Journal: bioRxiv

Article Title: Tonic inhibition of the chloride/proton antiporter ClC-7 by PI(3,5)P2 is crucial for lysosomal pH maintenance

doi: 10.1101/2021.10.07.463477

Figure Lengend Snippet: 3 channel colocalization images of a U2OS cell stained with Lysotracker blue, Magic-Red, and Oregon Green 488-dextran. Lysotracker blue and Magic Red stain cell’s acidic compartments and Cathepsin-B positive compartments, respectively.

Article Snippet: Isolated total RNA from WT and KO U2OS cells (QIAGEN RNeasy Mini Kit) reverse transcribed into cDNA (BIORAD iScript ™ cDNA Synthesis Kit) and primers (indicated as followed) were used to quantify mRNA expression levels by realtime PCR using iQ ™ SYBR ® Green Supermix (BioRad) and CFX96 Touch Real-time PCR Detection System (Bio-Rad).

Techniques: Staining

Journal: bioRxiv

Article Title: Tonic inhibition of the chloride/proton antiporter ClC-7 by PI(3,5)P2 is crucial for lysosomal pH maintenance

doi: 10.1101/2021.10.07.463477

Figure Lengend Snippet: Lysosomes swollen by PIKfyve inhibition are hyperacidic. (A) Protocol timeline. U2OS cells were “lysosome-loaded” with Oregon Green 488 dextran (OG loading) and treated for 3h with PIKfyve inhibitor apilimod (100nM, red) or its vehicle (0.25% DMSO, control) before imaging. (B - E) A representative experiment. (B) Images of cells acquired by 445nm laser excitation. Bright objects represent OG positive lysosomes in control (left) versus apilimod (right) conditions. Dotted lines delineate cell outlines. (C) pH calibration curves obtained in control (grey empty circle) or apilimod (red triangle) conditions. Each symbol represents the averaged lysosomal 488/445 ratio of one cell (4 cells per condition). (D) Lysosomal pH measured in control (left, grey empty circles, 16 cells, 4.40 ± 0.02) versus 100 μM apilimod (red triangles, 17 cells, 4.12 ± 0.03) for an individual experiment. Dark symbols represent averages over all cells in the experiment. Each pale symbol represents the averaged lysosomal pH of one cell. Unpaired t-test: p<0.0001. (E) Cumulative distribution of individual lysosomal pH for control (grey empty circle, 16 cells, 732 lysosomes) versus apilimod (red triangle, 17 cells, 628 lysosomes) conditions from the same experiment as in . Each symbol represents the fraction of lysosomes having a pH value below that represented in the abscissa. (F) Apilimod-induced pH shifts (−0.31 ± 0.04) from multiple independent experiments and corresponding fold changes in proton concentration (2.03 ± 0.17); 10 experiments (8-17 cells per condition in each experiment). Proton concentration change ([H + ] change ) was calculated from apilimod-induced pH shift (ΔpH) using the following relation: [H + ] change = 10 −ΔpH Paired t-test: p<0,0001. Dark symbols represent averages over all independent experiments. Each pale symbol represents the averaged lysosomal pH from multiple cells in one experiment. The dot delineated by a black box corresponds to the experiment presented in panels B-E. Data are displayed as mean ± SEM.

Article Snippet: Isolated total RNA from WT and KO U2OS cells (QIAGEN RNeasy Mini Kit) reverse transcribed into cDNA (BIORAD iScript ™ cDNA Synthesis Kit) and primers (indicated as followed) were used to quantify mRNA expression levels by realtime PCR using iQ ™ SYBR ® Green Supermix (BioRad) and CFX96 Touch Real-time PCR Detection System (Bio-Rad).

Techniques: Inhibition, Imaging, Concentration Assay

Journal: bioRxiv

Article Title: Tonic inhibition of the chloride/proton antiporter ClC-7 by PI(3,5)P2 is crucial for lysosomal pH maintenance

doi: 10.1101/2021.10.07.463477

Figure Lengend Snippet: ( A ) Protocol timeline to analyze the evolution of lysosomal pH and size during PIKfyve inhibition. U2OS cells were “lysosome-loaded” with OG then treated for 30min, 1h, 3h or 24h with apilimod (100nM, red) or its vehicle (0.25% DMSO, control) before imaging. ( B ) Representative images of cells acquired by 445nm laser excitation. Bright objects represent OG positive lysosomes. Dotted lines delineate cell outlines. ( C - D ) Comparison of lysosomal pH ( C ) and size ( D ) in control (grey empty circle) and after different apilimod treatment times (red triangle). Each symbol represents the averaged lysosomal pH of one experiment (4 independent experiments, 10-15 cells per condition per experiment). Red dashed line in ( C ) represents the average pH value of 1h to 24h apilimod time points. P-values are obtained from one-way ANOVA Dunnett’s multiple comparisons test. ( E ) Comparison of lysosomal pH in control condition (grey empty circle) and after different treatment times with the PIKfyve inhibitor WX8 (1μM, blue triangle). Each symbol represents the averaged lysosomal pH of one experiment (2 experiments, 10-13 cells per condition per experiment). Blue dashed line represents the average value of 1h to 24h WX8 time points. ( F ) Protocol timeline to analyze the recovery of lysosomal pH and size after washout of apilimod. U2OS cells were “lysosome-loaded” with OG, treated 2h with 100nM apilimod or its vehicle (0.25% DMSO, control) to induce lysosomal hyperacidification and swelling. Apilimod was subsequently washed out with fresh media and imaged 30min, 1h, 3h, 6h and 24h after the washout. ( G ) Representative images of cells acquired using 445nm laser excitation. Bright objects represent OG positive lysosomes. Dotted lines delineate cell outlines. The orange box at 30min time point highlights lysosomal tubulation events. ( H - I ) Comparison of lysosomal pH ( H ) and size ( I ) in control condition (grey empty circle), after 2h apilimod treatment (black triangle) and at different times after apilimod washout (red triangle). Each symbol represents the average lysosomal pH of one experiment (3-7 experiments, 8-15 cells per condition per experiment).

Article Snippet: Isolated total RNA from WT and KO U2OS cells (QIAGEN RNeasy Mini Kit) reverse transcribed into cDNA (BIORAD iScript ™ cDNA Synthesis Kit) and primers (indicated as followed) were used to quantify mRNA expression levels by realtime PCR using iQ ™ SYBR ® Green Supermix (BioRad) and CFX96 Touch Real-time PCR Detection System (Bio-Rad).

Techniques: Inhibition, Imaging

Journal: bioRxiv

Article Title: Tonic inhibition of the chloride/proton antiporter ClC-7 by PI(3,5)P2 is crucial for lysosomal pH maintenance

doi: 10.1101/2021.10.07.463477

Figure Lengend Snippet: ( A ) Protocol timeline. U2OS cells were “lysosome-loaded” with OG and pre-treated 30min with chloroquine (12μM, red), BafA1 (100nM, blue) or a vehicle (0.31% DMSO, grey), before 3h treatment with apilimod (100nM, bottom images) or a vehicle (control, DMSO 0.31%, upper images). ( B ) Representative images of cells acquired by 445nm laser excitation. Bright objects represent OG positive lysosomes. Dotted lines delineate cells outlines. ( C , D ) Comparison of lysosomal pH ( C ) or size ( D ) in control (grey), chloroquine (chloro., red) and BafA1 (bafilo., blue) conditions during untreated (api. -, empty symbols) versus apilimod treated (api. +, filled symbols) conditions. Each symbol represents the averaged lysosomal pH or size of one experiment (4 experiments; 8-11 cells per condition). In (C) Apilimod induces a lysosomal pH shift to more acidic value in chloroquine condition (Paired t-test, p=0.02).

Article Snippet: Isolated total RNA from WT and KO U2OS cells (QIAGEN RNeasy Mini Kit) reverse transcribed into cDNA (BIORAD iScript ™ cDNA Synthesis Kit) and primers (indicated as followed) were used to quantify mRNA expression levels by realtime PCR using iQ ™ SYBR ® Green Supermix (BioRad) and CFX96 Touch Real-time PCR Detection System (Bio-Rad).

Techniques:

Journal: bioRxiv

Article Title: Tonic inhibition of the chloride/proton antiporter ClC-7 by PI(3,5)P2 is crucial for lysosomal pH maintenance

doi: 10.1101/2021.10.07.463477

Figure Lengend Snippet: ( A ) Protocol timeline. U2OS cells were transfected with either mCherry alone, mCherry and PIKfyve, or mCherry and PIKfyve KYA and subsequently “lysosome-loaded” with OG. Cells were imaged after 24h expression to quantify lysosomal pH and size. ( B ) Western-blot indicating PIKfyve or PIKfyve KYA expression level for each condition after 24h expression. Note that PIKfyve endogenous level (control) was too low to be detected. ( C ) Representative cells imaged by 445nm laser excitation (OG channel, left image) and 640nm laser excitation (mCherry channel, right image). The red arrow indicates one cell containing lysosomes loaded with OG (bright dots in OG channel) and expressing mCherry (bright cell in mCherry channel). The white arrow indicates one cell from the same batch containing lysosomes filled with OG but not expressing mCherry. Dotted lines delineate cell shape. ( D , E ) Comparison of lysosomal pH ( D ) or size ( E ) in control (circle), PIKfyve-transfected (triangle) and PIKfyve KYA -transfected (diamond) conditions. For each condition, cells were separated into two populations based on the presence or absence of mCherry. Each symbol represents the averaged lysosomal pH or size of one experiment (6 experiments; 8-11 cells per condition per experiment). P-values: Paired t-test between PIKfyve-mCherry and PIKfyve KYA -mCherry conditions.

Article Snippet: Isolated total RNA from WT and KO U2OS cells (QIAGEN RNeasy Mini Kit) reverse transcribed into cDNA (BIORAD iScript ™ cDNA Synthesis Kit) and primers (indicated as followed) were used to quantify mRNA expression levels by realtime PCR using iQ ™ SYBR ® Green Supermix (BioRad) and CFX96 Touch Real-time PCR Detection System (Bio-Rad).

Techniques: Transfection, Expressing, Western Blot

Journal: bioRxiv

Article Title: Tonic inhibition of the chloride/proton antiporter ClC-7 by PI(3,5)P2 is crucial for lysosomal pH maintenance

doi: 10.1101/2021.10.07.463477

Figure Lengend Snippet: ( A ) Agarose gel electrophoresis of PCR product amplified from ClCN7 deletion site of wild type (ClC7 Wt, left) and CLCN7 knock-out (ClC-7 KO, right) U2OS cells. ( B ) EXON1 sequence from CLCN7 WT (black) and KO (purple and teal) alleles. ( C ) Protocol timeline. Cells were “lysosome-loaded” with OG and treated for 3h with apilimod (100nM, red) or its vehicle (0.25% DMSO, control) before imaging. ( D ) Images from a representative experiment: ClC-7 WT (left) or ClC-7 KO (right) cells acquired by 445nm laser excitation. Bright objects represent OG positive lysosomes in control (top) versus apilimod (bottom) conditions. Dotted lines delineate cell outlines. ( E , F ) Lysosomal pH ( E ) or size ( F ) from ClC-7 WT (grey circle) or ClC-7 KO (red triangle) cells in apilimod (filled symbols) versus control (empty symbols) in a representative experiment. Dark symbols are averages over all cells; each pale symbol represents the average lysosomal pH from one cell. P-values for apilimod effects are obtained from two-way ANOVA. ( G ) There is no significant difference in pH between untreated WT and untreated KO cells (P=0.5675, unpaired t-test). For G, H, I, dark symbols are averages over all experiments; each pale symbol represents the averaged lysosomal pH or size from one experiment (10 and 6 independent experiments for WT and KO conditions, respectively; each experiment represents 8-18 cells per condition). ( H , I ) Comparison of lysosomal pH-shift ( H ) or size-shift ( I ) induced by apilimod treatment in ClC-7 Wt (grey) versus ClC-7 KO (red) cells. Proton concentration change ( H , [H + ] change ) was calculated from apilimod-induced pH shift (ΔpH) using the following relation: [H + ] change = 10 −ΔpH . P-values from unpaired t-test.

Article Snippet: Isolated total RNA from WT and KO U2OS cells (QIAGEN RNeasy Mini Kit) reverse transcribed into cDNA (BIORAD iScript ™ cDNA Synthesis Kit) and primers (indicated as followed) were used to quantify mRNA expression levels by realtime PCR using iQ ™ SYBR ® Green Supermix (BioRad) and CFX96 Touch Real-time PCR Detection System (Bio-Rad).

Techniques: Agarose Gel Electrophoresis, Amplification, Knock-Out, Sequencing, Imaging, Concentration Assay

Journal: bioRxiv

Article Title: Tonic inhibition of the chloride/proton antiporter ClC-7 by PI(3,5)P2 is crucial for lysosomal pH maintenance

doi: 10.1101/2021.10.07.463477

Figure Lengend Snippet: ( A ) RT-qPCR amplification plot obtained from ClC-7 Wt (grey) and ClC-7 KO (red) U2OS cells whole RNA. GAPDH housekeeping RNA level (left-grey and left-red traces) is used as a control of RNA quantity. ClCN7 RNA level corresponds to right-grey and right-red traces. Δ = 3.5 cycles, indicating a 90% decrease in KO ClC-7 RNA level compared to Wt. ( B ) Agarose gel electrophoresis of qPCR product from ClC-7 Wt (grey) and ClC-7 KO (red) U2OS cells GAPDH or CLCN7 cDNA.

Article Snippet: Isolated total RNA from WT and KO U2OS cells (QIAGEN RNeasy Mini Kit) reverse transcribed into cDNA (BIORAD iScript ™ cDNA Synthesis Kit) and primers (indicated as followed) were used to quantify mRNA expression levels by realtime PCR using iQ ™ SYBR ® Green Supermix (BioRad) and CFX96 Touch Real-time PCR Detection System (Bio-Rad).

Techniques: Quantitative RT-PCR, Amplification, Agarose Gel Electrophoresis

Journal: bioRxiv

Article Title: Tonic inhibition of the chloride/proton antiporter ClC-7 by PI(3,5)P2 is crucial for lysosomal pH maintenance

doi: 10.1101/2021.10.07.463477

Figure Lengend Snippet: Left: pH calibration curves of U2OS ClC-7 Wt (WT) or ClC-7 KO (KO) lysosomes loaded with OG in untreated (control) or 3h apilimod-treated conditions (apilimod). Each dot represents the averaged lysosomal 488/445 ratio of one cell (4 cells per condition). Right: Comparison of cumulative distribution of U2OS ClC-7 Wt (grey, control, 9 cells, 427 objects) versus ClC-7 KO (blue, 10 cells, 548 objects) lysosomal pH population. Each symbol represents the proportion of lysosomes having a pH value below the pH value represented in the abscissa axis.

Article Snippet: Isolated total RNA from WT and KO U2OS cells (QIAGEN RNeasy Mini Kit) reverse transcribed into cDNA (BIORAD iScript ™ cDNA Synthesis Kit) and primers (indicated as followed) were used to quantify mRNA expression levels by realtime PCR using iQ ™ SYBR ® Green Supermix (BioRad) and CFX96 Touch Real-time PCR Detection System (Bio-Rad).

Techniques: