DCTN1/p150 (#610473), from BD Transduction Laboratories; PDI (#MA3-019) and Rhodamine-conjugated Phalloidin (#R415) were from Invitrogen; against TOM20 (#11802-1-AP) from Proteintech; and against Src (#OP07) from Calbiochem.

Techniques: Transfection, Plasmid Preparation, Staining, Clinical Proteomics, Membrane, Cell Counting, Control, Over Expression, Transformation Assay, Negative Control, Concentration Assay, Cell Culture

Journal: bioRxiv

Article Title: MCT4 and CD147 co-localize with MMP14 in invadopodia and autolysosomes and collectively stimulate breast cancer cell invasion by increasing extracellular matrix degradation

doi: 10.1101/2023.09.03.556100

Figure Lengend Snippet: MDA-MB-231 cells were transfected with siRNA targeting MCT4 (a-c) or CD147 (d-f) or with MCT4, CD147, both constructs or the corresponding empty vector (g-i). Alternatively, MDA-MB-231 cells with stable KD of MCT4 were employed (b-c). Cells were seeded in migration or invasion Boyden chamber inserts with 10% FBS as chemoattractant in lower chamber, and migration or invasion, as indicated, was determined 24 h after seeding of the cells in the upper chamber. Cells were fixed and stained with Giemsa solution and relative cell migration or invasion was determined by counting the cells which had moved through the inserts. (a, d, g) Representative images. Scale bar: 40 µm. (b, e, h) Relative cell migration as determined by the number of migrated cells relative to that in the control condition (mock, pLKO.1 or vector). (c, f, i) Relative cell invasion as determined by the number of cells invaded relative to that in the control condition (mock, pLKO.1 or vector). (a-i) n = 4, 3, 3, 6 in MCT4 siRNA KD, MCT4 stable KD, CD147 KD and overexpression, respectively. Statistics: Two-tailed, paired Students t -test (b, c) or one-way ANOVA with Dunnett’s post-test (e, f, h, i).

Article Snippet: Primary antibodies against GAPDH (#2118), LC3A/B (#4108), and Golgin-97 (#97537) were purchased from Cell Signaling Technology; against CD147/EMMPRIN (#MAB972) and CD147 (#AF972) from R&D Systems; against MCT4 (#376140) and LAMP-1 (#20011) from Santa Cruz; against MMP14 (#271840 and #192782), p62 (#56416), and p-cortactin (Y421) (#47768) from Abcam; against DCTN1/p150 (#610473), from BD Transduction Laboratories; PDI (#MA3-019) and Rhodamine-conjugated Phalloidin (#R415) were from Invitrogen; against TOM20 (#11802-1-AP) from Proteintech; and against Src (#OP07) from Calbiochem.

Techniques: Transfection, Construct, Plasmid Preparation, Migration, Staining, Control, Over Expression, Two Tailed Test

Journal: bioRxiv

Article Title: MCT4 and CD147 co-localize with MMP14 in invadopodia and autolysosomes and collectively stimulate breast cancer cell invasion by increasing extracellular matrix degradation

doi: 10.1101/2023.09.03.556100

Figure Lengend Snippet: MDA-MB-231 cells were transfected with siRNA targeting MCT4 (a-b) or CD147 (c-d) or with empty vector, or corresponding MCT4-, CD147-, or MCT4- and CD147 plasmids (e-f), seeded on Oregon-green conjugated gelatin-coated coverslips and grown for 4, 8 or 12 h. Cells were subsequently fixed and stained for F-actin (Rhodamine Phalloidin, RP), and nuclei (DAPI). Suppl. Fig. 5 and 6 show the corresponding full overview images of the cropped images in this figure, as well as the images for CD147 siRNA-2. (a, c, e) Representative images of cells growing on Oregon-green gelatin for the indicated times, presented as merged images of Oregon-green, F-actin (RP) and DAPI. White arrowheads indicate overlap of gelatin degradation with F-actin staining at cell protrusions and intracellular structures. Scale bars: 10 µm. (b, d, f) Quantification of gelatin degradation (performed in ImageJ) by MDA-MB-231 cells after 4, 8, and 12 h. Relative degradation is the degraded area fraction normalized to the cell count. The data points represent the mean relative degradation of 6-13 images per condition. n= 4-5, 4-6, 5-7 for MCT4 KD, CD147 KD and overexpression, respectively. Statistics: (b), (d), (f): Two-tailed paired Students t -test (b) or one-way ANOVA followed by Dunnett’s multiple comparisons post-test (d, f) for each time point. Statistical analysis was performed on log-transformed data. (g) Relative reC1M (MMP-generated fragment of collagen-I) concentration measured by ELISA in samples collected from the culture medium of MDA-MB-231 cells (transfected with vector, MCT4, CD147 or MCT4 and CD147 plasmid constructs) 48 h after seeding on collagen-I. reC1M concentrations are normalized to the vector control within each independent experiment. n=7. One-way ANOVA followed by Dunnett’s multiple comparisons post-test was performed on log-transformed data.

Article Snippet: Primary antibodies against GAPDH (#2118), LC3A/B (#4108), and Golgin-97 (#97537) were purchased from Cell Signaling Technology; against CD147/EMMPRIN (#MAB972) and CD147 (#AF972) from R&D Systems; against MCT4 (#376140) and LAMP-1 (#20011) from Santa Cruz; against MMP14 (#271840 and #192782), p62 (#56416), and p-cortactin (Y421) (#47768) from Abcam; against DCTN1/p150 (#610473), from BD Transduction Laboratories; PDI (#MA3-019) and Rhodamine-conjugated Phalloidin (#R415) were from Invitrogen; against TOM20 (#11802-1-AP) from Proteintech; and against Src (#OP07) from Calbiochem.

Techniques: Transfection, Plasmid Preparation, Staining, Cell Counting, Over Expression, Two Tailed Test, Transformation Assay, Generated, Concentration Assay, Enzyme-linked Immunosorbent Assay, Construct, Control

Journal: bioRxiv

Article Title: MCT4 and CD147 co-localize with MMP14 in invadopodia and autolysosomes and collectively stimulate breast cancer cell invasion by increasing extracellular matrix degradation

doi: 10.1101/2023.09.03.556100

Figure Lengend Snippet: MDA-MB-231 wt cells were seeded on oregon-green conjugated gelatin-coated coverslips, fixed after 8 h of growth and subsequently subjected to IFM analysis. Nuclei were stained with DAPI (blue). (a, b) Representative images of MDA-MB-231 cells on gelatin (green) showing the cellular localization of MCT4 (magenta) or CD147 (magenta), and pY421-cortactin (red, in a) or F-actin (red in b). White arrowheads indicate overlap of gelatin degradation with MCT4/CD147 and pY421-cortactin (a) or F-actin (b). Scale bar: 10 µm. n=3. (c,d) Representative Western blot (c) and corresponding quantification (d) of MMP14. DCTN1 was used as loading control. MMP14 band intensities were normalized to loading control and control (vector). n=6. (e) Representative images of MDA-MB-231 cells on gelatin (green, lower panel) showing the cellular localization of MMP14 (magenta) and F-actin (green or red). MMP14 is shown at a low (upper panel) and high (HE, lower panel) exposure. Scale bar: 10 µm. n=3. (f) Representative images of MDA-MB-231 cells on gelatin (green, lower panel) showing the cellular localization of CD147 (magenta) and MMP14 (red or green). MMP14 (green) is shown at a low (upper panel) and high (HE, lower panel) exposure. Scale bar: 10 µm. n=3.

Article Snippet: Primary antibodies against GAPDH (#2118), LC3A/B (#4108), and Golgin-97 (#97537) were purchased from Cell Signaling Technology; against CD147/EMMPRIN (#MAB972) and CD147 (#AF972) from R&D Systems; against MCT4 (#376140) and LAMP-1 (#20011) from Santa Cruz; against MMP14 (#271840 and #192782), p62 (#56416), and p-cortactin (Y421) (#47768) from Abcam; against DCTN1/p150 (#610473), from BD Transduction Laboratories; PDI (#MA3-019) and Rhodamine-conjugated Phalloidin (#R415) were from Invitrogen; against TOM20 (#11802-1-AP) from Proteintech; and against Src (#OP07) from Calbiochem.

Techniques: Staining, Western Blot, Control, Plasmid Preparation

Journal: bioRxiv

Article Title: MCT4 and CD147 co-localize with MMP14 in invadopodia and autolysosomes and collectively stimulate breast cancer cell invasion by increasing extracellular matrix degradation

doi: 10.1101/2023.09.03.556100

Figure Lengend Snippet: MDA-MB-231 wt cells were seeded on Oregon-green conjugated gelatin-coated coverslips, fixed after 8 h of growth and subsequently subjected to IFM analysis. Nuclei were stained with DAPI (blue). (a) Representative images of MDA-MB-231 cells on gelatin (green, upper panel) showing the cellular localization of MMP14 (magenta) and LAMP1 (red in upper panel, green in lower panel). Scale bar: 10 µm. n=3. (b) Representative images of MDA-MB-231 cells on gelatin (green, upper panel) showing the cellular localization of CD147 (magenta) and LC3 A/B (red in upper, green in lower panel). Scale bar: 10 µm. n=3. (c) . Representative images showing the localization of LC3 A/B (green) and MCT4 (magenta, top panel) and of LC3 A/B (green) and LAMP1 (magenta, lower panel). Scale bar: 10 µm. n=1. (d-e) Representative Western blot and corresponding quantification of LC3-II/I ratio 48 h after transfection with MCT4, CD147 or MCT4 and CD147 plasmids or empty pcDNA3.1 (+) vector as negative control. DCTN1 was used as loading control. n=3. (f) Representative Western blot of p62 protein level 48 h after transfection with MCT4, CD147 or MCT4 and CD147 plasmids or empty pcDNA3.1 (+) vector as negative control. DCTN1 was used as loading control. n=3. (g) Working hypothesis. MCT4-CD147-costructures are delivered to the plasma membrane along with MMP14 and from there the three proteins are retrieved together by endocytosis and delivered to invadopodia in large vesicles of late endosomal/lysosomal origin. Whether the role of MCT4-CD147 in ECM degradation involves a role in formation or recycling of the large vesicles and/or lactate-H + efflux in invadopodia remains to be determined. See text for further details.

Article Snippet: Primary antibodies against GAPDH (#2118), LC3A/B (#4108), and Golgin-97 (#97537) were purchased from Cell Signaling Technology; against CD147/EMMPRIN (#MAB972) and CD147 (#AF972) from R&D Systems; against MCT4 (#376140) and LAMP-1 (#20011) from Santa Cruz; against MMP14 (#271840 and #192782), p62 (#56416), and p-cortactin (Y421) (#47768) from Abcam; against DCTN1/p150 (#610473), from BD Transduction Laboratories; PDI (#MA3-019) and Rhodamine-conjugated Phalloidin (#R415) were from Invitrogen; against TOM20 (#11802-1-AP) from Proteintech; and against Src (#OP07) from Calbiochem.

Techniques: Staining, Western Blot, Transfection, Plasmid Preparation, Negative Control, Control, Clinical Proteomics, Membrane